Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1227

Studies on the Molecular Biology ofthe Mouse Pneumotropic

Polyomavirus

BY

SHOUTING ZHANG

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2003

Studies on the Molecular Biology of theMouse Pneumotropic Polyomavirus

SHOUTING ZHANG

Dissertation for the degree of Doctor of Philosophy, Faculty of Medicine, inMedical Virology presented at Uppsala University in 2003

ABSTRACT

Zhang, S. 2003. Studies on the Molecular Biology of the Mouse Pneumotropic Polyomavirus.Acta Unviersitatis Upsaliensis. Comprehensive summaries of Uppsala Dissertations from theFacuöty of Medicine 1227. 48pp. Upsala. ISBN 91-554-5541-7

The Murine Pneumotropic Virus (MPtV), in contrast to the other Murine Polyomavirus(MPyV), appears to be non-tumourigenic in its natural host. Instead, MPtV causes acutepneumonia and can serve as a model in studies of polyomavirus-induced disease. In initialexperiments, MPtV large T-antigen (LT) was expressed in a heterologous system. LT wascharacterized with regard to its metabolic stability and cell immortalizing activity and, afterpurification, to its specific DNA binding.

The absence of permissive cell culture system for MPtV has hampered its study. Wemade attempts to widen the host range of the virus by modifying the regulatory and lateregions of the genome. The enhancer substitution mutant (KVm1), having a transcriptionalenhancer substituted with a corresponding DNA segment from MPyV, was able to replicate inmouse 3T3 cells and form virus particles that were infectious in mice. However, efficientinfection of cells in vitro was not achieved with this mutant virus, possibly due to the absenceof virus-specific receptors on the cells. The capsid protein substitution mutants, having capsidprotein genes of MPyV, for which receptors are present on a variety of cell types, showed alsono cytopathic effect, despite an enhanced viral DNA replication and assembly of virusparticles.

MPtV-DNA extracted from virus in lung tissue of infected mice had a heterogeneousenhancer segment. A majority of the DNA molecules had a structure differing from thestandard-type. A 220 base-pair insertion at nucleotide position 142 with a concomitantdeletion of nucleotides 143 to 148 was a prominent variation. Other genome variants showedcomplete or partial deletions of the insertion and surrounding sequences in the viral enhancer.In relation to the standard-type, all variant genomes showed differences in the activities oftranscriptional promoters and the origin DNA replication. Analysis by DNA reassociationshowed that a large number of nucleotide sequences related to the 220 base-pair insert in theMPtV genome were present in mouse and human DNA, but not in Escherichia coli DNA.Together, the data suggest that the 220 base-pair insertion is related to a transposable elementof a novel type.

Key word: Murine pneumotropic virus, Kilham mouse polyomavirus, large T-antigen,enhancer, regulatory region rearrangement, gene expression regulation, DNA replication,transposable elements.

Shouting Zhang, Department of Medical Biochemistry and Microbiology,Biomedical center, Box 582, Uppsala University, SE-751 23 Uppsala, Sweden

”Shouting Zhang 2003

ISSN 0282-7476ISBN 91-554-5541-7

Printed in Sweden by Eklundsholf Grafiska AB, Uppsala 2003

To my family,

e s p e c ia l l y ,

to the memory of my father

This thesis is based on the following articles, which in the text are referred toby their roman numerals:

I. Shouting Zhang, Göran Magnusson. 2003. Large T Antigenfrom Murine Pneumotropic Virus: Expression and analysis ofDNA Binding. Manuscript.

II. Shouting Zhang, Göran Magnusson. 2002. KilhamPolyomavirus: Activation of Gene Expression in MouseFibroblast Cells by an Enhancer Substitution. Journal ofVirology, 75(21):10015-10023.

III. Shouting Zhang, Göran Magnusson. 2003. Cellular MobileGenetic Elements in the Regulatory Region of the PneumotropicMouse Polyomavirus Genome: Structure and Function in ViralGene Expression and DNA Replication. Journal of Virology,77(6) (In press).

Reprints were made with the permission from the publisher: the Americanfor Microbiology.

TABLE OF CONTENTS INTRODUCTION ....................................................................................................7

VIRUS .....................................................................................................................7 POLYOMAVIRUSES..................................................................................................7

Introduction.......................................................................................................7 Polyomaviruses as paradigms...........................................................................8 Virus genome ....................................................................................................9 Replication cycle .............................................................................................10 Early proteins..................................................................................................14

POLYOMAVIRUS REGULATORY REGIONS...............................................................19 Structure..........................................................................................................19 Rearrangements ..............................................................................................19

MOUSE PNEUMOTROPIC VIRUS .............................................................................21 CELLULAR MOBILE DNA ELEMENTS ....................................................................22

Classification ..................................................................................................22 Functions ........................................................................................................24 TEs and polyomaviruses .................................................................................24 TEs and cancer ...............................................................................................25

PRESENT INVESTIGATION ..............................................................................26

IMPETUS AND PROJECTS........................................................................................26 CHARACTERIZATION OF MPTV LARGE T-ANTIGEN ..............................................27 MPTV HOST RANGE MUTANT- (KVM1) ENHANCER SUBSTITUTION ......................28 MPTV HOST RANGE MUTANT- LATE REGION SUBSTITUTION.................................29 CELLULAR TRANSPOSABLE ELEMENTS IN MPTV GENOME....................................31

FAQS .......................................................................................................................33

ACKNOWLEDGEMENTS ...................................................................................36

REFERENCE .........................................................................................................38

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Abbreviations

MPtV Murine pneumotropic virus (previously called Kilham mouse polyomavirus, KV)

KV Kilham mouse polyomavirus, currently called MPtV MPyV Mouse polyomavirus BKPyV BK polyomavirus JCPyV JC polyomavirus PML Progressive multifocal leukoencephalopathy SV-40 Simian virus 40 BFDV Budgerigar fledgling disease virus LT Large tumor antigen st Small tumor antigen RR or rr Regulatory region pRb Retinoblastoma protein Hsc Heat shock cognate protein kd kilo-dalton kb kilobase-pair bp Base-pair nt nucleotide ori DNA replication origin PP2A Protein phosphatase 2A ses SV-40 encapsidation signal TE Transposable element MITEs Miniature inverted-repeat transposable elements SINEs Short interspersed nuclear elements LINEs Long interspersed nuclear elements

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Introduction Virus

Viruses are very small, infectious, obligate intracellular (molecular) parasites and are the causes of many diseases. It is possible that viruses have been present on earth since the existence of the first form of life, but knowledge of their existence has had a much shorter life span (117). Viruses are far simpler than even the smallest cellular microorganisms and lack the complex energy-generating and biosynthetic systems necessary for independent existence. On the other hand, viruses are not the simplest biologically active agents: even the smallest virus, built from a very limited genome and a single type of protein, is significantly more complex than other parasitic molecular pathogens, such as viroids, which only comprise a single small molecule of RNA, and prions, which are believed to be single molecules of protein (63).

Viruses have played, and still play, a central role in the unraveling of general principles in modern biology. Since their genomes are small, they provide simple genetic systems that are useful for studying the structure and function of genes. The viral oncogenes provide some of the best model systems to reveal the mechanisms of cellular growth control and oncogenic transformation of mammalian cells. A vast amount of the knowledge that we now have has benefited from the studies of certain viruses, for example, regulation of transcription and transcription factors, messenger RNA regulation, polyadenylation and translation (14, 57, 95, 100). Viruses will continue to be indispensable tools in the deciphering of regulatory mechanisms that control gene oncogene expression.

Polyomaviruses

Introduction The polyomaviruses are a subfamily of the Papovaviridae, a family of

small, nonenveloped viruses with icosahedral capsids. They are widely distributed in nature. Twelve members of this family have now been identified. All have capsids that are of the same size and are constructed from three capsid proteins. All have genomes of approximately 5,000 bp and display a similar genomic organization. Many regions of their genomes are highly conserved, demonstrating that the polyomaviruses have descended from a common ancestor. Most of these viruses display a narrow host range and do not productively infect other species. However, infection of cells in which these viruses do not grow productively often leads to malignant transformation of the infected cells. Infection with polyomaviruses usually

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occurs during infancy and causes few acute symptoms, and then a life-long, persistent infection is established. Immunosuppressive treatment of persistently infected hosts frequently results in reactivation of the virus and may result in severe disease, at least in the case of human JC polyomavirus infection. Infection with avian polyomavirus (BFDV) results in the death of affected birds (21). Table 1 lists the member’s natural hosts and the main characteristics of infections (61).

TABLE 1. Natural hosts and main characteristics of polyomavirus infections (Adapted from ref 60 & 61) Host Virus Characteristics Human BK polyomavirus

(BKPyV) Common infection of early childhood; persists in

kidney epithelium and lymphocytes JC polyomavirus

(JCPyV) Common infection of late childhood; persists in

kidney epithelium, lymphocytes, and brain; produces PML in immunocompromised hosts

Monkeya Simian virus 40 (SV-40) Natural infection of macaques in Asia; persists in kidney; produce PML-like disease in immunocompromised macaques

Baboon polyomavirus 2 (PPyV)

Natural infection of baboons in Africa

African green monkey polyomavirus (AGMPyV)

Virus of Africa green monkey; multiply in B lymphoblasts

Cattle Bovine polyomavirus (BPyV)

Common infection of cattle; probably persists in Kidney

Rabbit Rabbit kidney vacuolating virus (RKV)

Infection of cottontail rabbits

Mouse Mouse polyomavirus (MPyV)

Natural infection of mice in wild; may infect laboratory mouse colonies; persists in Kidney

Murine pneumotropic virus (MPtV)

Natural infection of mice; infects lung endothelium

Hamster Hamster polyomavirus (HaPyV)

Produces cutaneous tumors in hamster

Athymic rat

Rat polyomavirus Affects parotid gland

Parakeet Budgerigar fledgling disease virus (BFPyV)

Produces acute fatal illness in fledgling budgerigars

aA virus initially described as originating from stump-tailed macaques was subsequently identified as bovine polyomavirus (Parry et al, 1983).

Polyomaviruses as paradigms The initial impetus for the studies of these viruses was their oncogenic

potential. Later, because they have small genomes and are easy to cultivate in tissue culture and purify, polyomaviruses have been simple model systems for diverse studies in molecular biology. Over the past 30 years these viruses have been used to examine many fundamental questions in eukaryotic molecular biology. Some major discoveries from work on these

9

viruses are the finding of the supercoiled DNA structure, the identification of eukaryotic DNA replication origins, the discovery of enhancers, elucidation of the organization of promoters involved in transcriptional regulation, and the detailed understanding of negative and positive control of gene expression. In addition, replication of SV-40 origin-containing DNA in vitro has served as a model for understanding eukaryotic chromosomal DNA replication.

Studies on these oncogenic viruses as agents for cell transformation have led to important discoveries with relevance to human cancer. The ras, myc, fos, abl, and src oncogenes and the p53, erbB2, and Wnt-APC tumor suppressor gene pathway are a few prominent examples of genes and pathways the discovery of which is grounded in research on tumor viruses (12). MPyV is one of the few oncogenic viruses amenable to study both in culture and in its natural host. The polyoma-mouse system affords opportunities to test the relevance of transforming functions in the context of tumor formation in a variety of tissue and also to explore the role of the host genetic background in determining susceptibility or resistance to tumor development.

Virus genome All members of the polyomavirus family have DNA genomes with a

size of approximately 5 kb. In the cell and in the capsid, this DNA is wound around nucleosomes to form a “mini-chromosome”. Most viral DNA molecules contain a full complement of nucleosomes, but some lack nucleosomes over viral regulatory sequences. Histone H1 is not present in virion mini-chromosomes, whereas the mini-chromosomes found within infected cells contains it (200). The circular genomes are superficially arrayed in a similar fashion, and three distinct functional regions can be identified: the regulatory region (RR), the early coding region, and the late

FIG.1. SV-40 genomic organization of the alternatively spliced early (large T-antigen [LT], small t-antigen [St], and 17K T-antigen) and late (VP1, VP2, and VP3) proteins. The early (PE) and late (PL) promoters exist in opposite orientations that flank the SV-40 origin (Ori) of replication. (Redrawn from ref. 191).

SV-40 genome� 5243 bp�

PL�

Ori�

PE�

VP1�

VP3�

VP2�

St� 17kT�

LT�

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coding region. The RR comprises enhancer, promoters and a single unique origin for DNA replication (ori). Transcription extends bi-directionally from initiation sites near the origin, with early and late mRNAs being transcribed from opposite strands of the viral genome. The early region encodes the viral regulatory proteins, the tumor or T antigens, so called because they can be detected with antisera derived from animals bearing tumors induced by these viruses or by cells transformed by these viruses (60). SV-40 and other primate polyomavirus encode two T antigens, designated large T- and small t-antigens on the basis of size. Mouse polyomavirus (MPyV) and hamster polyomavirus (HaPyV) encode three T antigens—large T, middle T, and small t. A tiny t-antigen was recently identified in mouse polyoma and SV40. All of the T antigens of each virus share N-terminal sequences and contain different C-terminal regions resulting from alternative splicing of a common pre-mRNA. Figure 1 shows the genome structure of SV-40. The late regions encode three capsid proteins VP1, VP2, and VP3. As with the early mRNAs, late mRNAs are generated from a common pre-mRNA by alternative splicing. SV-40 and the human polyomaviruses encode a fourth late protein, called the agnoprotein.

Replication cycle Productive infection of cells by polyomaviruses can be divided into

early and late stages. The early stage starts with attachment of virus to cells

FIG.2. Replication cycle of polyomaviruses. Steps in the replication cycle are indicated by numbers as follows: 1, adsorption of virions to the cell surface; 2,entry by endocytosis; 3, transport to the cell nucleus; 4, uncoating; 5, transcription to produce early mRNAs; 6,

11

translation to produce early proteins (T-antigens); 7, viral DNA replication; 8, transcription to produce late mRNAs; 9, translation to produce late proteins (capsid proteins); 10, assembly of progeny virions in the nucleus; 11, entry of virions into cytoplasmic vesicles; 12, release of virions from the cell by fusion of membrane vesicles with the plasma membrane; 13, released virion. Virions are most likely also released from cell at cell death, when virions have an opportunity to leak out of the nucleus. In non-permissive cells the first six steps occur normally, but viral DNA replication cannot occur and subsequently events do not take place. (From ref. 60 &61) and continues until the beginning of viral DNA replication. The late stage extends from the onset of viral DNA replication to the end of the infection cycle and involves the replication of viral DNA, expression of the viral late genes encoding the capsid proteins, the assembly of progeny virus particles in the nucleus of the infected cells, and the release of virus. Virions are most likely released from cells at cell death, when they have an opportunity to leak out of the nucleus. In non-permissive cells the first six steps occur normally, but viral DNA replication cannot occur and subsequent events do not take place. Figure 2 illustrates the steps in the infectious cycle of MPyV and SV-40.

Depending upon the kind of cells infected, the response to infection by polyomaviruses can take either of two courses. Productive infection results in virus replication, production of infectious progeny virus particles and death of the host cell. Nonproductive infection results in interruption of the virus life cycle and failure to produce infectious progeny virus. MPyV causes a lytic infection of mouse cells and a nonproductive infection of hamster or rat cells. SV-40 causes a lytic infection of monkey cells but causes a nonproductive infection of mouse cells. The time course of lytic infection by MPyV and SV-40 depends upon the multiplicity of infection and the growth state of host cells. A single cycle of infection by MPyV takes 24-48 hours and by SV-40 48-72 hours.

Adsorption and receptors The initial step in the establishment of virus infection is the interactions between the virus and receptors present on the surface of cells and tissues. Virus-receptor interactions play a major role in determining virus tropism and tissue-specific pathology associated with virus infection (120). In some instances virus tropism is strictly determined by these interactions (9, 43, 86, 203) and in other instances, successful entry into a cell is necessary but not sufficient for virus growth (7, 11, 134, 167). In these cases, additional permissive factors that interact with viral regulatory elements are required. In general, viruses that have a very narrow host range and tissue tropism, such as JC polyomavirus, are often shown to interact with high affinity with limited number of specific receptors present on susceptible cells (85, 128). For MPyV, the primary receptors or attachment proteins are a heterogeneous

12

class of sialoglycoproteins that are abundantly and broadly expressed on cell surface. The virus is thus able to establish a rapid and widely disseminated infection in the newborn mouse (12, 45). Competition experiments indicate that SV-40 and MPyV receptors are distinct (10). Uptake of SV-40 occurs specifically by the caveolar pathway (145, 152, 154). After binding to the plasma membrane via major histocompatibility (MHC) class I antigen (23, 187), virus particles diffuse laterally along the membrane until trapped in caveolar (153). A secondary receptor may exist to mediate the tight binding of the virus to the caveolar membrane and trigger the signal needed to induce the endocytic process (152). The JCPyV does not share receptor specifically with SV-40 on human glial cells (119). Instead of the caveolar pathway, JCPyV enters human glial cells by clathrin-dependent receptor-mediated endocytosis (155). The attachment to a cell surface is mediated by sialoglycoproteins, including alpha-acid glycoprotein, fetuin, and transferring receptor, and this attachment depends on alpha-2-3-linked sialic acids and N-linked sugar chains (108), whereas neoglycoprotein containing alpha-(2-6)-linked sialic acid has the highest affinity (108, 120). For MPyV and SV-40, it was suggested that VP2 also interacts with cellular membranes (41, 171, 188), whereas VP1, the outer capsid protein, binds to the specific receptors present in the cell surface to establish the infection (6, 12, 35, 187).

Onset of early transcription Once virions have been uncoated, the viral mini-chromosome can be

used as a template for transcription by RNA polymerase II to produce early viral mRNAs, derived from the early region of the viral genome. Viral early promoters and the enhancers govern the production of early mRNAs. Since they contain multiple sites to which transcription factors can bind productively, the enhancers play a pivotal role in regulating viral transcription and DNA replication, as well as in controlling the viral host range in tissue culture systems, in particular the ability to replicate in cells at different stages of differentiation (4, 5, 87). The early promoters of all polyomaviruses are autoregulated by their LTs (39, 103, 165). Early after infection, when LT levels are much lower, LT stimulates transcription from its own promoter by a mechanism that does not require direct binding of T-antigen to viral DNA (205). However, when LT levels are relatively high, the activity of early promoter will be inhibited (39).

DNA replication T-antigen stimulates resting cells to enter the cell cycle and replicate

their DNA, thereby creating an environment optimal for viral DNA replication. Soon after T-antigen is detected within cells, the levels of many cellular enzymes increase, reflecting the ability of T-antigen to stimulate

13

expression from a variety of viral and cellular promoters (71, 81, 163). T-antigen also stimulates the expression of ribosomal RNA synthesis (182). Replication of viral DNA within infected cells requires a functional origin of DNA replication, LT with its DNA-binding and helicase activities intact, and the set of cellular proteins involved in replicative DNA synthesis. Ori regions are an inverted repeat of 14 bp on the early gene side of ori, a GC-rich palindrome of 23-34 bp in the center, and an AT sequence of 15-20 bp on the late side. These common structures participate in binding proteins and in initiation of viral DNA replication. In the presence of ATP, T-antigen assembles into a double hexamer by binding to the ori (18, 47, 50, 97, 132) and causes structural changes in the flanking DNA sequences. The DNA at the early palindrome becomes melted and the AT-rich region is untwisted (17, 18, 49, 176). Then T-antigen functions as a helicase to bidirectionally unwind the DNA in the presence of replication protein A (RPA), a single-stranded DNA-binding protein, and topoisomerase I (48, 56, 206). Other cellular factors, including DNA polymerase α-primase, RFC, PCNA, and DNA polymerase δ, are also recruited to form the replication fork (3, 24, 141, 142, 201, 208). The association of the viral ori, the T-antigen double hexamer and RPA is referred to as the preinitiation complex. This complex converts to an initiation complex by recruiting cellular DNA polymerase α-primase. It is this step in viral DNA replication that defines the very limited host range for polyomaviruses (60).

Onset of late transcription Following the onset of DNA replication, late transcripts are synthesized

to a detectable level and increase rapidly until the end of the lytic infection (33), whereas the early transcription is repressed during the late phase (59, 110). Two primary mechanisms involved in the production of high levels of the late transcripts are amplification of templates for late transcription by viral DNA replication and transactivation of transcription from the viral late promoter by LT. The structure of replicated minichromosomes probably also plays a key role in activation of late transcription (194). Besides, studies on MPyV implied a role for middle T and /or small t-antigen in the activation of the early-to-late transcriptional switch (34).

Virus assembly Virion proteins VP1, VP2, and VP3 are synthesized in the cytoplasma of

infected cells and transported to the nucleus for assembly into virions. Viral packaging is a complex biological process that involves interactions between proteins as well as between proteins and DNA, leading to a three-dimension structure. Packaging occurs by gradual addition and organization of capsid proteins around the viral chromatin (67). In SV-40, a specific DNA element,

14

the encapsidation signal (ses), is required for packaging (146). ses is present within SV-40 RR, in close proximity to the origin of replication, encompassing a 200 bp region containing the ori, six repeats of GGGCGC (GC boxes), 26-bp of the enhancer (44) and recognition signals for several cellular regulatory proteins, including Sp1, AP-2 and p53. The function of ses is suggested to be to act as a sensor for the level of the viral late proteins and to allow the SV40 minichromosome to form the correct higher order structure which is required for packaging (44). Co-expression of the three capsid proteins in insect cells led to spontaneous assembly of SV-40-like particles. This process, which utilizes naked DNA, is not dependent on the ses (173). However, with ses, the specific viral DNA packaging occurs with a far higher affinity than that for cellular DNA (74). Since there are several DNA elements in ses that may bind to Sp1, it is believed that Sp1 recruits the capsid proteins to ses, where they form the nucleation center for capsid assembly.

Early proteins The functions of tumor antigens are illustrated below (12, 156, 164,

191). The domain structures of SV-40 T-antigen are depicted in figure 3.

FIG.3. The domain structure of SV-40 early proteins – T antigens. SV-40 large T-antigen is a large 708-amino-acid multidomain protein. It consists of an amino-terminal J domain (that directly contacts Hsc70), followed by an pRB family binding motif (LXCXE) which binds to all three pRB family members, pRB, p107, and p130; a nuclear localization signal (NLS); a

15

specific DNA binding domain (ori binding); a zinc finger motif (Zn); ATPase domain; a bipartite p53 binding domain that is also essential for mediating interaction with the transcriptional adapter protein p300; and an HR specificity region. 17kT antigen is comprised of the first 131 amino acids of large T antigen (including the J domain [J]) and a pRB-binding (LXCXE) motif, plus an additional four unique amino acids. The amino terminus of small t antigen is comprised of the J domain (amino acids 1 to 82), plus an additional carboxyl-terminal domain that binds to the multimeric protein phosphatase 2A (pp2A) comprised of a catalytic and a regulatory peptide. (From ref. 191).

Large T-antigen The LTs of the polyomaviruses are complex multifunctional proteins

that have multiple enzymatic activities, interact with several cellular proteins and perform several different roles during infection. In addition to their roles in regulating viral transcription, DNA replication and viral assembly, they are also responsible for tumor induction by these viruses. The LTs of polyomaviruses are closely related and contain identical or similar sequences over most of their lengths (60). LTs encoded by MPyV, HaPV, LPV, MPtV, BPyV and BFPyV contains insertions within the amino-terminal domain relative to that of SV-40, BKPyV and JCPyV, whereas LTs encoded by these three polyomaviruses contain approximately 70 amino acids at their carboxyl-terminus that have been shown to encode an activity in viral assembly.

LTs are modified posttranslationally in several ways. SV-40 LT contains two clusters of phosphorylation sites (174) and is modified by O-glycosylation (175), acylation (107), adenylation (22), poly(ADP)-ribosylation (73) and amino-terminal acetylation (135). Little is known about the functions of any of these modifications except for phosphorylation, which is known to play a major role in controlling the activities and functions of the protein.

The N-terminal parts of LTs, except that of Bovine PyV, are homologous to the conserved domain of the DnaJ family of molecular chaperone (185) and function as a chaperone J protein. Chaperone proteins are a group of proteins that promote the folding of proteins and prevent protein aggregation during periods of cellular stress (84). DnaJ and its homologous proteins (J proteins) are co-chaperone regulators that stimulate the ATPase activity of Hsc70 and promote substrate interactions with Hsc70s (29, 32, 42). All J proteins contain a domain of approximately 70 amino acids known as the J domain that binds directly to Hsc70. Mutational analysis of SV-40 LT has demonstrated that the LT J domain is essential for multiple viral activities, including viral DNA replication, transformation, transcriptional activation and virion assembly (191).

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oriΒΑ2 1 A B C

5295/0 1604975Mouse polyoma virus regulatory region

LT binding regionEnhancer

IIIIII

SV-40 regulatory region

72 72 21 21 22 ori

0/5243 5163320

FIG.4. The structure of the SV-40 and MPyV regulatory region. Shown are the core origin of viral DNA replication, enhancer region and large T-antigen binding region. Pentanucleotides involved directly in binding are indicated by arrowheads that point the directions of the pentanucleotides, 5’-GRGGC-3’. For SV-40 the locations that serve as enhancers and of the three nearly perfect 21-bp GC-rich repeats of the two 72-bp repeats are also shown. (Redrawn from ref 60 & 61) LTs are complex DNA-binding proteins that can bind double-, as well as single –stranded DNA (123, 207, 208). LTs interact specifically with several pentameric sequences (GRGGC) at the origin of DNA replication and in the early promoter, except in BFDV, where this motif is not found in the ori region (124). Several binding sites have been identified in SV-40 and MPyV (Fig.4). Binding to DNA permits LTs to autoregulate production of early mRNAs and to play a central role in initiation of viral DNA replication. The ability of LT to initiate DNA replication is regulated positively or negatively by its state of phosphorylation (202).

Helicase activity of LTs (48) is crucial for viral DNA replication and requires the ability to bind to viral DNA and the functional ATPase activity (3). The ATPase activity domain is the most highly conserved domain among all polyomavirus LTs (156).

LT binds to the active and hypophosphorylated pRb to release E2F, a transcription factor that activates the transcription of S-phase genes and, in some instances also induces immortalization of primary embryo fibroblasts (112, 113). It has been reported recently that LT induces apoptosis, an activity completely dependent on binding to pRb, but independent of the J-domain (177). Like the E1A protein of adenovirus, SV-40 and MPyV LTs can bind to p300 (177). p300 and the related protein CBP (CREB-binding protein) are transcriptional adapter (or co-activator) proteins that coordinate the formation of specific transcription factor complexes (190) and enhance the transcriptional activity of many transactivators, including p53. Expression of LTs inactivates p300 transcriptional activation and changes

17

the phosphorylation state of p300, consequently inhibiting p53 transactivation activity and fostering a cellular environment conducive to viral replication (89). Figure 5 depicts the inhibition of LT to both pRb and p53 pathways.

Both genetic and biochemical data indicate that a wild-type LT is required for virion assembly for at least two independent steps of virion maturation. In SV-40, some LT mutants in either the extreme carboxyl-terminal or the J domain replicated DNA and produced VP1-3, but failed to assemble mature virions (183, 184). The two mutants can complement each other to infect cells as well as wild-type virus, suggesting that they code for two separate functions (151). The virion assembly defect of these mutants may be indirect, for example, due to failed induction of a cellular peptide that is necessary for proper virion assembly. Alternatively, the defect may result from physical inability of LT to function directly in the assembly process.

p16INK

Cyclin D/cdk4,6

pRb

E2F

apoptosis

MDM2

baxp53

DNA PK

DNA damageUnscheduledDNA synthesis

Hsc70

Hsc70p21

S phase genes,cell cycleprogression

??

p19ARF

TAg

TAg

FIG. 5. T antigen inhibits both the pRB and p53 tumor suppressor pathways. Mitogenic stimulation triggers phosphorylation of pRB by cyclin D/CDK4,6 complexes. This releases pRB-mediated repression of E2F transactivation, thus allowing the synthesis of the enzymes necessary for cell cycle progression and DNA replication. T antigen induces free E2F. p53, "the guardian of the genome," inhibits cell cycle progression; one way this is accomplished is via p21, which inhibits phosphorylation of pRB. Additionally, p53 induces apoptosis when activated by genotoxic stresses. T antigen inhibits multiple activities of p53. Hsc70 inhibits apoptosis and may play an additional role in regulating the activities of both pRB and p53. (From ref. 191).

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Studies also revealed that mouse polyoma virus LT can mediate high-frequency recombination and that this recombination-promoting activity that can be dissociated from its replicative function (186). Recombination mediated by LT requires the viral replication origin and some homology within the integrated sequences. Homology is usually provided by complete or partial tandem insertions of viral DNA (19, 40, 114, 137), and this homologous recombination promoted by LT does not require recombination hot spots in the viral genome other than the replication origin. This finding may explain why the expression of mouse polyoma virus LT in the presence of a functional viral origin can be toxic to some cells. The toxicity may due to its capability to promote high-frequency intra-chromosomal recombination in the vicinity of a functional replication origin.

Middle T-antigen Middle T-antigen is the major transforming protein (36, 109, 111). It

alone can transform some established fibroblasts (196). In SV-40 and human polyomaviruses, which do not encode middle T-antigen, the LT is the transforming protein. Middle T-antigen is not necessary for initiating viral DNA replication, but in resting cells middle T-antigen enhances viral DNA replication (129).

Small t-antigen The polypeptide of small t-antigen is present both in the cytoplasm and

in the nucleus. It is necessary for the formation of mature virions (68, 129). Although LT is sufficient for viral DNA replication, small t-antigen can enhance this process (129). The SV-40 small t-antigen has also been reported to play an essential role in the development of SV-40-induced mesotheliomas (38).

Small t-antigen associates with one cellular protein, the regulatory A subunit of PP2A. Small t-antigen thereby displaces the regulatory B subunit and inhibits the phosphatase activity of the protein (210).

Tiny t-antigen Tiny T-antigen is found to be located diffusely in the cytoplasma and

functions as a DnaJ-domain (164) which stimulates the ATPase activity of the Hsc70 protein (193). The tiny T-antigen is expressed during infection in small amounts relative to other T antigens and has a very short lifetime (20 minutes). It is also proposed that SV-40 tiny T-antigen may function in fine-tuning SV-40-mediated cell cycle control (213).

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Polyomavirus regulatory regions

Structure The critical features of the mouse polyoma and SV-40 RR are shown in

figure 5. In SV-40, three binding sites (I, II, and III) for SV-40 LT are located in this region. Upstream from the start sites for transcription is located an AT-rich region that contains a TATA-box-like element and functions to direct transcription initiation to a site approximately 30 nucleotides downstream. Upstream from the AT-rich region is a GC-rich cluster that contains three 21-bp repeats which are important promoter elements, but are not absolutely essential for early transcription (13, 64). Lying further upstream are two copies of a 72-bp repeat elements that function as enhancers, which act to increase transcription initiation in an orientation-independent mechanism and show little dependence on distance for their enhancing effects (96). Besides the regulatory function in transcription, enhancers influence the activity of adjacent origins of DNA replication (52). The SV-40 enhancer is quite strong and functions well in cells of a variety of species and types. It has been widely used as an enhancer to drive expression of heterologous genes.

The organization of MPyV RR is similar to that of SV-40, but has some important differences (Fig.5). The MPyV enhancer has been defined as a 244-nucleotide fragment between the BclI and PvuII sites (nt 5023 to 5267) (46) and can be subdivided by using a convenient restriction endonuclease site into two major domains, A (nt 5023 to 5130) and B (nt 5131 to 5267) (4), which can function independently and confer different cell type specificities (87).

Five independent DNA-binding sites for LT, designated 1-2 and A-C, have been identified in mouse polyoma virus DNA (54, 157). Strong binding region (Sites A-C) contains the early promoter. Binding of LT to one or more sites in this region inhibits the activity of the early promoter (15), whereas binding to site 1 and site A is essential for initiation of viral DNA replication.

Rearrangements RR structural diversity has been found in many members of the

polyomavirus family. In comparison to other parts of polyomavirus genomes, their RRs are extremely variable in structure. A large number of RR variants have been identified among various polyomaviruses [human polyomavirus BK (139, 143), JC (212), and SV-40 (116)]. Most of the variations occur in a short segment of the genome first identified in mouse polyoma virus DNA as an enhancer of early transcription (46). Natural SV-40 isolates from simian brain or kidney tissues typically have an archetypal

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RR arrangement with a single 72-bp enhancer element. The so-called protoarchetypal SV-40 lacks a duplicated sequence in the GC-rich region. Occasionally, SV-40 strain variants arise de novo with complex RRs, which typically contain sequence reiterations, rearrangements, and/or deletions (116).

JCPyV DNAs recovered from different patients suffered from progressive multifocal leukoencephalopathy (PML), a fatal progressive demyelinating disease, show variations in their RRs (92) (Fig.6). PML isolates typically show a duplication of domain A and a deletion of domain B of the RR (211). A high frequency of rearrangement JCPyV RR has also been detected in brain tissues, cerebrospinal fluid, peripheral blood leukocytes from individuals with different clinical conditions (37). BKPyV RR, like those of SV-40 and JCPyV, contains a lot of rearrangements in its natural isolates. The rearrangements consist of duplications, deletions and, occasionally, point mutations.

UrineIsolates

PML brainIsolates

Lat e ge nesEarly gen esDomain A Domain B

Archetypal regulatory region

FIG.6. Representation of the regulatory region rearrangements in the JCPyV isolates from the urine of normal individuals or from the brain tissue of patients with PML. The unrearranged archetypal regulatory region is found in most kidney or urine isolates from individuals without PML (examples shown in the top two lines). For the PML brain isolates, the lines represent sequences shared with the archetype, parallel lines represent duplications and gapes indicated deletions relative to the archetype. Rearrangements in the regulatory region are generally found in the viral DNA of isolates from the JCPyV infected brains with PML and from lymphocytes. These rearrangements involve duplications of the region corresponding to domain A and deletions involving the sequences in domain B. The regulatory regions schematically represented here have been redrawn from figures published in ref 60, 61 & 92.

How these rearrangements are generated is not clear. It is unknown

whether these rearrangements were selected from a natural virus mixture with different RRs or whether they are arose by an unknown mechanism from viruses with archetypal RRs during infection in individuals or during

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viral passage in primary cells in culture (58, 116). The patterns of RR rearrangements display high correlation, suggesting that rearrangements are generated in the host from a basic archetypal sequence within which there are preferred targets for strand breaks, allowing deletion or duplication of sequence blocks (8). Most of the polyomavirus rearrangements occur in the enhancer regions. The enhancer contains a number of transcription factor-binding sites (12), which are functional elements of a variety of viral and eukaryotic DNA replication systems (198). The number, type, and rearrangement of transcription factor-binding sites in the RR of variants help to determine their replication efficiency. The complexity of enhancers relates to the need of regulation and cell-type specificity of gene expression. Only limited information is available about the functional significance of the different RR rearrangements. Polyomavirus infections of immunocompetent hosts are inapparent, with the persistence of small amounts of slow-growing viruses. It is likely that in natural infections the faster-growing viruses are cleared quickly by the immune system, whereas some slower-growing viruses may escape immune elimination and persist. It is not surprising that wild isolates, having archetypal RRs, grow more slowly in tissue culture than do those with complex RRs (116). The RR rearrangements thus may be associated with the persistent state of viral infection (58). However, the activation of persistent virus is not likely to be dependent on the selection of cell specific promoter elements. Instead, it may be controlled by conditions closely related to the immunological competence of the viral host (58). The activation of viral replication may result in the JCPyV PML variants which probably grow better in brain tissue than the archetypal form (1). The RR rearrangements may alter the virus-cell interaction in the course of infection (122) and play a major role in cell-type-specific or organ-specific adaptation of the virus (122, 178). The rearranged variants and archetypal form may exhibit overlapping tissue-tropic properties that determine their abilities to establish infections at distinct secondary sites after their hematogenous spread from a primary site (31).

Mouse pneumotropic virus The mouse pneumotropic virus (MPtV), previously called Kilham strain of mouse polyomavirus (KV), is a second murine member of the polyomavirus family, first isolated by Kilham in 1953 (106). MPtV, in contrast to other mammalian polyomaviruses, is associated with severe disease. Infection of newborn mice causes interstitial pneumonia with a high fatality (75, 76), whereas exposure of fully immunocompetent mice to MPtV leads to a persistent and inapparent infection. In primary infections, MPtV replicates mostly in vascular endothelial cells of the lung, liver and spleen (75-77, 138). However, during the persistent phase, infected cells are mainly

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found in renal tubules (80). Therefore, although MPtV and MPyV show differences in organ and cell tropism during primary infection, they appear to share specificity for tubular epithelium during persistent infection (78). A major difference between MPtV and MPyV is that inoculation of newborn mice with MPtV does not result in tumors (79, 150, 195). However, cells transformed with MPtV can form transplantable tumors (195). Both the virulence upon primary infection of suckling mice and the nontumorogenic phenotype separate MPtV from MPyV, justifying a comparative investigation of the two viruses. The genome of MPtV is a circular, 4756 bp double-stranded DNA molecule (133, 215). As predicted from the nucleotide sequence, MPtV DNA encodes two early proteins (large and small T-antigen) and three late proteins (VP1, VP2 and VP3) (133). Although analysis of cloned DNA definitely established that MPtV belongs to the polyomaviruses (115), it is not closely related to the previously characterized MPyV (16, 133). Unlike MPyV and the hamster polyomavirus (HaPyV), MPtV does not encode any middle T-antigen. Besides, although the deduced amino acid sequences of MPtV LT and VP1 contain the most prominent clusters of homology in all polyomavirus strains (133), these two proteins are distantly related to the proteins encoded by other polyomaviruses. However, comparison of the functional domains shows that MPtV LT resembles SV-40 LT more than that of MPyV (216).

MPtV exhibits a stringent host and cell specificity, and the absence of a permissive tissue culture system has hampered its study. A possible determinant of cell tropism is the enhancer segment of the RR of the genome. Numerous mutants of MPyV with altered host ranges have been described (65, 101, 126). Most of these have point mutations or rearrangements of the enhancer segment, and these mutations have been directly linked to the host range phenotype. Similar observations have been made with the primate polyomaviruses (199). Although the MPtV RR shares some general features with those of other polyomaviruses, it does not have an enhancer similar to other polyomavirus enhancers with long repeated elements. Besides, with its unique number and location of the pentanucleotide GRGGC consensus sequences, the number of DNA-binding sites would be unique within the polyomavirus family (133).

Cellular mobile DNA elements

Classification Cellular mobile genetic elements, or transposable elements (TEs), also

called transposons, are distinguished by their ability to insert at new genomic locations. There are two major classes of transposons (62). Class I elements,

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such as the Alu elements in primates (90) and B1 sequences in rodents (166), are retroelements that use reverse transcriptase to transpose by means of an RNA intermediate, a process called retrotransposition. In contrast, class II elements transpose directly from one location in the DNA to another. In mammalian genomes, two types of class I retroelements predominate: long and short interspersed nuclear elements (LINEs and SINEs) (214).

Approximately one-quarter of the human genome is composed of SINEs and LINEs (83). SINEs are short (≤400 bp) genetic elements that have replicated to extremely high copy numbers in the genomes of many species (51). The predominant human SINE, the Alu repeat, comprises approximately 10% of the genome, occurring on average once every 3 to 6 kb, although the distribution is non-uniform (136). It is not clear what fraction of Alu repeats are replication-competent, but most of them are probably permanently inactivated (83). LINE elements, L1 being the most prominent, comprise an even larger portion of the genome than SINEs (180). There are approximately half a million truncated and 3000 to 5000 full-length L1 elements, approximately 6 to 7 kb, in the human genome (102, 180).

Besides the class I and II TEs, another type of TEs, called miniature inverted-repeat transposable elements (MITEs), has been found mainly in plant genomes (204). Although a family member typically present in thousands of copies in plant, MITEs are still too poorly understood to classify. They were first described in plants, but the growing numbers of new examples from other organisms, including fungi, xenopus, mosquitoes, and human (140), indicate that they are widespread, if not ubiquitous, components of the genomes of higher eukaryotes (93, 181). To date, no MITE family has been shown to be actively transposing (217), but a report showed that the plant MITE mPing becomes mobilized in another culture (105). The structures and sequences of MITEs are clearly distinct from those of LINEs and SINEs (204). Various MITEs have common features, including short length (74–490 bp), terminal inverted repeats (10–15 bp), target site preference, low G+C content (average, 34%) and high copy number (204, 214). MITEs share some features with both DNA transposons and retroposons, indicating that these elements might share some aspects of their transpositional mechanisms (93). However, the high copy numbers and nonautonomous nature of MITEs suggest that, similarly to retroposons, they use a basic cellular mechanism for their amplification (93). It is proposed that the stable secondary structures of MITEs are crucial to their amplification, which occurs as an error of the DNA replication machinery, and MITEs appear to be genomic parasites that borrow all of the necessary factors for their amplification from products encoded in the genomes in which they reside.

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Functions The most obvious function performed by TEs is to cause mutations (62, 104). Because of the mechanism of the insertion and excision, TEs cause a variety of short additions and deletions of nucleotide sequences as they move in or out of chromosomes (30). This function of TEs may not appear beneficial in the short term, but TEs cause some changes that are difficult to generate by other means and which might be advantageous in the long term (62). Approximately half of the human genome was recently shown to consist of TE sequences and a growing body of evidence indicates that TEs have been major players in the evolution of eukaryotic complexity by providing novel regulatory or coding sequences (20, 98, 127). TEs may have contributed substantially to the evolution of both gene-specific and global patterns of human gene regulation (98).

Integration of TEs might alter both the level of expression and the spatial expression pattern of adjacent genes. TEs might also contain tissue-specific enhancers. The TE insertion may lead to a new expression pattern: increasing levels in one organ, decreasing them in another, and having no effect on a third (72). Some MITE families contain cis-acting domains that regulate the expression of nearby genes (161) and may play varying roles in chromosome structure and regulation of gene expression (104).

TEs and polyomaviruses In SV-40, a variant was found to have a 157 bp Alu-element-like

insertion immediately upstream of the early coding sequences (53). The Alu-family of interspersed repeated sequences belongs to the retrotransposons. There are additional reports of cellular DNA segments integrated into the RR of polyomavirus genomes (55, 147). However, in these cases, genomes with inserts of cellular DNA were either found as a small minority in a population of normal viral genomes, or in a larger proportion of defective viral genomes after repeated high multiplicity passage of virus in cell culture. In our present investigation, DNA segments (220 bp) apparently derived from repeated sequences in cellular DNA were found integrated in the enhancer of the MPtV genome. Moreover, these inserted genetic elements did change the viral potential of gene expression and DNA replication. In comparison to most transposons of mammalian cells these elements are short, have no coding potential and apparently have a high target site specificity. These features and their A-T content suggest a relationship with a recently discovered class of elements, (MITEs). Although most, but not all, MITEs contain inverted terminal repeats for their transposition (25-28, 197), the MPtV insert and the flanking virus sequence do not contain obvious terminal inverted repeats. Instead, the 220 bp insert has short direct repeats like a retrotransposon. There is an earlier observation (53) of a SV-40 mutant with a 175 bp segment related to the Alu-family of

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interspersed repeated sequences in primate DNA. These elements belong to the retrotransposons.

TEs and cancer TEs have been shown to be involved in carcinogenesis and tumor progression (51, 83, 118, 121, 172, 209). A lot of evidence indicates that transcripts from TEs are much more abundant in cancer tissues than in normal tissues (91, 99). Effects of TE insertion on gene expression are common, partly because the movement of a TE will generally carry with it new sequences that act as a binding sites for sequence-specific DNA-binding proteins. These sequences can thereby act as regulatory sequences and enhancers to affect the transcription of nearby genes, effects that commonly contribute to the evolution of cancer cells, where oncogenes can be created by the transposition of such regulatory sequences into the neighborhood of a proto-oncogene (2). Exposure of cells to a variety of DNA-damaging agents, including several common chemotherapeutic drugs and γ-radiation, is associated with dramatic induction of SINE transcription and activation of a cluster of retrotransposable elements in response to DNA damage, a process involving the formation of secondary malignancies in the cancer survivors (83). Although the mechanism regulating transcription of Alu element, a SINE in human, is not clear, it has been shown that Alu transcription increased following a number of cellular stresses, including heat shock and translational inhibition (118, 121), infection by herpes simplex virus (94, 148) or adenovirus type II and V (149, 170). The possible mechanism of the involvement of retrotransposable elements in the secondary malignancies might be: i) the DNA breaks resulting from DNA damage by genotoxic agents not only stimulate induction of apoptotic cell death pathways in the cells, but also serve as substrates for genomic recombination and transposition (82); ii) TEs serve as templates for homologous and non-homologous recombination events(189, 192); iii) genotoxic exposure stimulates TEs mobility in the genome (168). Besides cancer, mobile genetic elements have been associated with a variety of human diseases through multiple mechanisms. A number of genetic diseases have been attributed to recombination events between SINEs and LINEs. The recombination events result in deletions, duplications and rearrangements in the disease-related genes (51). Interestingly, human disease may, in some cases, be correlated with the absence of a mobile element (172). TEs may thus affect human health through a variety of proximal effects on disease-related genes.

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Present investigation Impetus and projects Studies on MPtV revealed that it can be considered as a model system for studying polyomavirus persistence and reactivation in vivo. Polyomaviruses are strictly host specific and produce lifelong persistence after acute infection in their natural hosts. Immunosupressive treatment of persistently infected hosts frequently results in reactivation of the virus, at least in the cases of human polyomaviruses (JCPyV and BKPyV) infection. The reactivation may cause severe disease. Data on the human polyomaviruses indicate that establishment and maintenance of persistence are probably influenced by the expression of viral genes. To get further insight into the mechanisms restricting viral gene expression of polyomavirus in vivo, a model system that allows persistent infection without being impaired by tumor induction is needed. Although this is the case with human polyomavirus infections and the natural infection of SV-40 in macaques, the experimental approach is obviously highly restricted. Therefore, MPtV, as a non-oncogenic virus with a murine host, might serve as a model system for studies on the mechanisms of polyomavirus persistence and reactivation, including studies of pathogenesis by human polyomaviruses. LTs of polyomavirus play a central role in the viral life cycle. For further study of MPtV, it seemed essential to reveal the characteristics of its LT, which had only been predicted by comparison of its deduced amino acids with those of other polyomaviruses. Therefore, we cloned and expressed MPtV LT in heterologous systems and characterized LT with regard to its metabolic stability, cell immortalizing activity and, after purification, to its specific DNA binding (paper I). In contrast to other polyomaviruses, MPtV exhibits a stringent host and cell specificity, and the absence of a permissive tissue culture system has hampered its study. To investigate its biological properties, we tested a number of cell lines potentially permissive to MPtV infection and made attempts to widen its host range to non-permissive cell lines, such as mouse fibroblast cells, by genetic modification of the regulatory and late regions of its genome. The enhancer substitution mutant (KVm1), having a transcriptional enhancer substituted with a corresponding DNA segment from MPyV, was able to replicate in mouse 3T3 cells and form virus particles (Paper II) that were infectious in mice. However, efficient infection of cells in vitro was not achieved with these virus mutants. As we speculated on the basis of the stringency of the MPtV host range, the MPtV RR is not homogeneous. Our data confirmed that MPtV genomes extracted from virus in lung tissue – like genomes of JCPyV, BKPyV and

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SV-40 isolated from infected individuals – contained a number of variations in the RR. Studies on the function and structure of the rearrangements led to the discovery of mobile DNA elements in MPtV RR (paper III). A general illustration of the results from the present investigations described above is presented below.

Characterization of MPtV large T-antigen The intron-deleted MPtV LT gene was cloned and MPtV LT was expressed in heterologous systems – baculovirus, Semliki Forest virus, and pcDNA3 expression system – due to different purposes of protein characterization. The protein expressed in insect cells by recombinant baculovirus was purified with immuno-affinity chromatography and the activity of the protein was verified by ATPase activity. The binding sites of this protein in viral DNA were first screened by DNA immunoprecipitation (McKay assay) and followed by a more precise delineation using DNase I footprinting. The binding properties were similar to those of MPyV and SV-40, although the arrangement of binding sites in MPtV DNA was unique. Two regions were identified for DNA binding with one site (probably for early promoter regulation) showing strong binding and another (for viral DNA replication) much weaker. Since the DNA-binding of LT plays an essential role in DNA replication and an important role in transcription regulation, the difference of functional in the number and organization of LT-binding motifs in the RR of MPtV DNA, as compared to other polyomavirus genomes remains unclear. Table 2.Immortalization of secondary rat embryo fibroblasts (REF) cells. _________________________________________________ No. of established linesb/ Plasmidsa No. of isolated colonies _________________________________________________ pSV2NEO 1 / 24 pSV2NEO/KV 23 / 24 pSV2NEO/dl1061 24 / 24 pcDNA3 1 / 24 pcDNA3/KVLT 3 / 24 _________________________________________________ a pSV2NEO, expression vector contains the neomycin phosphotransferase gene [Southern, 1982 #187]; pSV2NEO/KV,contains whole MPtV genome with the large T-antigen and the neomycin phosphotransferase gene in opposite polarity; pSV2neo/dl1061, contains MPyV mutant dl1061 that only expresses MPyV large T-antigen [Nilsson, 1983 #24]; pcDNA3/KVLT, only expresses MPtV large T-antigen under CMV promoter. pSV2NEO and pcDNA3(InVitrogen) were used as negative control for immortalization activity, while pSV2NEO/dl1061 was employed as positive control. b Primary REF Cells were thawed and transfected with 4 µg of plasmid DNA per 60-mm dish following the LipofectAmine procedure. At 48 hr after transfection, the cells were detached and reseeded into two 100-mm dishes and medium with 0.5 mg/ml of G418 was added. After

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2-3 weeks, a number of G418 resistant colonies were isolated by the cloning cylinder technique and transferred to multiwell dishes. Clones that could grow to confluence after one additional transfer were scored as established [Söderbärg, 1993 #10]. MPyV LT can confer on primary embryo fibroblasts the ability to grow in long-term culture without entering crisis (162). Interestingly, MPtV LT has a very weak immortalizing activity in comparison to SV-40 and MPyV LT, although MPtV LT showed a metabolic stability similar to that of mouse polyoma virus. MPtV small t-antigen can enhance the immortalization activity of MPtV LT to almost the same level as MPyV LT (Table 2), in agreement with the previous conclusion for SV-40 that small t antigen assists in transforming some cell type (159, 160, 169). This activity of LTs is associated with their binding abilities to pRb by a polypeptide segment within their N-terminal parts (LXCXE). The MPtV LT amino acid sequence appears to contain this motif, suggesting that it interacts with pRb, and raising the question concerning why MPtV LT functions so weakly in cell immortalization. The explanation might be that it could not inactivate pRb function properly or that it becomes too cytotoxic by interacting with other cellular proteins, such as p53. Experiments are in progress to clarify the interaction of MPtV LT with the cellular proteins.

MPtV host range mutant- (KVm1) enhancer substitution MPtV DNA replication in mouse fibroblast cells cannot be detected when only the viral genome is transfected. However, expression of MPtV LT in trans enables viral DNA replication at the detection level, but without successful virion assembly, suggesting that the weak early transcription is not the only checkpoint for MPtV cell specificity, and the weak late transcription might have blocked the virion formation.

MPyV Enhancer domains have been shown to play pivotal roles in controlling the host range in tissue culture systems (4, 5, 87). MPyV, with a powerful enhancer and cell specificity towards mouse fibroblast cells, is supposed to be an functional donor of enhancer for MPtV. Accordingly, we constructed a MPtV substitution mutant (KVm1), having a part of the MPtV RR DNA substituted with a segment of the polyomavirus transcriptional enhancer. The substitution mutant was able to replicate in transfected 3T3 cells, and the newly replicated viral DNA succeeded to associate with proteins to form viral particles. However, these particles were noninfectious when tested on 3T3 cells, probably owing to failure of absorption or uptake of virus by these cells. Analysis of early and late promoter activity by luciferase reporter gene expression showed that the enhancer substitution enables MPtV replication in mouse fibroblast cells by enhancing the

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transcription, especially the late transcription for capsid proteins, in this cell line. A comparison of the MPtV and MPy V LTs showed that they were not interchangeable in the initiation of MPtV, KVm 1 and MPyV DNA synthesis. Furthermore, the standard-type MPtV origin of DNA replication was less active than the mutant structure in the presence of saturating amounts of MPtV LT. In conclusion, these data demonstrate several differences between the two types of LTs in their interactions with cellular proteins. Meanwhile, these data had confirmed the predicted location of the MPtV enhancer (133). Data from this study also suggested that MPtV RR harbors a negative element that weakens MPtV function in transcription in 3T3 cells.

FIG.7. Analy sis of MPtV virus distribution in infected mouse organs by Southern-blot DNA hybridization. Virus DNA (s tKV, standard type MPtV; m 1KV, MPtV mutant no.1) was extracted from equal amount of mouse organs and samples were hybridized with 32P labeled MPtV specific fragment. Signals were quantified and relative intensity was calculated for comparison.

To investigate the infectivity of the assembled virion, KV m1 transfected cell supernatant was inoculated into newborn mice. In contrast to the mice inoculated with standard-type MPtV suspension, mice inoculated with KVm 1 supernatant showed no mortality, possibly because of its much lower titre for inoculation or the lower pathological effect of KVm 1 infection. The existence of KVm 1 virion in i nfected mouse organ was confirmed by southern-blot (Fig.7) and PCR. Sequence analysis i ndicated that the RR of KVm 1 is quite stable, suggesting a sufficient RR function in this cell ty pe. Experiments concerning the genetic stability and pathological effect of KVm 1 are being performed by infecting cells or mice with controlled virus titre.

MPtV host range mutant- late region substitution The failure of KVm 1 to infect 3T3 cells suggests the absence of virus-

specific receptors on the cells. Therefore, we constructed capsid protein substitution mutants that have capsid protein genes of MPyV , that has receptors on a variety of cell types. The late coding region of MPtV and

0

50

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250

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n

stKV

m1KV

Lung Kidney Liver Spleen

mouse organ

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KVm1 (in MPtV: nt 2805 SacI to nt 4754 XhoI) were substituted with the corresponding region of MPyV (nt 2885 NdeI to nt 5132 PvuII). The late substitution mutants were tested for DNA replication and virion assembly in mouse 3T3 cells. The substitution activated both MPtV and KVm1 DNA replication significantly, even in the absence of MPtV LT (Fig.8, lane PyV/stKV and PyV/KVm1), strongly suggesting that the late coding region A

MPtV-LT:

Viral DNA:

DpnI resistant:

stKV m1KV

_ ___

B FIG.8. (A) Southern-blot analysis of viral DNA replication of MPtV late region substitution mutans (PyV/stKV, PyV/m1KV), stKV and m1KV in NIH 3T3 mouse fibroblasts. Transfected viral DNA and the amount of MPtV-LT expression plasmid were indicated at the top. Extraction and analysis of viral DNA were done as described previously [Zhang, 2001 #186]. (B) Fraction of DpnI-resistant viral DNA normalized to the fraction of m1KV DNA synthesized in the absence of separate large T-antigen expression.

may influence viral DNA replication significantly by an unclear mechanism. Primary experiments in virion assembly showed negative results, possibly resulting from the absence or impairment of the MPyV packaging signal in the MPtV mutants. The location of the MPyV virion packaging signal is unknown, but studies on SV-40 suggest a location close to the replication

0

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Transfected MPtV-LT (ng

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origin. Therefore, another MPtV mutant was constructed by substituting the MPtV late region (nt 2805-nt 4754) by the late region of MPyV that contains sequence closer to its origin (nt 2885-nt 5269 PvuII) than the previous one. This mutant showed enhanced DNA replication compared to others and efficient virion assembly. Experiments are ongoing to test its infectivity in mouse 3T3 cells.

Cellular transposable elements in MPtV genome The stringency of the MPtV host range implies that it may contain a RR with narrow cell specificity that might show heterogeneities due to rearrangement followed by virus selection. Our data confirmed that MPtV genomes extracted from virus in lung tissue – like genomes of JCPyV, BKPyV and SV-40 isolated from infected individuals – contained a number of variations in the RR, whereas the protein-coding part of the genome had a uniform length. A majority of the MPtV RR rearranged DNA molecules had a structure differing from the standard-type. A 220 base-pair insertion at nucleotide position 142 with a concomitant deletion of nucleotides 143 to 148 was a prominent variation. There were two variants of the 220 bp insertion differing at two nucleotide positions at one of the termini. Other DNA molecules showed complete or partial deletions of these structures and surrounding sequences in the viral enhancer. However, the end of the insertion at nucleotide 142 was frequently preserved. In relation to the standard-type, all variant genomes, including a G272T mutant which contains a point mutation in the LT binding region, showed differences in the activities of transcriptional promoters and the origin DNA replication. Analysis by DNA reassociation showed that a large number of nucleotide sequences related to the 220 base-pair insert in the MPtV genome were present in mouse and human DNA, but not in Escherichia coli DNA. Moreover, analysis by PCR indicated that there were multiple copies in the mouse genome of sequences that were identical or closely related to the 220 bp viral DNA segment. Together, the data suggest that the 220 base-pair insertion is related to a TE of a novel type.

However, in a BLAST search of the mouse DNA database (18 November 2002), only sixty-four entries with significant similarity were detected and the BLAST search did not reveal more than five sequences with short (20-23 bp), perfect or nearly perfect similarity in the entire nucleic acid database. Experiments are ongoing to locate the MPtV-insert-specific signal in the mouse genome.

Besides the insertion-deletions, another frequently occurring variation in the RR of the MPtV genome was the G272T base substitution, which is located in the LT binding site and has a global positive effect on the activity of both viral transcriptions and DNA replication. Since both the base substitution and the insertion-deletions may occur together or alone and are

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very close (130 bp), based on the assumption that the G↔C transversion occurred very rarely, the site-specific recombination of the insertion-deletion might occur at a high frequency (Figure 9).

342 (E)

272T

04632 (L)KV

130bp

272Gst

dl143-148

inA/B (1-220)dl143-148

inA/B (1-220)

Fig.9. Site-specific recombination at high frequency? Both insert A and B may occur with either 272G or T with a short distance as 130 bp between them. The standard type MPtV regulatory region is displayed at the top.

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FAQs Is mouse the natural host of MPtV?

MPtV was first identified by Kilham and Murphy in 1952 in laboratory mice (106). MPtV is clearly classified as a member of the polyomavirus genus on the basis of its physical structure and genomic structural similarity to other polyomaviruses, its stability towards ether, its nuclear location in host cells (150) and recognition by antibodies shared by all members of the polyomavirus group (16, 195). Serological study of epidemiology on MPtV among wild mice implies mouse the natural host of MPtV (88). Our findings that MPtV failed to infect all tested mouse cell lines, including two mouse endothelial cell lines, raised the question whether mouse is the natural host for MPtV. In fact, human JCPyV also displays very stringent host specificity and its propagation in cell culture has been limited to human glial cells (125, 178). To investigate the natural host of MPtV, experiments are in progress to test viral infectivity and DNA replication in cell types derived from other species. Investigation of MPtV serological epidemiology in other species will be difficult to carry out without knowledge of its prevalence.

Is the MPtV large T-antigen involved in the high-frequency recombination?

Intramolecular homologus recombination of MPyV DNA is dependent upon promoter structure or function (69). MPyV LT has been shown to be able to mediate high-frequency recombination, and this recombination-promoting activity can be dissociated from its replicative function (186). Accumulated evidence has revealed that LT can bind directly to topoisomerase I (66, 70, 130, 131, 158, et al, 179), which can introduce double-strand DNA breaks utilized in site-specific recombination. Our data demonstrate that the preferred target sequence of MPtV insertion is exactly the preference of topoisomerase I. Put together, it is quite possible that MPtV LT plays a role in the site-specific high-frequency recombination. What is the MPtV insert classification? Although the direct repeat in the insert terminal implies its class as a retrotransposon, the cut-paste fashion (deletion-insertion) clearly suggests that it transposes in a way similar to DNA transposons. However, evidence has to be provided to prove the mobility of the insert. As indicated above, it probably belongs to a category similar to MITEs. It has all of the characteristics shared by most MITEs, but a definite classification requires further investigation of its properties. Quite few MITEs have been identified in the mammalian genome. If MPtV inserts belong to MITEs, they would be the only MITEs found in mammalian genomes with obviously active transposition. The study of MITEs in gene regulation has been limited by the

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complexity of the phenotype resulting from the diploid genotype. Therefore, MPtV, if identified as a MITE target, will be an excellent model system for the study of MITEs gene regulation and mechanism of transposition. How do the MPtV inserts exist in the cellular genome? This is still a puzzling issue. No solid evidence can be obtained for the presence of MPtV insert identical sequences in cellular genome when we ran the search against the genome database. One possibility is that these sequences are so active that they exist in individuals and play a role in the individual diversity. If they are ubiquitous in all individual genomes, this explanation would be challenged by our difficulties to amplify the sequence by PCR from genome DNA extracted from tissue culture cells. Another possibility is that it transposes via RNA intermediates with complex secondary structure that generate a great diversity of the nucleotide sequences. Experiments are ongoing to locate the insert-specific DNA hybridized signals in mouse genome. Can MPtV without insertion gain one during infection, or vise versa?

This is an issue we are planning to address. After the clones of the variants with and without the inserts are obtained by molecular cloning, they will be inoculated into newborn mice separately or in combination. Replicated virus will be analyzed with respect to rearrangements, the distribution and proportion of possible variations, the existence of MPtV inserts, etc. They may also be directly co-transfected into tissue culture cells with MPtV LT and the replicated DNA analyzed for occurrence of rearrangements or transposition. Is transposable element a unique phenomenon in MPtV or common in DNA viruses?

Variation of the genome is a quite frequent phenomenon among DNA viruses. Since BKPyV and JCPyV showed similar RR rearrangements, it is possible that they also utilize similar mechanisms. Preliminary investigations did not reveal any possibility of the existence of TE in the genome or any evidence for the presence of JCPyV and BKPyV deletion segments in the cellular genome. What is the role of MPtV insert in virus persistence and reactivation? Data from studies of BKPyV, JCPyV, and SV-40 demonstrated the role of variations in virus persistence and reactivation, although it is generally acknowledged that the immune system plays a major role. Our distribution study of MPtV variants in mouse organs implied that they had different organ specificities or proportions. We speculate that beside a direct role in gene expression and viral DNA replication regulation, the MPtV inserts may

35

also function to generate mutations by frequent transposition. Each of the mutants thus struggles by itself to modulate its infectivity for persistence and reactivation or cell specificity by the introduction of mutations. Another possibility, which is more likely, is that the variants orchestrate together for the pathogenesis of viral infection. Since the function of MPtV variations in viral transcription and DNA replication are complex, the variants may work both for themelves and for the others as help virus. For example, the virus with cell specific signals may need the help of MPtV LT expressed efficiently from another one which needs help to compete for cognate DNA-binding proteins. Because a genome having superior fitness will suppress the activity of genomes with less fitness (144) leading to the rapid disappearance the inferior genome, the co-existence of the variants suggests that none of variants has inferior fitness. The different prevalence of variants in different organ that we found may indicate that MPtV modulates its cell specificity and infectivity either by modulation of the variants proportions or by creating new variations, or by both. It would be interesting to establish the prevalence of MPtV variants during acute infection, persistent infection and reactivation. MPtV is recognized as non-tumorigenic virus in mice, although MPtV- transformed cells can cause tumors (described above). With very strict cell specificity, it is not advantageous for MPtV to eliminate its host cells by either lytic infection or tumorigenesis. Besides acting as modulators for viral infectivity, the MPtV insert may thus behave as anti-tumorigenic factors, a role similar to other mobile elements reported elsewhere (172). What is the significance of the identification these cellular mobile elements in MPtV?

The significance is difficult to define in the absence of additional data. The present results suggest that MPtV might be an excellent, unique (in some cases) model system for studies on mammalian DNA transposition, gene regulation by MITEs, viral persistence and reactivation and tumorigenesis. We hope that future data will prove that this finding is not only interesting but also significant.

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Acknowledgements This work was carried out at the department of Medical Biochemistry and Microbiology, Section of Microbiology. The project was supported by grants from the Swedish Cancer Society.

I wish to take this chance to thank every one of you who has encouraged and supported me this far, especially:

Göran Magnusson, for being a supervisor I can deeply trust and respect, for providing me this opportunity to extend my academic career, for countless discussions that taught me about your scientific and witty thinking on work, English presentation and importantly, views about life (I will miss those moments), and for your friendly concern for my motherland.

Göran Akusjärvi, my examiner, for your encouraging word: “Aha, so you are soon getting finished with your Ph.D”, when I still felt desperate with it, for your endurance of the loud radio and my disturbing whistling (Your office faced my lab bench, unfortunately).

Professors in Institute of Virology, Beijing: Gu Shuyan, my former supervisor, for your endless patience in pushing me to science even when I was very erratic about work. Pi Guohua, for joyful discussions; Gong Xinchang, for kindly helping me with work and living; Duan Shuxue, for always being kind and helpful; Bi Shengli, for being my reference.

My present group, Lena Möller, for good lab cooperation and being happy family members; Anders Melander, for efficient cooperation, especially in refilling the liquid nitrogen (4 tanks in 30 seconds) and for always being ready to be my language translator.

My former group: Alexis Fuentes, for your enormous help that you always said “for nothing”; Hamid Houshmand, for teaching me the small but important tricks to make the experiments work perfectly; Jorge Blackhall and Mariann Muñoz for so much fun we had; Barbara Ryan, for also sharing the experience of bringing kids up; Carin Söderhäll, Ingrid Törnberg, Anders Bergqvist, Ola Rönn, Karin Söderbärg, Mats Nilsson, Magnus Molin, Lina Dimberg and Anki Magnusson, for have been kindly and helpfully around.

Li Hongyun, for having been my colleague, conference partner and good friend for so long time both in Beijing and in Uppsala and for introducing me to Göran; Wang Ruigong, for so much delightful time our two families shared, such as playing cards (day and night), traveling to the fantastic north; and Duo-duo, for calling me “Jiu-Jiu”(uncle) sweetly.

All the present and former people in the groups of this corridor, Catharina Svensson’s, Göran Akusjärvi’s, Peter Kreivi’s and Stefan Schwartz’s, for making the environment of this corridor exciting for staying and efficient for working. Jinping Li, for valuable suggestions and discussions. Members of Jinping’s group for being helpful when I did my last experiments for my thesis.

37

All the present and former staff of IMBIM: Maud Petterson, for having been the most helpful secretary since I came to Sweden. You can always quickly help me out with those forms and formalities that I totally don’t understand (The trick is that you do everything). Erika Enström, for answering my endless questions with your lovely smile (You are so helpful that you are the first one I would go to when I think I need help). Olav Nordli, for fixing my computer in time even when you are totally occupied with your assignments (Sorry, my computer had problems surprisingly often and big). Kerstin Lidholt, for helping me with the programs and trying to teach me the tricks about computer (I usually forgot immediately). Tony Grundin, for the things I got from my mailbox. Ylva Jansson, for making my daily experiments easier. The late Lilian Ekström, for having been a secretary so helpful and kind to me.

David Eaker, for your prompt and great proofreading of my thesis.

Liu Zhurong and Xu Xiaoyang, for sharing the ambition and views on life and for having shown me the way to BMC. Zhao Hongxin, for nice discussions about life in our department.

Hongbin Henriksson & Gunnar Henriksson, Zou Xiang & Hu Lijuan, Yang Haitao and Zong Fang, for being helpful and sincere friends. Wang Bin and Wang Changan, for the short pleasant time we had.

Yu Jiang & Hongmei & Julie, Zhanchun & Lijun & Hao, Gao Lan & Yuanyue & Lily, Enyin & Lingli & Sisi, Yupeng & Li Sa, Guo Qi & Zheng Yan, Haiyan, Liu Liang and Shuxia, Dong Jiajun and Hu Guozhen, Tao Ran & Yuchen, families of Xu Chongyu’s, Cheng Tingzhu’s, Shao Xingwu’s, Wu Ping’s, Zhang Guangming’s, without your friendship and the pleasure we shared, life in Uppsala would have been so lonely.

Friends in the Chinese football team and BMC lunch group for the great pleasure.

All the rest of my friends in Uppsala, for making my life in this small city interesting and happy.

My family, for your guaranteed love and support, without which I would hardly succeed with my PhD and with my life. You are the foundation upon which my character and self-confidence are built.

My family-in-law, for your trust and your encouragement.

Jing, my dear wife, for sharing my life so thoroughly and so delightedly with love and understanding, and for having been believed in me all the time, not the least for your support with my thesis. Diandian, my sweetheart, for your love and for letting me be such a happy and proud father. With your love, I will never be afraid of any difficulties and desperations.

My dear father, for having been my best friend, my best supervisor and best teacher. Time seems to unable to diminish my pain of losing you. I miss you so much, especially at this moment you had dreamed about. Your inspiration has carried me through the hardest times. I will try to prove your belief that your children are great. I am doing that.

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Reference 1. Agostini, H. T., C. F. Ryschkewitsch, E. J. Singer, and G. L. Stoner. 1997. JC virus

regulatory region rearrangements and genotypes in progressive multifocal leukoencephalopathy: two independent aspects of virus variation. J Gen Virol 78:659-64.

2. Alberts, B., D. Bray, J. Lewis, M. Raff, K. Roberts, and J. D. Watson. 1994. Molecular biology of the cell, 3rd Edition ed. Garland Publishing, Inc., New York & London.

3. Alexandrov, A. I., M. R. Botchan, and N. R. Cozzarelli. 2002. Characterization of Simian Virus 40 T-antigen Double Hexamers Bound to a Replication Fork. THE ACTIVE FORM OF THE HELICASE. The Journal of Biological Chemistry 277:44886-97.

4. Amalfitano, A., L. G. Martin, and M. M. Fluck. 1992. Different roles for two enhancer domains in the organ- and age-specific pattern of polyomavirus replication in the mouse. Molecular and Cellular Biology 12:3628-35.

5. Amati, P. 1985. Polyoma regulatory region: a potential probe for mouse cell differentiation. Cell 43:561-2.

6. Anderson, H. A., Y. Chen, and L. C. Norkin. 1996. Bound simian virus 40 translocates to caveolin-enriched membrane domains, and its entry is inhibited by drugs that selectively disrupt caveolae. Mol Biol Cell 7:1825-34.

7. Atwood, W. J. 1991. Major histocompatibility complex encoded HLA class I proteins are cell surface receptors for the simian virus 40. Ph.D. Dissertation.

8. Ault, G. S., and G. L. Stoner. 1993. Human polyomavirus JC promoter/enhancer rearrangement patterns from progressive multifocal leukoencephalopathy brain are unique derivatives of a single archetypal structure. J Gen Virol 74:1499-507.

9. Bai, M., B. Harfe, and P. Freimuth. 1993. Mutations that alter an Arg-Gly-Asp (RGD) sequence in the adenovirus type 2 penton base protein abolish its cell-rounding activity and delay virus reproduction in flat cells. J Virol 67:5198-205.

10. Basak, S., H. Turner, and R. W. Compans. 1992. Expression of SV40 receptors on apical surfaces of polarized epithelial cells. Virology 190:393-402.

11. Bass, D. M., and H. B. Greenberg. 1992. Strategies for the identification of icosahedral virus receptors. J Clin Invest 89:3-9.

12. Benjamin, T. L. 2001. Polyoma virus: old findings and new challenges. Virology 289:167-73.

13. Benoist, C., and P. Chambon. 1981. In vivo sequence requirements of the SV40 early promotor region. Nature 290:304-10.

14. Berget, S. M., C. Moore, and P. A. Sharp. 1977. Spliced segments at the 5' terminus of adenovirus 2 late mRNA. Proc Natl Acad Sci U S A 74:3171-5.

15. Bergqvist, A., M. Nilsson, K. Bondeson, and G. Magnusson. 1990. Loss of DNA-binding and new transcriptional trans-activation function in polyomavirus large T-antigen with mutation of zinc finger motif. Nucleic Acids Res. 18:2715-2720.

16. Bond, S. B., P. M. Howley, and K. K. Takemoto. 1978. Characterization of K virus and its comparison with polyoma virus. J. Virol. 28:337-343.

17. Borowiec, J. A., F. B. Dean, and J. Hurwitz. 1991. Differential induction of structural changes in the simian virus 40 origin of replication by T antigen. Journal of Virology 65:1228-35.

18. Borowiec, J. A., and J. Hurwitz. 1988. ATP stimulates the binding of simian virus 40 (SV40) large tumor antigen to the SV40 origin of replication. Proc Natl Acad Sci U S A 85:64-8.

19. Botchan, M., W. Topp, and J. Sambrook. 1979. Studies on simian virus 40 excision from cellular chromosomes. Cold Spring Harb Symp Quant Biol 43:709-19.

39

20. Bowen, N. J., and I. K. Jordan. 2002. Transposable elements and the evolution of eukaryotic complexity. 4:65-76.

21. Bozeman, L. H., R. B. Davis, D. Gaudry, P. D. Lukert, O. J. Fletcher, and M. J. Dykstra. Characterization of a papovavirus isolated from fledgling budgerigars. Avian Diseases 25:972-80.

22. Bradley, M. K., J. Hudson, M. S. Villanueva, and D. M. Livingston. 1984. Specific in vitro adenylylation of the simian virus 40 large tumor antigen. Proc Natl Acad Sci U S A 81:6574-8.

23. Breau, W. C., W. J. Atwood, and L. C. Norkin. 1992. Class I major histocompatibility proteins are an essential component of the simian virus 40 receptor. Journal of Virology 66:2037-45.

24. Brown, G. W., T. Melendy, and D. S. Ray. 1993. Replication protein A from the trypanosomatid Crithidia fasciculata is inactive in the primosome assembly step of SV40 DNA replication. Molecular and Biochemical Parasitology 59:323-5.

25. Bureau, T. E., P. C. Ronald, and S. R. Wessler. 1996. A computer-based systematic survey reveals the predominance of small inverted-repeat elements in wild-type rice genes. Proc. Natl. Acad. Sci. U S A 93:8524-8529.

26. Bureau, T. E., and S. R. Wessler. 1994. Mobile inverted-repeat elements of the Tourist family are associated with the genes of many cereal grasses. Proc. Natl. Acad. Sci. U S A 91:1411-1415.

27. Bureau, T. E., and S. R. Wessler. 1994. Stowaway: a new family of inverted repeat elements associated with the genes of both monocotyledonous and dicotyledonous plants. Plant Cell 6:907-916.

28. Bureau, T. E., and S. R. Wessler. 1992. Tourist: a large family of small inverted repeat elements frequently associated with maize genes. Plant Cell 4:1283-1294.

29. Caplan, A. J., D. M. Cyr, and M. G. Douglas. 1993. Eukaryotic homologues of Escherichia coli dnaJ: a diverse protein family that functions with hsp70 stress proteins. Mol Biol Cell 4:555-63.

30. Capy, P., R. Vitalis, T. Langin, D. Higuet, and C. Bazin. 1996. Relationships between transposable elements based upon the integrase- transposase domains: is there a common ancestor? J Mol Evol 42:359-68.

31. Chatterjee, M., T. B. Weyandt, and R. J. Frisque. 2000. Identification of archetype and rearranged forms of BK virus in leukocytes from healthy individuals. J Med Virol 60:353-62.

32. Cheetham, M. E., and A. J. Caplan. 1998. Structure, function and evolution of DnaJ: conservation and adaptation of chaperone function. Cell Stress Chaperones 3:28-36.

33. Chen, L., and M. Fluck. 2001. Kinetic analysis of the steps of the polyomavirus lytic cycle. J Virol 75:8368-79.

34. Chen, L., and M. M. Fluck. 2001. Role of middle T-small T in the lytic cycle of polyomavirus: control of the early-to-late transcriptional switch and viral DNA replication. J Virol 75:8380-9.

35. Chen, M. H., and T. Benjamin. 1997. Roles of N-glycans with alpha2,6 as well as alpha2,3 linked sialic acid in infection by polyoma virus. Virology 233:440-2.

36. Cherington, V., B. Morgan, B. M. Spiegelman, and T. M. Roberts. 1986. Recombinant retroviruses that transduce individual polyoma tumor antigens: effects on growth and differentiation. Proceedings of the National Academy of Sciences of the United States of America 83:4307-11.

37. Ciappi, S., A. Azzi, R. De Santis, F. Leoncini, G. Sterrantino, F. Mazzotta, and L. Mecocci. 1999. Archetypal and rearranged sequences of human polyomavirus JC transcription control region in peripheral blood leukocytes and in cerebrospinal fluid. J Gen Virol 80:1017-23.

38. Cicala, C., F. Pompetti, and M. Carbone. 1993. SV40 induces mesotheliomas in hamsters. Am J Pathol 142:1524-33.

40

39. Cogen, B. 1978. Virus-specific early RNA in 3T6 cells infected by a tsA mutant of polyoma virus. Virology 85:223-30.

40. Colantuoni, V., L. Dailey, G. D. Valle, and C. Basilico. 1982. Requirements for excision and amplification of integrated viral DNA molecules in polyoma virus-transformed cells. J Virol 43:617-28.

41. Cole, C. N., T. Landers, S. P. Goff, S. Manteuil-Brutlag, and P. Berg. 1977. Physical and genetic characterization of deletion mutants of simian virus 40 constructed in vitro. J Virol 24:277-94.

42. Cyr, D. M., T. Langer, and M. G. Douglas. 1994. DnaJ-like proteins: molecular chaperones and specific regulators of Hsp70. Trends Biochem Sci 19:176-81.

43. Dalgleish, A. G., P. C. Beverley, P. R. Clapham, D. H. Crawford, M. F. Greaves, and R. A. Weiss. 1984. The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature 312:763-7.

44. Dalyot-Herman, N., O. Ben-nun-Shaul, A. Gordon-Shaag, and A. Oppenheim. 1996. The simian virus 40 packaging signal ses is composed of redundant DNA elements which are partly interchangeable. J Mol Biol 259:69-80.

45. Dawe, C. J., R. Freund, G. Mandel, K. Ballmer-Hofer, D. A. Talmage, and T. L. Benjamin. 1987. Variations in polyoma virus genotype in relation to tumor induction in mice. Characterization of wild type strains with widely differing tumor profiles. Am J Pathol 127:243-61.

46. de Villiers, J., and W. Schaffner. 1981. A small segment of polyoma virus DNA enhances the expression of a cloned beta-globin gene over a distance of 1400 base pairs. Nucleic Acids Res. 9:6251-6264.

47. Dean, F. B., J. A. Borowiec, T. Eki, and J. Hurwitz. 1992. The simian virus 40 T antigen double hexamer assembles around the DNA at the replication origin. The Journal of Biological Chemistry 267:14129-37.

48. Dean, F. B., P. Bullock, Y. Murakami, C. R. Wobbe, L. Weissbach, and J. Hurwitz. 1987. Simian virus 40 (SV40) DNA replication: SV40 large T antigen unwinds DNA containing the SV40 origin of replication. Proc Natl Acad Sci U S A 84:16-20.

49. Dean, F. B., and J. Hurwitz. 1991. Simian virus 40 large T antigen untwists DNA at the origin of DNA replication. The Journal of Biological Chemistry 266:5062-71.

50. Deb, S. P., and P. Tegtmeyer. 1987. ATP enhances the binding of simian virus 40 large T antigen to the origin of replication. J Virol 61:3649-54.

51. Deininger, P. L., and M. A. Batzer. 1999. Alu repeats and human disease. Mol Genet Metab 67:183-93.

52. DePamphilis, M. L. 1993. Eukaryotic DNA replication: anatomy of an origin. Annu. Rev. Biochem. 62:29-63.

53. Dhruva, B. R., T. Shenk, and K. N. Subramanian. 1980. Integration in vivo into simian virus 40 DNA of a sequence that resembles a certain family of genomic interspersed repeated sequences. Proc. Natl. Acad. Sci. U S A 77:4514-4518.

54. DiMaio, D., and D. Nathans. 1982. Regulatory mutants of simian virus 40. Effect of mutations at a T antigen binding site on DNA replication and expression of viral genes. Journal of Molecular Biology 156:531-48.

55. Ding, D., M. D. Jones, A. Leigh-Brown, and B. E. Griffin. 1982. Mutant din-21, a variant of polyoma virus containing a mouse DNA sequence in the viral genome. EMBO J. 1:461-466.

56. Dodson, M., F. B. Dean, P. Bullock, H. Echols, and J. Hurwitz. 1987. Unwinding of duplex DNA from the SV40 origin of replication by T antigen. Science 238:964-7.

57. Dynan, W. S., and R. Tjian. 1983. The promoter-specific transcription factor Sp1 binds to upstream sequences in the SV40 early promoter. Cell 35:79-87.

58. Elsner, C., and K. Dorries. 1998. Human polyomavirus JC control region variants in persistently infected CNS and kidney tissue. J Gen Virol 79:789-99.

41

59. Farmerie, W. G., and W. R. Folk. 1984. Regulation of polyomavirus transcription by large tumor antigen. Proc Natl Acad Sci U S A 81:6919-23.

60. Fields, B. N., D. M. Knipe, P. M. Howley, and e. al. (ed.). 1996. Fields Virology, Third Edition ed. Linppincott-Raven Publishers, Philadephia.

61. Fields, B. N., D. M. Knipe, P. M. Howley, and e. al. (ed.). 1996. Fields Virology, third Edition ed. Linppincott-Raven Publishers, Philadephia.

62. Finnegan, D. J. 1992. Transposable elements. Curr. Opin. Genet. Dev. 2:861-867. 63. Flint, S. J., L. W. Enquist, R. W. Krug, V. R. Racaniello, and A. M. Skalka. 2000.

Principles of Virology. ASM press, Washington, D.C. 64. Fromm, M., and P. Berg. 1982. Deletion mapping of DNA regions required for SV40

early region promoter function in vivo. J Mol Appl Genet 1:457-81. 65. Fujimura, F. K., and E. Linney. 1982. Polyoma mutants that productively infect F9

embryonal carcinoma cells do not rescue wild-type polyoma in F9 cells. Proc. Natl. Acad. Sci. U S A 79:1479-1483.

66. Gai, D., R. Roy, C. Wu, and D. T. Simmons. 2000. Topoisomerase I associates specifically with simian virus 40 large-T- antigen double hexamer-origin complexes. J Virol 74:5224-32.

67. Garber, E. A., M. M. Seidman, and A. J. Levine. 1980. Intracellular SV40 nucleoprotein complexes: synthesis to encapsidation. Virology 107:389-401.

68. Garcea, R. L., D. A. Talmage, A. Harmatz, R. Freund, and T. L. Benjamin. 1989. Separation of host range from transformation functions of the hr-t gene of polyomavirus. Virology 168:312-9.

69. Gendron, D., L. Delbecchi, D. Bourgaux_Ramoisy, and P. Bourgaux. 1996. An enhancer of recombination in polyomavirus DNA. Journal of Virology 70:4748-60.

70. Giacherio, D., and L. P. Hager. 1980. A specific DNA unwinding activity associated with SV40 large T antigen. J Biol Chem 255:8963-6.

71. Gilinger, G., and J. C. Alwine. 1993. Transcriptional activation by simian virus 40 large T antigen: requirements for simple promoter structures containing either TATA or initiator elements with variable upstream factor binding sites. J Virol 67:6682-8.

72. Girard, L., and M. Freeling. 1999. Regulatory changes as a consequence of transposon insertion. Dev. Genet. 25:291-296.

73. Goldman, N., M. Brown, and G. Khoury. 1981. Modification of SV40 T antigen by poly ADP-ribosylation. Cell 24:567-72.

74. Gordon_Shaag, A., O. Ben_Nun_Shaul, V. Roitman, Y. Yosef, and A. Oppenheim. 2002. Cellular transcription factor Sp1 recruits simian virus 40 capsid proteins to the viral packaging signal, ses. Journal of Virology 76:5915-24.

75. Greenlee, J. E. 1981. Effect of host age on experimental K virus infection in mice. Infect. Immun. 33:297-303.

76. Greenlee, J. E. 1979. Pathogenesis of K virus infection in newborn mice. Infect. Immun. 26:705-713.

77. Greenlee, J. E., S. H. Clawson, R. C. Phelps, and W. G. Stroop. 1994. Distribution of K-papovavirus in infected newborn mice. J. Comp. Pathol. 111:259-268.

78. Greenlee, J. E., and W. K. Dodd. 1984. Reactivation of persistent papovavirus K infection in immunosuppressed mice. J. Virol. 51:425-429.

79. Greenlee, J. E., and M. F. Law. 1985. Interaction of K papovavirus with hamster cells: transformation of glial cells in vitro but failure of the virus to produce central nervous system tumors in vivo. Arch. Virol. 83:207-215.

80. Greenlee, J. E., R. C. Phelps, and W. G. Stroop. 1991. The major site of murine K papovavirus persistence and reactivation is the renal tubular epithelium. Microb. Pathog. 11:237-247.

81. Gruda, M. C., J. M. Zabolotny, J. H. Xiao, I. Davidson, and J. C. Alwine. 1993. Transcriptional activation by simian virus 40 large T antigen: interactions with multiple components of the transcription complex. Mol Cell Biol 13:961-9.

42

82. Gu, Y., H. Alder, T. Nakamura, S. A. Schichman, R. Prasad, O. Canaani, H. Saito, C. M. Croce, and E. Canaani. 1994. Sequence analysis of the breakpoint cluster region in the ALL-1 gene involved in acute leukemia. Cancer Res 54:2326-30.

83. Hagan, C. R., and C. M. Rudin. 2002. Mobile genetic element activation and genotoxic cancer therapy: potential clinical implications. Am J Pharmacogenomics 2:25-35.

84. Hartl, F. U. 1996. Molecular chaperones in cellular protein folding. Nature 381:571-9. 85. Haun, G., O. T. Keppler, C. T. Bock, M. Herrmann, H. Zentgraf, and M. Pawlita.

1993. The cell surface receptor is a major determinant restricting the host range of the B-lymphotropic papovavirus. J Virol 67:7482-92.

86. Haywood, A. M. 1994. Virus receptors: binding, adhesion strengthening, and changes in viral structure. J Virol 68:1-5.

87. Herbomel, P., B. Bourachot, and M. Yaniv. 1984. Two distinct enhancers with different cell specificities coexist in the regulatory region of polyoma. Cell 39:653-62.

88. Holt, D. 1959. Presence of K virus in wild mice in Australia. Aust. J. Exp.Biol. 37:183-192.

89. Hottiger, M. O., and G. J. Nabel. 2000. Viral replication and the coactivators p300 and CBP. Trends Microbiol 8:560-5.

90. Houck, C. M., F. P. Rinehart, and C. W. Schmid. 1979. A ubiquitous family of repeated DNA sequences in the human genome. J. Mol. Biol. 132:289-306.

91. Hsieh, L. L., W. L. Hsiao, C. Peraino, R. R. Maronpot, and I. B. Weinstein. 1987. Expression of retroviral sequences and oncogenes in rat liver tumors induced by diethylnitrosamine. Cancer Research 47:3421-4.

92. Iida, T., T. Kitamura, J. Guo, F. Taguchi, Y. Aso, K. Nagashima, and Y. Yogo. 1993. Origin of JC polyomavirus variants associated with progressive multifocal leukoencephalopathy. Proc Natl Acad Sci U S A 90:5062-5.

93. Izsvak, Z., Z. Ivics, N. Shimoda, D. Mohn, H. Okamoto, and P. B. Hackett. 1999. Short inverted-repeat transposable elements in teleost fish and implications for a mechanism of their amplification. J Mol Evol 48:13-21.

94. Jang, K. L., and D. S. Latchman. 1989. HSV infection induces increased transcription of Alu repeated sequences by RNA polymerase III. FEBS Lett 258:255-8.

95. Jang, S. K., M. V. Davies, R. J. Kaufman, and E. Wimmer. 1989. Initiation of protein synthesis by internal entry of ribosomes into the 5' nontranslated region of encephalomyocarditis virus RNA in vivo. J Virol 63:1651-60.

96. Jones, N. C., P. W. Rigby, and E. B. Ziff. 1988. Trans-acting protein factors and the regulation of eukaryotic transcription: lessons from studies on DNA tumor viruses. Genes Dev 2:267-81.

97. Joo, W. S., H. Y. Kim, J. D. Purviance, K. R. Sreekumar, and P. A. Bullock. 1998. Assembly of T-antigen double hexamers on the simian virus 40 core origin requires only a subset of the available binding sites. Molecular and Cellular Biology 18:2677-87.

98. Jordan, I., I. Rogozin, G. Glazko, and E. Koonin. 2003. Origin of a substantial fraction of human regulatory sequences from transposable elements. Trends Genet 19:68-72.

99. Jurgens, B., B. J. Schmitz_Drager, and W. A. Schulz. 1996. Hypomethylation of L1 LINE sequences prevailing in human urothelial carcinoma. Cancer Research 56:5698-703.

100. Kates, J., and J. Beeson. 1970. Ribonucleic acid synthesis in vaccinia virus. II. Synthesis of polyriboadenylic acid. J Mol Biol 50:19-33.

101. Katinka, M., M. Yaniv, M. Vasseur, and D. Blangy. 1980. Expression of polyoma early functions in mouse embryonal carcinoma cells depends on sequence rearrangements in the beginning of the late region. Cell 20:393-399.

43

102. Kazazian, H. H., Jr. 2000. Genetics. L1 retrotransposons shape the mammalian genome. Science 289:1152-3.

103. Khoury, G., and E. May. 1977. Regulation of early and late simian virus 40 transcription: overproduction of early viral RNA in the absence of a functional T- antigen. J Virol 23:167-76.

104. Kidwell, M. G., and D. Lisch. 1997. Transposable elements as sources of variation in animals and plants. Proc Natl Acad Sci U S A 94:7704-11.

105. Kikuchi, K., K. Terauchi, M. Wada, and H. Hirano. 2003. The plant MITE mPing is mobilized in anther culture. Nature 421:167-170.

106. Kilham, L., and H. W. Murphy. 1953. A pneumotropic virus isolated from C3H mice carrying the Bittner milk agent. Proc. Soc. Exp. Biol. Med. 82:133-137.

107. Klockmann, U., and W. Deppert. 1983. Acylated simian virus 40 large T-antigen: a new subclass associated with a detergent-resistant lamina of the plasma membrane. Embo J 2:1151-7.

108. Komagome, R., H. Sawa, T. Suzuki, Y. Suzuki, S. Tanaka, W. Atwood, and K. Nagashima. 2002. Oligosaccharides as receptors for JC virus. J.Virol. 76:12992-13000.

109. Kornbluth, S., F. R. Cross, M. Harbison, and H. Hanafusa. 1986. Transformation of chicken embryo fibroblasts and tumor induction by the middle T antigen of polyomavirus carried in an avian retroviral vector. Molecular and Cellular Biology 6:1545-51.

110. Kumar, M., and G. G. Carmichael. 1997. Nuclear antisense RNA induces extensive adenosine modifications and nuclear retention of target transcripts. Proc Natl Acad Sci U S A 94:3542-7.

111. Kypta, R. M., A. Hemming, and S. A. Courtneidge. 1988. Identification and characterization of p59fyn (a src-like protein tyrosine kinase) in normal and polyoma virus transformed cells. The Embo Journal 7:3837-44.

112. Larose, A., N. Dyson, M. Sullivan, E. Harlow, and M. Bastin. 1991. Polyomavirus large T mutants affected in retinoblastoma protein binding are defective in immortalization. J Virol 65:2308-13.

113. Larose, A., L. St-Onge, and M. Bastin. 1990. Mutations in polyomavirus large T affecting immortalization of primary rat embryo fibroblasts. Virology 176:98-105.

114. Laurent, S., V. Frances, and M. Bastin. 1995. Intrachromosomal recombination mediated by the polyomavirus large T antigen. Virology 206:227-33.

115. Law, M. F., K. K. Takemoto, and P. M. Howley. 1979. Characterization of the genome of the murine papovavirus K. J. Virol. 30:90-97.

116. Lednicky, J. A., and J. S. Butel. 2001. Simian virus 40 regulatory region structural diversity and the association of viral archetypal regulatory regions with human brain tumors. Semin Cancer Biol 11:39-47.

117. Levine, A. J. 1996. The origins of virology, 3 rd ed ed, vol. 1, Philadelphia. 118. Li, T. H., C. Kim, C. M. Rubin, and C. W. Schmid. 2000. K562 cells implicate

increased chromatin accessibility in Alu transcriptional activation. Nucleic Acids Res 28:3031-9.

119. Liu, C. K., A. P. Hope, and W. J. Atwood. 1998. The human polyomavirus, JCV, does not share receptor specificity with SV40 on human glial cells. Journal of Neurovirology 4:49-58.

120. Liu, C. K., G. Wei, and W. J. Atwood. 1998. Infection of glial cells by the human polyomavirus JC is mediated by an N-linked glycoprotein containing terminal alpha(2-6)-linked sialic acids. J Virol 72:4643-9.

121. Liu, W. M., W. M. Chu, P. V. Choudary, and C. W. Schmid. 1995. Cell stress and translational inhibitors transiently increase the abundance of mammalian SINE transcripts. Nucleic Acids Res 23:1758-65.

122. Loeber, G., and K. Dorries. 1988. DNA rearrangements in organ-specific variants of polyomavirus JC strain GS. J Virol 62:1730-5.

44

123. Lorimer, H. E., E. H. Wang, and C. Prives. 1991. The DNA-binding properties of polyomavirus large T antigen are altered by ATP and other nucleotides. Journal of Virology 65:687-99.

124. Luo, D., H. Muller, X. B. Tang, and G. Hobom. 1994. Expression and DNA binding of budgerigar fledgling disease virus large T antigen. J Gen Virol 75:1267-80.

125. Lynch, K. J., S. Haggerty, and R. J. Frisque. 1994. DNA replication of chimeric JC virus-simian virus 40 genomes. Virology 204:819-22.

126. Maione, R., C. Passananti, V. De Simone, P. Delli-Bovi, G. Augusti-Tocco, and P. Amati. 1985. Selection of mouse neuroblastoma cell-specific polyoma virus mutants with stage differentiative advantages of replication. EMBO J. 4:3215-3221.

127. Majewski, J., and J. Ott. 2002. Distribution and characterization of regulatory elements in the human genome. Genome Res. 12:1827-1836.

128. Marsh, M., and A. Helenius. 1989. Virus entry into animal cells. Adv Virus Res 36:107-51.

129. Martens, I., S. A. Nilsson, S. Linder, and G. Magnusson. 1989. Mutational analysis of polyomavirus small-T-antigen functions in productive infection and in transformation. J Virol 63:2126-33.

130. Marton, A., D. Jean, L. Delbecchi, D. T. Simmons, and P. Bourgaux. 1993. Topoisomerase activity associated with SV40 large tumor antigen. Nucleic Acids Res 21:1689-95.

131. Marton, A., B. Marko, L. Delbecchi, and P. Bourgaux. 1995. Topoisomerase activity associated with polyoma virus large tumor antigen. Biochim Biophys Acta 1262:59-63.

132. Mastrangelo, I. A., P. V. Hough, J. S. Wall, M. Dodson, F. B. Dean, and J. Hurwitz. 1989. ATP-dependent assembly of double hexamers of SV40 T antigen at the viral origin of DNA replication. Nature 338:658-62.

133. Mayer, M., and K. Dorries. 1991. Nucleotide sequence and genome organization of the murine polyomavirus, Kilham strain. Virology 181:469-480.

134. Mei, Y. F., and G. Wadell. 1995. Molecular determinants of adenovirus tropism. Curr Top Microbiol Immunol 199:213-28.

135. Mellor, A., and A. E. Smith. 1978. Characterization of the amino-terminal tryptic peptide of simian virus 40 small-t and large-T antigens. J Virol 28:992-6.

136. Mighell, A. J., A. F. Markham, and P. A. Robinson. 1997. Alu sequences. FEBS Lett 417:1-5.

137. Miller, J., P. Bullock, and M. Botchan. 1984. Simian virus 40 T antigen is required for viral excision from chromosomes. Proc Natl Acad Sci U S A 81:7534-8.

138. Mokhtarian, F., and K. V. Shah. 1980. Role of antibody response in recovery from K-papovavirus infection in mice. Infect. Immun. 29:1169-1179.

139. Monini, P., A. Rotola, D. Di Luca, L. De Lellis, E. Chiari, A. Corallini, and E. Cassai. 1995. DNA rearrangements impairing BK virus productive infection in urinary tract tumors. Virology 214:273-279.

140. Morgan, G. T. 1995. Identification in the human genome of mobile elements spread by DNA- mediated transposition. J Mol Biol 254:1-5.

141. Murakami, Y., and J. Hurwitz. 1993. DNA polymerase alpha stimulates the ATP-dependent binding of simian virus tumor T antigen to the SV40 origin of replication. The Journal of Biological Chemistry 268:11018-27.

142. Murakami, Y., and J. Hurwitz. 1993. Functional interactions between SV40 T antigen and other replication proteins at the replication fork. The Journal of Biological Chemistry 268:11008-17.

143. Negrini, M., S. Sabbioni, R. R. Arthur, A. Castagnoli, and G. Barbanti-Brodano. 1991. Prevalence of the archetypal regulatory region and sequence polymorphisms in nonpassaged BK virus variants. J. Virol. 65:5092-5095.

45

144. Nilsson, M., M. Osterlund, and G. Magnusson. 1991. Analysis of polyomavirus enhancer-effect on DNA replication and early gene expression. J. Mol. Biol. 218:479-483.

145. Norkin, L. C., H. A. Anderson, S. A. Wolfrom, and A. Oppenheim. 2002. Caveolar endocytosis of simian virus 40 is followed by brefeldin A-sensitive transport to the endoplasmic reticulum, where the virus disassembles. Journal of Virology 76:5156-66.

146. Oppenheim, A., Z. Sandalon, A. Peleg, O. Shaul, S. Nicolis, and S. Ottolenghi. 1992. A cis-acting DNA signal for encapsidation of simian virus 40. J Virol 66:5320-8.

147. Oren, M., S. Lavi, and E. Winocour. 1978. The structure of a cloned substituted SV40 genome. Virology 85:404-421.

148. Panning, B., and J. R. Smiley. 1994. Activation of RNA polymerase III transcription of human Alu elements by herpes simplex virus. Virology 202:408-17.

149. Panning, B., and J. R. Smiley. 1993. Activation of RNA polymerase III transcription of human Alu repetitive elements by adenovirus type 5: requirement for the E1b 58-kilodalton protein and the products of E4 open reading frames 3 and 6. Mol Cell Biol 13:3231-44.

150. Parsons, D. F. 1963. Morphology of K virus and its relation to the papova group of viruses. Virology 20:385-388.

151. Peden, K. W., S. L. Spence, L. C. Tack, C. A. Cartwright, A. Srinivasan, and J. M. Pipas. 1990. A DNA replication-positive mutant of simian virus 40 that is defective for transformation and the production of infectious virions. J Virol 64:2912-21.

152. Pelkmans, L., and A. Helenius. 2002. Endocytosis via caveolae. 3:311-20. 153. Pelkmans, L., J. Kartenbeck, and A. Helenius. 2001. Caveolar endocytosis of simian

virus 40 reveals a new two-step vesicular-transport pathway to the ER. Nature Cell Biology 3:473-83.

154. Pelkmans, L., D. Puntener, and A. Helenius. 2002. Local actin polymerization and dynamin recruitment in SV40-induced internalization of caveolae. Science 296:535-9.

155. Pho, M. T., A. Ashok, and W. J. Atwood. 2000. JC virus enters human glial cells by clathrin-dependent receptor-mediated endocytosis. Journal of Virology 74:2288-92.

156. Pipas, J. M. 1992. Common and unique features of T antigens encoded by the polyomavirus group. J Virol 66:3979-85.

157. Pomerantz, B. J., C. R. Mueller, and J. A. Hassell. 1983. Polyomavirus large T antigen binds independently to multiple, unique regions on the viral genome. J. Virol. 47:600-610.

158. Pommier, Y., G. Kohlhagen, C. Wu, and D. T. Simmons. 1998. Mammalian DNA topoisomerase I activity and poisoning by camptothecin are inhibited by simian virus 40 large T antigen. Biochemistry 37:3818-23.

159. Porras, A., J. Bennett, A. Howe, K. Tokos, N. Bouck, B. Henglein, S. Sathyamangalam, B. Thimmapaya, and K. Rundell. 1996. A novel simian virus 40 early-region domain mediates transactivation of the cyclin A promoter by small-t antigen and is required for transformation in small-t antigen-dependent assays. J Virol 70:6902-8.

160. Porras, A., S. Gaillard, and K. Rundell. 1999. The simian virus 40 small-t and large-T antigens jointly regulate cell cycle reentry in human fibroblasts. J Virol 73:3102-7.

161. Pozueta-Romero, J., G. Houlne, and R. Schantz. 1996. Nonautonomous inverted repeat Alien transposable elements are associated with genes of both monocotyledonous and dicotyledonous plants. Gene 171:147-53.

162. Rassoulzadegan, M., A. Cowie, A. Carr, N. Glaichenhaus, R. Kamen, and F. Cuzin. 1982. The roles of individual polyomavirus early proteins in oncogenic transformation. Nature 300:713-718.

163. Rice, P. W., and C. N. Cole. 1993. Efficient transcriptional activation of many simple modular promoters by simian virus 40 large T antigen. J Virol 67:6689-97.

46

164. Riley, M. I., W. Yoo, N. Y. Mda, and W. R. Folk. 1997. Tiny T antigen: an autonomous polyomavirus T antigen amino-terminal domain. J Virol 71:6068-74.

165. Rio, D. C., and R. Tjian. 1983. SV40 T antigen binding site mutations that affect autoregulation. Cell 32:1227-40.

166. Rogers, J. H. 1985. The origin and evolution of retroposons. Int. Rev. Cytol. 93:187-279.

167. Rossmann, M. G. 1994. Viral cell recognition and entry. Protein Sci 3:1712-25. 168. Rudin, C. M., and C. B. Thompson. 2001. Transcriptional activation of short

interspersed elements by DNA- damaging agents. Genes Chromosomes Cancer 30:64-71.

169. Rundell, K., S. Gaillard, and A. Porras. 1998. Small-t and large-T antigens cooperate to drive cell proliferation. Dev Biol Stand 94:289-95.

170. Russanova, V. R., C. T. Driscoll, and B. H. Howard. 1995. Adenovirus type 2 preferentially stimulates polymerase III transcription of Alu elements by relieving repression: a potential role for chromatin. Mol Cell Biol 15:4282-90.

171. Sahli, R., R. Freund, T. Dubensky, R. Garcea, R. Bronson, and T. Benjamin. 1993. Defect in entry and altered pathogenicity of a polyoma virus mutant blocked in VP2 myristylation. Virology 192:142-53.

172. Samani, N. J., J. R. Thompson, L. O'Toole, K. Channer, and K. L. Woods. 1996. A meta-analysis of the association of the deletion allele of the angiotensin-converting enzyme gene with myocardial infarction. Circulation 94:708-12.

173. Sandalon, Z., N. Dalyot-Herman, A. B. Oppenheim, and A. Oppenheim. 1997. In vitro assembly of SV40 virions and pseudovirions: vector development for gene therapy. Hum Gene Ther 8:843-9.

174. Scheidtmann, K. H., B. Echle, and G. Walter. 1982. Simian virus 40 large T antigen is phosphorylated at multiple sites clustered in two separate regions. J Virol 44:116-33.

175. Schmitt, M. K., and K. Mann. 1987. Glycosylation of simian virus 40 T antigen and localization of glycosylated T antigen in the nuclear matrix. Virology 156:268-81.

176. SenGupta, D. J., and J. A. Borowiec. 1994. Strand and face: the topography of interactions between the SV40 origin of replication and T-antigen during the initiation of replication. Embo J 13:982-92.

177. Sheng, Q., T. M. Love, and B. Schaffhausen. 2000. J domain-independent regulation of the Rb family by polyomavirus large T antigen. J Virol 74:5280-90.

178. Shinohara, T., M. Matsuda, K. Yasui, and K. Yoshiike. 1989. Host range bias of the JC virus mutant enhancer with DNA rearrangement. Virology 170:261-3.

179. Simmons, D. T., T. Melendy, D. Usher, and B. Stillman. 1996. Simian virus 40 large T antigen binds to topoisomerase I. Virology 222:365-74.

180. Smit, A. F. 1996. The origin of interspersed repeats in the human genome. Curr Opin Genet Dev 6:743-8.

181. Smit, A. F., and A. D. Riggs. 1996. Tiggers and DNA transposon fossils in the human genome. Proc Natl Acad Sci U S A 93:1443-8.

182. Soprano, K. J., N. Galanti, G. J. Jonak, S. McKercher, J. M. Pipas, K. W. Peden, and R. Baserga. 1983. Mutational analysis of simian virus 40 T antigen: stimulation of cellular DNA synthesis and activation of rRNA genes by mutants with deletions in the T-antigen gene. Mol Cell Biol 3:214-9.

183. Spence, S. L., and J. M. Pipas. 1994. Simian virus 40 large T antigen host range domain functions in virion assembly. J Virol 68:4227-40.

184. Spence, S. L., and J. M. Pipas. 1994. SV40 large T antigen functions at two distinct steps in virion assembly. Virology 204:200-9.

185. Srinivasan, A., A. J. McClellan, J. Vartikar, I. Marks, P. Cantalupo, Y. Li, P. Whyte, K. Rundell, J. L. Brodsky, and J. M. Pipas. 1997. The amino-terminal transforming region of simian virus 40 large T and small t antigens functions as a J domain. Mol Cell Biol 17:4761-73.

47

186. St-Onge, L., L. Bouchard, and M. Bastin. 1993. High-frequency recombination mediated by polyomavirus large T antigen defective in replication. J Virol 67:1788-95.

187. Stang, E., J. Kartenbeck, and R. G. Parton. 1997. Major histocompatibility complex class I molecules mediate association of SV40 with caveolae. Mol Biol Cell 8:47-57.

188. Streuli, C. H., and B. E. Griffin. 1987. Myristic acid is coupled to a structural protein of polyoma virus and SV40. Nature 326:619-22.

189. Strout, M. P., G. Marcucci, C. D. Bloomfield, and M. A. Caligiuri. 1998. The partial tandem duplication of ALL1 (MLL) is consistently generated by Alu-mediated homologous recombination in acute myeloid leukemia. Proc Natl Acad Sci U S A 95:2390-5.

190. Struhl, K. 1998. Histone acetylation and transcriptional regulatory mechanisms. Genes Dev 12:599-606.

191. Sullivan, C. S., and J. M. Pipas. 2002. T antigens of simian virus 40: molecular chaperones for viral replication and tumorigenesis. Microbiol Mol Biol Rev 66:179-202.

192. Super, H. G., P. L. Strissel, O. M. Sobulo, D. Burian, S. C. Reshmi, B. Roe, N. J. Zeleznik-Le, M. O. Diaz, and J. D. Rowley. 1997. Identification of complex genomic breakpoint junctions in the t(9;11) MLL-AF9 fusion gene in acute leukemia. Genes Chromosomes Cancer 20:185-95.

193. Szabo, A., T. Langer, H. Schroder, J. Flanagan, B. Bukau, and F. U. Hartl. 1994. The ATP hydrolysis-dependent reaction cycle of the Escherichia coli Hsp70 system DnaK, DnaJ, and GrpE. Proc Natl Acad Sci U S A 91:10345-9.

194. Tack, L. C., and P. Beard. 1985. Both trans-acting factors and chromatin structure are involved in the regulation of transcription from the early and late promoters in simian virus 40 chromosomes. J Virol 54:207-18.

195. Takemoto, K. K., and P. Fabisch. 1970. Transformation of mouse cells by K-papovavirus. Virology 40:135-143.

196. Treisman, R., U. Novak, J. Favaloro, and R. Kamen. 1981. Transformation of rat cells by an altered polyoma virus genome expressing only the middle-T protein. Nature 292:595-600.

197. Tu, Z., and S. P. Orphanidis. 2001. Microuli, a family of miniature subterminal inverted-repeat transposable elements (MSITEs): transposition without terminal inverted repeats. Mol. Biol. Evol. 18:893-895.

198. Turner, W. J., and M. E. Woodworth. 2001. DNA replication efficiency depends on transcription factor-binding sites. J Virol 75:5638-45.

199. Vacante, D. A., R. Traub, and E. O. Major. 1989. Extension of JC virus host range to monkey cells by insertion of a simian virus 40 enhancer into the JC virus regulatory region. Virology 170:353-361.

200. Varshavsky, A. J., V. V. Bakayev, P. M. Chumackov, and G. P. Georgiev. 1976. Minichromosome of simian virus 40: presence of histone HI. Nucleic Acids Res 3:2101-13.

201. Waga, S., and B. Stillman. 1994. Anatomy of a DNA replication fork revealed by reconstitution of SV40 DNA replication in vitro. Nature 369:207-12.

202. Wang, E. H., S. Bhattacharyya, and C. Prives. 1993. The replication functions of polyomavirus large tumor antigen are regulated by phosphorylation. J Virol 67:6788-96.

203. Weiss, R. A., and C. S. Tailor. 1995. Retrovirus receptors. Cell 82:531-3. 204. Wessler, S. R., T. E. Bureau, and S. E. White. 1995. LTR-retrotransposons and

MITEs: important players in the evolution of plant genomes. Curr. Opin. Genet. Dev. 5:814-821.

205. Wildeman, A. G. 1989. Transactivation of both early and late simian virus 40 promoters by large tumor antigen does not require nuclear localization of the protein. Proc Natl Acad Sci U S A 86:2123-7.

48

206. Wold, M. S., J. J. Li, and T. J. Kelly. 1987. Initiation of simian virus 40 DNA replication in vitro: large-tumor- antigen- and origin-dependent unwinding of the template. Proc Natl Acad Sci U S A 84:3643-7.

207. Wu, C., D. Edgil, and D. T. Simmons. 1998. The origin DNA-binding and single-stranded DNA-binding domains of simian virus 40 large T antigen are distinct. Journal of Virology 72:10256-9.

208. Wu, C., R. Roy, and D. T. Simmons. 2001. Role of single-stranded DNA binding activity of T antigen in simian virus 40 DNA replication. Journal of Virology 75:2839-47.

209. Xu, T. H., and K. J. Deng. 2002. Transposable elements and tumor progression. Medical Hypotheses 58:293-6.

210. Yang, S. I., R. L. Lickteig, R. Estes, K. Rundell, G. Walter, and M. C. Mumby. 1991. Control of protein phosphatase 2A by simian virus 40 small-t antigen. Mol Cell Biol 11:1988-95.

211. Yogo, Y., T. Kitamura, C. Sugimoto, T. Ueki, Y. Aso, K. Hara, and F. Taguchi. 1990. Isolation of a possible archetypal JC virus DNA sequence from nonimmunocompromised individuals. J Virol 64:3139-43.

212. Yogo, Y., T. Matsushima-Ohno, T. Hayashi, C. Sugimoto, M. Sakurai, and I. Kanazawa. 2001. JC virus regulatory region rearrangements in the brain of a long surviving patient with progressive multifocal leukoencephalopathy. J. Neurol. Neurosurg. Psychiatry 71:397-400.

213. Zerrahn, J., U. Knippschild, T. Winkler, and W. Deppert. 1993. Independent expression of the transforming amino-terminal domain of SV40 large I antigen from an alternatively spliced third SV40 early mRNA. Embo J 12:4739-46.

214. Zhang, Q., J. Arbuckle, and S. R. Wessler. 2000. Recent, extensive, and preferential insertion of members of the miniature inverted-repeat transposable element family Heartbreaker into genic regions of maize. Proc. Natl. Acad. Sci. U S A 97:1160-1165.

215. Zhang, S., and G. Magnusson. 2003. Cellular Mobile Genetic Elements in the Regulatory Region of the Pneumotropic Mouse Polyomavirus Genome. Structure and Function in Viral Gene Expression and DNA Replication. Accepted by J. Virol.

216. Zhang, S., and G. Magnusson. 2003. Expression and DNA Binding of Murine Pneunotropic Virus (MPtV) Large T Antigen. Manuscript.

217. Zhang, X., C. Feschotte, Q. Zhang, N. Jiang, W. B. Eggleston, and S. R. Wessler. 2001. P instability factor: an active maize transposon system associated with the amplification of Tourist-like MITEs and a new superfamily of transposases. Proc. Natl. Acad. Sci. U S A 98:12572-12577.