POSTTRANSLATIONAL MODIFICATIONS OF POTATO VIRUS A MOVEMENT RELATED PROTEINS CP AND VPg

Pietri Puustinen

Institute of Biotechnology and Department of Applied Biology

Faculty of Forestry and Agriculture, and Department of Biological and Environmental Sciences

Division of Genetics, Faculty of Biosciences University of Helsinki

ACADEMIC DISSERTATION

To be presented with the permission of the Faculty of Biosciences of the University of Helsinki for public criticism in auditorium 1041

of Biocenter II, Viikinkaari 5, Helsinki, 20 may 2005, at 12.00 noon.

HELSINKI 2005

Supervisor

Docent Kristiina Mäkinen Department of Applied BIology University of Helsinki Finland

Reviewers

Docent Tero AholaInstitute of BiotechnologyUniversity of Helsinki

And

Dr. Lesley TorranceScottish Crop Research Institute Dundee, Scotland United Kingdom

Opponent

Professor Timo HyypiäDepartment of VirologyUniversity of TurkuFinland

ISSN 1239-9469 ISBN 952-10-2420-8University Printing HouseHelsinki 2005

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Original publications:

I. Ivanov K., Puustinen P, Merits A., Saarma M. and Mäkinen K. (2001): Phosphorylation down-regulates the RNA binding function of the coat protein of potato virus A. Journal of Biological Chemistry. 276, 13530 – 13540.

II. Puustinen P, Rajamäki M-L, Ivanov K, Valkonen J.P.T, and Mäkinen K. (2002): Detection of the potyviral genome linked protein VPg in virions and its phosphorylation by host kinases. Journal of Virology. 76, 12703 – 12711.

III. Ivanov K., Puustinen P., Gabrenaite R., Vihinen H., Rönnstrand L., Valmu L., Kalkkinen N and and Mäkinen K. (2003): Phosphorylation of the potyvirus capsid protein by protein kinase CK2 and its relevance for virus infection. Plant Cell, 15, 2124 – 2139.

IV. Puustinen P. and Mäkinen K. (2004): Uridylylation of the potyvirus VPg by viral replicase NIb Correlates with the nucleotide binding capacity of VPg. Journal of Biological Chemistry. 279, 38103 – 38110.

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Abbreviations

aa: amino acidATP: adenosine triphosphatebp: base pairBSA: bovine serum albuminCD: circular dichroismCIP: cylindrical inclusion protein CK2: casein kinase IICP: coat proteind: deoxydd: dideoxyDMC: divalent metal cationDNA: deoxyribonucleic acidDRB: 5,6-dicloro-1-(β-D-ribofuranosyl)benzimidazole ds: double strandedEDTA: ethylenediaminetetraacetic acidEM: electron microscopyER: endoplasmic reticulumGTP: guanosine triphosphateHC-pro helper component proteinaseicDNA: infectious complementary DNAIPTG: isopropyl- β -D-thiogalactopyranosidekb: kilobasekDa: kilodaltonMAPK: mitogen-activated protein kinasesMP: movement proteinMPR: movement related proteinMW: molecular weightnt: nucleotideNIa: proteinase part of NIaNIa –tot: VPg + proteinase part intermediateNIb: RNA dependent RNA polymeraseNLS: nuclear localization signalNTP: nucleoside triphosphatemCKII maize CKIIORF: open reading framePAGE: polyacrylamide gel electrophoresisPVA: Potato virus APV: poliovirusRC: replication complexRdRP: RNA-dependent RNA polymeraseRNA: ribonucleic acidRNAi: RNA interferencermCK2: recombinant maize casein kinase 2 subunitRNase: ribonucleaseSAP: shrimp alkaline phosphataseSAR: systemic acquired resistancetCK2: tobacco CK2TEV: tobacco etch virusTMV: tobacco mosaic virusUMP: uridine monophosphateUTP: uridine triphosphate VPg: viral genome linked proteinwt: wild type

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“I’ve learned to think like a virus; no neurons, a lot of sex, and a lot of errors.”

John J. Holland. (from Annu. Rev. Phytopathol. 1997. 35:191–209)

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Contents

Original publications .................................................................................................3Abbreviations ............................................................................................................4ABSTRACT ..............................................................................................................71.0 INTRODUCTION ................................................................................................8 1.1 The viruses .......................................................................................8 1.2 Positive-strand RNA viruses .............................................................8 1.3 Potyviruses .......................................................................................9 1.4 Transmission of potyviruses ...........................................................10 1.5 Polyprotein processing ...................................................................10 1.6 Replication and translation .............................................................11 1.7 Viral cell-to-cell movement .............................................................12 1.8 Long-distance movement ...............................................................13 1.9 Protein phosphorylation .................................................................14 1.10 Protein kinases .............................................................................15 1.11 The CK kinase family ..................................................................16 1.12 Protein kinase II (CK2) .................................................................17 1.13 Function and comparison of plant CK2 –subunits ........................172.0 AIMS OF THE STUDY .....................................................................................183.0 MATERIALS AND METHODS .........................................................................194.0 RESULTS .........................................................................................................20 4.1 N.tabacum protein extracts phosphorylates PVA CP and VPg ......20 4.2 Identification of CK2 activity phosphorylating PVA CP ...................20 4.3 Identification of CK2 phosphorylation in vivo .................................21 4.4 Phosphorylation of PVA CP regulates its RNA-binding activity ......22 4.5 Phosphorylation of PVA VPg ..........................................................22 4.6 Nucleotidylation of PVA VPg .........................................................23 4.7 Identification of nucleotide binding site in PVA VPg .......................245.0 DISCUSSION ...................................................................................................25 5.1 Phosphorylation of the VPg protein ................................................25 5.2 Polar localization of VPg ...............................................................27 5.3 Nucleotidylation of the VPg ...........................................................27 5.4 The NTP-binding domain of VPg ...................................................29 5.5 Phosphorylation of the CP molecule ..............................................29 5.6 RNA-binding function of CP is regulated by phosphorylation ........30 5.7 PVA CP is phosphorylated by CK2 ................................................316.0 CONCLUDING REMARKS ..............................................................................34ACKNOWLEDGEMENTS ......................................................................................35REFERENCES .......................................................................................................36

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ABSTRACT

Protein kinases are known to be involved in the regulation of the virion assembly, rep-lication, and dissociation of numerous animal viruses. In contrast, there is a knowl-edge of scarce regarding the role of protein phosphorylation in the infection cycle. Potato virus A (PVA) is a positive-strand RNA virus, which belongs to the genus Poty-virus; family Potyviridae. The present work describes how the PVA movement-related proteins CP and VPg, when phosphorylated by protein kinase, function in the viral movement and replication cycle.

In this study, coat protein (CP) and viral genome linked protein (VPg) are shown to be under the regulation of tobacco protein kinases in vitro and in vivo. This finding sug-gests a mechanism to regulate the formation and stability of viral ribonucleoprotein complexes. This study also shows that VPg bound to virions can interact with host kinases. The VPg is known to support vascular movement with a different efficiency in tobacco and in potato. Detailed biochemical analysis reveals variations between different PVA-strain VPgs phosphorylated in vitro by tobacco and by potato kinases. This finding supports the idea that tobacco and potato kinases can recognize move-ment related virus proteins, and either support or inactivate movement functions.

A major objective of this work has been to study the phosphorylation of PVA CP with casein kinase II (CK2). CK2 is the key kinase responsible for phosphorylating in vitro and in vivo the triple consensus sequence in PVA CP. The function of the CK2 phos-phorylation site in CP was studied with point mutations introduced in GFP-tagged icDNA. The resulting mutants were movement deficient. Biochemical studies showed that the RNA binding affinity of CK2-phosphorylated CP was decreased. These find-ings together suggest that CK2 phosphorylation is functionally important for the infec-tion cycle of potyviruses.

Picornaviruses use a dedicated protein primer to initiate the synthesis of the viral RNA genome. The role of certain poliovirus (PV) VPg amino acids is essential for the initiation of viral replication. To understand the mechanism of PVA replication initiation, we analyzed VPg and NIb polymerase interaction in vitro. We found that recombinant VPg is nucleotidylated by NIb-RNA polymerase in an Mn2+ dependent reaction. Our in vitro reaction was RNA-template independent but was sensitive for VPg NTP-binding domain deletion. Intriguingly, VPg is an NTP-binding protein, and may require Mn2+ to coordinate and stabilize the incoming nucleotide. The VPg NTP binding region is located in a 7-amino acid-long domain, which shares a limited ho-mology with poliovirus VPg. The NTP binding of VPg was inactivated by a deletion of this 7-amino acid region. These findings suggest that picornaviruses and potyviruses may share a conserved replication initiation mechanism.

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1.0 INTRODUCTION

1.1 The virusesViruses are intracellular parasites that are classified according to their genetic mate-rial, which is either DNA or RNA. Further classification of viruses follows structural similarities of the particle, sequence, segmentation, and polarity of the genome, and the absence or presence of an envelope consisting of lipids and proteins (Fields et al., 1996). All life forms are invaded by viruses. Written history has recorded many catastrophes caused by viruses; even the modern world today with its controlled infrastructure cannot totally stop the spread of new viruses generating new threats, and direct and indirect economic damage (Domingo et al., 1999). As an example, in eastern Asia a new mutated influenza virus strain originated when bird influenza ap-parently jumped from chickens to humans. Viruses, being obligatory parasites, are capable of invading subcellular machinery, and forcing the host cell to support their replication, infection, and spread. The sim-plicity of viruses leads to underestimation of the complexity of the molecular pro-cesses involved in replication and translation of the virus genome. In particular, the replication as a molecular mechanism for copying the genome is under extensive study. Coordination of translation and replication is a key factor, which requires nu-merous interactions between viral elements and host-encoded factors (Noueiry et al., 2003). Evidence for long adaptation of viruses to hosts is seen in the specific host-virus interactions which play essential roles in support of the virus life cycle (De Jong & Ahlquist et al., 1995; Gamarnik and Adino. 1996).

1.2 Positive-strand RNA virusesPositive-stranded RNA viruses are the most abundant molecular parasites (Murphy, 1996; Lazarowitz, 2001). Evidently, 80% of all the virus species consist of RNA virus-es, the majority of them positive-strand RNA viruses, which comprise also the major-ity of plant viruses (Domingo et al., 1999). The genomic RNA is enclosed in a capsid working as a protective structure. After entry into the host cell, the messenger-sense RNA genome is released from the capsid, and the host proteins support the start of replication. The virus genome is translated into a polyprotein, which is processed by virally-encoded proteinases into both structural and non-structural proteins. The genomic RNA serves as a template to produce negative-strand RNA replication inter-mediates within a membrane-associated RNA replicase complex, and this produces positive-strand genomic RNA (Noueiry et al., 2003). Taken together, the high mutation rate and the error-prone nature of RNA-dependent RNA polymerase (RdRp) catalyzed RNA synthesis allow viruses to use continuous production of mutants as a means for adaptation to environmental changes. Viruses are able to respond to a selective pressure. Adaptability of RNA viruses is capable of producing mutations neutralizing at he effect of a number of antiviral inhibitors used against human immunodeficiency virus (HIV) during treatment of HIV patients (Harrigan and Alexander, 1999). The molecular mechanisms for adaptation are ge-nomic point mutations and recombination, both being major evolutionary forces for RNA viruses (Robertson et al., 1995; Sharp et al., 1994) Recombination serves as

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a mechanism to transfer foreign RNA segments into viral genomes (Chetverin et al., 1999; Moya et al., 2000; Domingo et al., 1997b). Another well-known example is an-nually occurring pandemic influenza, the segmented genome of which can reassort and overcome the human immune system, which produces antibodies against virus envelope proteins (Webster et al., 1995). The main factor for this high mutation rate is the lack of RNA-dependent RNA polymerase (RdRp) proofreading activity. Errors during RNA replication are estimated to be in the range of 10-5 to 10-3 substitutions per nucleotide and per round of copying. This in turn leads to a high mutation rate, and to a limited genome size (Domingo and Holland, 1997a; Domingo et al., 1997 b; Drake and Holland, 1999).RNA viruses have a limited possibility to carry information in the genome, and there-fore the number of genes is small. Different virus families usually have different ge-nome organizations, and distinct genome strategies of replication and gene expres-sion (Fields et al., 1996; Flint et al., 2000). Interestingly, one protein can serve several different functions during the viral life cycle (Fields et al. 1996; Flint et al., 2000).

1.3 PotyvirusesPotyviruses have filamentous virions with ca. 2000 copies of a single type of coat protein (CP) that encapsidates the positive-strand genomic RNA. RNA length ranges from 9 500 to 10 000 bp depending on the virus subtype. The potyvirus particles are 680 to 900 nm long and 11 to 15 nm in diameter when studied with electron micros-copy (EM). The x-ray crystallography of the constituent proteins of the virus particle, CP, and VPg has not been successful, but the secondary structure of potato virus A (PVA, genus Potyvirus, family Potyviridae) CP has been modelled using tritium bom-bardment, giving a prediction of the overall protein topology (Baratova et al., 2001). The VPg provides polarity to the positive-strand genome (Murphy et al., 1991). The nontranslated (NTR) region of the 5´-terminus enhances translation and contains an active internal ribosome entry site (Carrington and Freed, 1990; Basso et al., 1994). The 3´-NTR is polyadenylated and cooperates with the 5´-NTR sequence for efficient translation (Gallie et al., 1995). Potyviruses are of considerable biological importance, representing ca. 30% of the known plant viruses (Shukla et al., 1994; Urcuqui-Inchima et al., 2001). Potyviruses resemble picornaviruses in their genome organization and polyprotein processing (Sadowy et al., 2001). Phylogenetic analyses of potyvirus protein se-quences reveal a close evolutionary relationship among picorna-like viruses (Koonin and Dolja, 1993). The superfamily of the picorna-like viruses includes many viruses human infecting with similar genetic organization and somewhat homologous repli-case proteins. Similarities of genome organization indicate that these virus groups, picornaviruses and potyviruses, may have a common ancestor. Environmental fac-tors have forced plant viruses to generate specific movement mechanisms such as movement through the plant cell wall and systemic infection through phloem systems (Mushegian et al., 1993; Lucas et al., 1993; Lee et al., 2001). The level of identity across the genome of potyviruses is fairly conserved; amino acid similarity is less than 70%, which reflects rather high evolutionary flexibility and sug-gests that potyviruses continue to adapt to environmental changes (Dolja et al., 1991; Koonin and Dolja, 1993; Callaway et al. 2001). Like other RNA viruses, potyviruses

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accumulate mutations at a higher rate than do DNA viruses (Domingo and Holland, 1997; Smith et al., 1997).

1.4 Transmission of potyvirusesA mechanically strong cell wall surrounds plant cells; it consists of a complex mixture of polysaccharides and phenolic compounds making it resistant to attacks from out-side. Potyviruses use aphids as vectors to breach the cell wall and to inoculate new host cells. Potyviruses have adapted to aphid transmission by using two viral pro-teins, CP and helper-component proteinase (HC-Pro), that interacts with the aphid stylet interior. Potato virus Y (PVY) and tobacco vein mottling virus (TVMV) CP are shown to associate with HC-Pro in an EM study, and this protein bridge holds the virus in the aphid interior mouthparts (Ammar et al., 1994). Direct evidence for the requirement of a specific interaction between CP and HC-Pro in aphid transmission is demonstrated by in vitro binding studies. HC-Pro interacts with dot-blotted zucchini yellow mosaic virus (ZYMV) virions. Studies reveal interacting domains within the TVMV CP N-terminus and HC-Pro. Based upon these observations, it is suggested that HC-Pro interacts with the virion by using several short domains which form a bridge between particle and the unknown protein serving as a receptor in the aphid epicuticle mouthparts (Blanc et al., 1998; Blanc et al., 1997; Peng et al., 1998).

1.5 Polyprotein processingEfficient translation in eukaryotes requires 3´-polyadenylation and a 5´-cap structure in the mRNA. Potyviruses have developed an alternative mechanism to overcome these requirements (Gallie, 1998). Cap-independent translation for Tobacco etch virus (TEV) originates when the 5´-nontranslated region interacts with the 3´-poly(A), and the translation protein complex localizes upstream in the open reading frame. VPg interaction with the transcription initiation factor eIF(iso)4E is required for infectivity (Lellis et al., 2002; Wittmann et al., 1997). Potyvirus translation is supported by VPg interacting with eIF(iso)4E during translation initiation (Leonard et al., 2000). How-ever, the 5´-nontranslated RNA region of potyviruses is capable of supporting direct cap-independent translation enhancement without VPg (Carrington et al., 1990).

The potyviral proteins are translated as a polyprotein precursor from the (+)-strand RNA. The proteins are proteolytically cleaved to produce non-structural proteins and the CP. Processing of the precursors is catalyzed by virus-encoded proteases (Fig. 1). The N-terminal proteinases are: P1, a chymotrypsin-like serine protease and HC-Pro, a papain-like proteinase functional domain (Carrington et al., 1989; Verchot et al., 1991). The P1 and HC-Pro proteinases catalyze autoproteolytic cleavage of their C-termini in cis (Thornbury et al., 1993). HC-Pro catalyzes autoproteolytic cleavage at the Gly–Gly dipeptide between itself and the P3 protein (Carrington and Herndon, 1992; Kasschau et al., 1997; Merits et al., 2002; Ruiz-Ferrer et al., 2004). Nuclear inclusion proteinase (NIa), resembling picornavirus 3C proteinase in architecture, di-gests the rest of the proteins out from the polyprotein (reviewed in Riechmann et al., 1992; Merits et al., 2002). NIa and 3C both resemble members of the chymotrypsin family of serine proteinases. However, catalytic core of these viral chymotrypsin pro-

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teinases includes a cysteine as a nucleophil rather than serine (Allaire et al., 1994; Blazan et al., 1988; Matthews et al., 1994). NIa has two functionally distinct domains: a C-terminal proteinase domain and an N-terminal VPg domain. These domains are separated by an internal cleavage site (Dougherty et al. 1991; Merits et al., 2002). Processing at the NIa internal site is inefficient, due to a suboptimal cleavage site motif. Cleavage site mutations, having either an inactivating or more efficient pro-cessing sequence, cause amplification debilitation. This suggests that slow internal processing is an important regulatory feature (Li et al., 1997). Polyprotein processing may regulate activity of processed protein intermediates, since different cleavage sites are processed with different efficiencies depending on their position in the poly-protein (Schaad et al., 1996; Riechmann et al., 1995; Carrington et al., 1993; Merits et al., 2002).Because a complete polyprotein is never detected, it is suggested that polyprotein processing starts co-translationally, producing short half-life processing intermedi-ates. The stable processing intermediates P3-6K1, CI-6K2, and NIa (VPg-NIa-Pro) may play a role during replication. Although longer polyprotein intermediates have not been detected in pulse chase experiments, these may play a role in the potyvirus life cycle (Schaad et al., 1996; Merits et al., 2002). In alphaviruses, some of the process-ing intermediates have distinct functions during replication. It has been show that the polyprotein processing triggers those individual proteins taking part in replication to change specificity from minus-strand synthesis towards plus-strand and subgenomic RNA synthesis (Shirako et al., 1994; Vasiljeva et al., 2003).

Fig 1. Schematic representation of PVA genome. The polyprotein precursor of the non-structural and struc-tural proteins is produced from the (+) strand RNA (10 kb). The processing of the polyprotein precursor is mainly catalyzed by the NIa-Pro proteinase domain. Protease activities of P1 protease and HC-Pro cleave only their respective C-termini.

1.6 Replication and translationPotyvirus replication takes place in the cytoplasm in association with the endoplasmic reticulum-like membranes (Martin et al., 1995; Shaad et al., 1997a). Most potyviral proteins bind RNA in an unspecific manner in vitro, and thus may interact with the replication complex (Merits et al., 1998). The membrane-associated proteins 6K1 and 6K2 may anchor the replication complex to the membranous environment (Re-strepo-Hartwig and Carrington, 1994). The role of CP in RNA replication is unclear. CP-encoding RNA contains a cis-acting RNA element (Mahajan et al., 1996; Halde-man-Cahill et al., 1998) that forms a stem loop structure similar to that of the poliovirus cre-element located at the coding region of 2C in the poliovirus genome (Murray and Barton, 2003). In a yeast two-hybrid experiment, the CP was shown to interact with RNA-dependent RNA polymerase (NIb) and VPg. NIb interaction with CP is sensitive to changes in the highly conserved Gly-Asp-Asp amino acid motif of polymerase, but may involve regulation of viral RNA synthesis (Hong et al., 1995). HC-Pro is comple-mentation deficient, and it must act in cis in replication. The P3 protein, as well as P1 and HC-Pro, is necessary for virus replication as revealed by the debilitating effects

P1 HC-Pro P3 6K1 CI (Hel) 6K2 NIa NIb (Rep) CPpoly(A)

VPg

VPg

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of several insertion mutations (Klein et al., 1995), although, Merits et al. (1998) show that P3 does not bind RNA. The least conserved protein P1 provides an activity that stimulates genome amplification in trans (Verchot and Carrington. 1995).The potyviral replication complex is difficult to study. Potyvirus translation and repli-cation occur simultaneously in the same cellular compartment. The CI-6 K-NIa-NIb regions of the polyprotein have a functional similarity to the 2C-3A-3B-3C-3D region of the picornavirus polyprotein (Wimmer et al., 1993). These proteins derived from the central region of the genome are proposed to function in RNA replication. The cy-lindrical inclusion protein (CI) contains nucleoside triphosphatase and RNA unwind-ing activity and shares sequence similarity with other RNA helicases (Fernandez et al., 1997). The key enzyme in replication is NIb RdRp (Hong and Hunt et al., 1996; Li et al., 1997). Polymerase activity of NIb is stimulated by in vitro interaction with NIa (Fellers et al., 1998). The interaction between TEV NIa and RNA polymerase NIb plays an important role during viral RNA replication. Mutations in the NIa domain to which the genomic RNA is covalently attached eliminate Nib-NIa interaction (Car-rington et al., 1987; Daros et al., 1999; Hong et al., 1995). The 6 kDa protein is the only known TEV protein that possesses ER localization information for anchoring the genome amplification site (RC) to replication supporting membranes derived from ER (Restrepo-Hartwig and Carrington 1994). It is proposed that a primary determi-nant for targeting TEV replication complexes to membranous sites of RNA synthesis involves direct interaction of the 6 kDa protein with the ER. Induction of ER vesicle accumulation may be a general mechanism to increase the available surface area for RNA synthesis. The membrane may provide a scaffolding function, as well as provide a mechanism to limit diffusion and increase local concentrations of viral RNA and proteins (Schaad et al., 1997). The involvement of host proteins in potyviral replication an infected cell is obvious. The pea seed borne mosaic virus (PSbMV) replication front alters host gene ex-pression and protein synthesis in the infected plant cells (Wang and Maule 1995). Expression of ubiquitin and the HSP70 chaperone class are activated co-ordinately at the PSbMV infection front (Aranda et al., 1996). Moreover, potyvirus VPg binds cellular cap-binding protein eIF(iso)4E. The potential role of eIF(iso)4E in virus in-fection is connected with translation initiation, since it is a translation initiation factor (Rodriguez et al., 1998, Ruud et al., 1998). Later, eIF(iso)4E function was connected with potyvirus genome expression and replication through interaction with VPg, since the deletion of eIF(iso)4E reduced virus susceptibility (Lellis et al., 2002). Potyvirus infection is blocked in Arabidopsis mutants lacking the genes encoding eIF(iso)4E. The eIF(iso)4G interacts with microtubules and with eIF(iso)4E (Browning et al. 1996; Bokros et al. 1995). The potyviral genome may interact with microtubules through a VPg interaction with eIF(iso)4E-eIF(iso)4G; this interaction may serve as an intracel-lular localization function (Lellis et al., 2002).

1.7 Viral cell-to-cell movementPlasmodesma is a plant-specific structure that spans the cell wall between plant cells. Such structures are plasma membrane extensions with a modified ER -interior. These membranes form a space which is believed to be the main route for cell-to-cell transport for signaling proteins, RNA, and small metabolites. The plasmodesma

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diameter is highly regulated and can range from being closed to all molecules, to the interior passage of small metabolites, and to expansion allowing the passage of virus particles or viral RNP complexes (Crawford and Zambryski, 2000; Haywood et al., 2002). Plasmodesmata provide a barrier to viral local and systemic movement, but viruses have evolved movement proteins (MPs) to manipulate these channels to gain access to the neighbouring cells.

In contrast to other plant viruses, the potyviruses do not encode a dedicated move-ment protein; potyvirus movement requires viral proteins that also perform additional roles in the virus life cycle (Carrington et al., 1996). Many potyvirus proteins are in-volved in the incompletely understood movement process. These movement-associ-ated potyvirus proteins are HC-Pro, CI, 6K2, VPg, and CP (reviewed in Rajamäki et al. 2004). Potyviral CP and HC-Pro function as viral MPs; these proteins are able to move cell-to-cell through the plasmodesmata and possess RNA-binding properties. Microinjection experiments with recombinant CP, HC-Pro, and high molecular weight dextran show that the proteins raise the plasmodesmal size exclusion limit (SEL), and thus facilitate cell-to-cell trafficking (Rojas et al., 1997). The role of CP in viral movement is more diverse than merely structural. TEV CP mutation studies revealed both cell-to-cell, and long-distance movement-deficient mutants; however, some CP mutants form apparently normal virions (Dolja et al., 1994; 1995). The wild type CPs of three potyviruses localize to plasmodesmata. These results suggest that the po-tyvirus CP interacts with unknown plant proteins on the plasmodesmata to facilitate viral movement (Rodriguez-Cerezo et al. 1997; Rojas et al. 1997).

Mutational analysis of CI identified an essential role in potyvirus movement. Func-tionally, the N- terminal region of CI serves in cell-to-cell movement since several alanine-scanning mutants resulted in a movement-deficient virus, and data indicate that cell-to-cell movement function is genetically separable from its RNA replication functions (Carrington et al., 1998). An immunogold labeling EM study localized CI at an early stage of infection on the cell wall in close proximity to the plasmodesmata. The CI is believed to form organized structures, which attach to plasmodesmata after infection. These structures appear to have central channels that align with plasmo-desmal openings and that are associated with CP. Results suggest that CI protein have a direct but transient role in the transfer of potyvirus genetic material through vi-rally modified plasmodesmata (Rodriguez-Cerezo et al., 1997; Roberts et al., 1998).

1.8 Long-distance movementIn order to infect the whole plant, the replicating virus must move from mesophyll through bundle sheath cells, parenchyma, and companion cells into phloem sieve el-ements. In the sieve elements, the virus moves passively with photoassimilates until it reaches uninfected developing sink tissues. Long-distance movement and cell-to-cell movement are distinct functions (Carrington et al., 1996; Van Bel. 2003). Three potyviral proteins, CP, HC-Pro, and VPg, are involved in long distance movement. Transgenic plants expressing TEV wild type CP are able to complement several vi-ruses having their CP mutated, but complementation efficiency varied among the CP mutants (Dolja et al., 1994 and 1995). Mutation at the Asp-Ala-Gly domain of the

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TVMV CP N-terminal domain changes the long-distance movement capability of the virus (Andersen and Johansen, 1998). Formation of virions may not be necessary for potyvirus cell-to-cell movement, but a mutant that moves without virion formation has yet to be reported.The role of VPg in long-distance movement has been studied with chimeric virus strains of TEV-HAT, and -oxnar and an N.tabacum cultivar exhibiting a strain-specific defect in supporting long distance movement (Schaad et al., 1997b). Cultivars of to-bacco containing the va-resistance gene can restrict long-distance movement of the replicative TVMV (Nicolas et al., 1997). PVA VPg plays a role in the vascular move-ment. The immunolocalization and in situ hybridization RNA detection method local-izes VPg in infected S.commersonii plants in all vein classes. Detection of VPg also in companion cells in the absence of other viral proteins or RNA suggests that free VPg mediates its own movement in companion cells and sieve elements. Research-ers suggest that accumulated VPg in sink-leaf companion cells may be important for virus loading and may regulate virus accumulation in phloem cells (Rajamäki and Valkonen, 1999). HC-Pro is proposed to function in long-distance movement, replication, and as a pathogenicity enhancer (Shi et al., 1997). The evidence for these was found when a HC-Pro-expressing Potato virus X (PVX) vector replicated to a higher level com-pared to wild type PVX (Anandalakshami et al. 1998). Mutations in the central region of HC-Pro diminish long-distance movement either directly by inhibiting interaction with other viral movement factors or indirectly by interacting with host factors (Cro-nin et al., 1995; Kasschau et al., 1997). Plants trigger transcriptional gene silencing (PTGS), which is a general defence mechanism against viruses. PTGS is capable of restricting virus accumulation in primary infected cells and suppress virus movement (Baulcombe, 1996; Pruss et al., 1997). HC-Pro interferes with the PTGS mechanism, suppresses it, and enhances viral replication. The HC-Pro domain acting in PTGS in-terference is located on the central part of the protein. The presence of P1 protein en-hances HC-Pro PTGS interference efficiency (Kasschau and Carrington, 1998; Maia et al., 1996). Mutational analysis suggests that HC-Pro protease activity participates or indirectly has an effect on PTGS suppression (Kasschau et al., 1997; 2001).

1.9 Protein phosphorylationProtein phosphorylation is one of the most critical and widespread regulatory mecha-nisms of complex organisms. Phosphorylation is the most abundant posttranslational covalent modification of proteins. Human phosphoproteome size is estimated to cov-er 105 sites with multiple phosphorylation sites in many proteins (Hunter, 1995; Venter et al., 2001; Manning et al., 2002). Recent advances in genome sequencing have resulting a better understanding of protein kinase functions. Within the Arabidopsis genome, there are approximately 1000 protein kinases and ca 200 phosphatases (Xing et al., 2002). Historically, protein kinases have evolved early during eukaryotic evolution. We believe that cellular mechanisms are conserved, and thus comparison of protein kinase function is possible between animals and plants (Hardie, 1999; Tena et al., 2001).

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1.10 Protein kinasesA network of protein kinases and phosphatases receive signals from the external environment through receptor proteins and change the output response of the cell by use of targeted protein phosphorylation. Protein kinases mediate most of the sig-nal transduction in eukaryotic cells by modification of substrate protein activity, thus controlling many cellular processes (Fig. 2) (Manning et al., 2002). The kinases play an important role in development and differentiation by transmitting information from messenger molecules located on the outer surface of neighbouring cells. Particularly transmembrane domain-containing kinases may interact with other protein kinases, thus producing signal cascades (Hardie, 1999).

Fig 2. Phosphorylation of viral proteins is a key regulator. Virus protein activity is controlled by reversible phosphorylation by host kinases and phosphatases. Post-translational modification of protein regulates its activities by changing biological activity, subcellular location, interaction with other proteins, and half-life. Phosphorylation is an essential regulatory mechanism within the cells, and the complex signaling cascade machinery is a part of the plant defence mechanism.

Phosphorylation of proteins plays an important role in response to pathogen attacks. Several classes of kinases are involved in plant defence responses to different patho-gens. Pathogens are recognized as foreign molecules, and thus induce signal cas-cades to activate an early pathogen response (Zhou et al., 1997; Mundy and Schneiz, 2002). The mitogen-activated protein kinase (MAPK) pathways have several roles in plant signal transduction, including responsiveness to pathogens, wounding, and abiotic stress. This suggests that stress-induced transcription of genes for MAPKs in plants evolved early, before the divergence of dicots and monocots. The MAPK-fam-ily plays an important role in plant defence responses to multiple stresses (Hirt, 1997; Tena et al., 2001). MAPKs control a subset of defence responses shared between different stress stimuli. In yeast and mammals, MAPK signal cascades are often as-sociated close by. These arrangements help cross talk and feedback between differ-ent MAPK cascades, and allow a kinase to function in more than one module without affecting the specificity of the MAPK response (Widmann et al., 1999).

Biological

activity

Subcellular

location

Protein

half-life

Protein-protein

interaction

Protein

catalytic

activity

Protein

Signalling

cascade

Kinases

Phosphatases

P P

ProteinExtend and

Duration of the

Response

Cross talk

Signals

Stimulus

Receptors

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Several protein kinase activities are associated with plasmodesmal and cell wall-en-riched fractions (Citovsky et al. 1993; Karpova et al. 1999; Waigmann et al., 2000; Yahalom et al., 1998). The receptor-complex involved in the binding of the viral RNA-protein complex to the plasmodesma remains to be isolated. However, microinjection experiments with viral MPs revealed that proteins interact with a common receptor site in the pathway for cell-to-cell transport (Kragler et al. 1998). Among the known regulators of the processes involved in cell-to-cell trafficking, the role of protein phos-phorylation is significant. Phosphorylation events act as important steps in modulat-ing the specific protein-protein binding affinities or recognition events of plasmodes-mata. Unique protein kinases and phosphorylation events will similarly play a central role in this process. Movement proteins of viruses mediate transport of infectious viral nucleic acid. A complex of viral nucleic acids and MPs interacts with plasmodesmata and increase the size-exclusion limit (SEL) to advance the transport into new cells (Lazarowich and Beachy. 1999). Several movement proteins are shown to be phos-phorylated during the infection process (Haley et al. 1995; Kawakami et al., 1999; Matsushita et al. 2000; Sokolova et al. 1997). In vitro and in vivo studies have shown that the TMV MP can be phosphorylated at a number of residues (Watanabe et al. 1992; Citovsky et al. 1993; (Citovsky et al., 1993). It has been speculated, however, that the phosphorylated MP may be targeted for sequestration within the median cav-ity of the plasmodesma. The plasmodesma associated MP phosphorylating kinase may play a critical role in regulating the amount of MP available for viral RNP trans-port (Karpova et al., 1997).Microinjection studies of TMV MP mutant forms verify that phosphorylation influences its action. Non-phosphorylatable forms of MP fail to raise the plasmodesma size ex-clusion limit, but this is not essential for MP accumulation in plasmodesma (Lee and Lucas, 2001; Waigmann et al., 2000).Phosphorylation may regulate viral infectivity by affecting the stability of MP RNA complexes. Phosphorylation of the MP is thought to be an essential step, upon its exit from the plasmodesma, in driving the dissociation of the MP from the RNP (Kar-pova et al., 1997). Results of Karpova et al., (1999) show that TMV RNA complexes change into translatable form after transport through plasmodesmata, and in the ab-sence of this putative RNP-kinase, the viral RNA bound within the MP (RNA-MP complex) remains nontranslatable.

1.11 The CK kinase family The casein kinase family consists of two subfamilies, casein kinase I (CK1), and casein kinase II (CK2). There is a historical reason for calling these kinases as CK1 and CK2, since their activity was first detected by use of milk protein casein as the substrate (Meggio and Pinna, 2003). Both kinases use serine and threonine as sub-strates in the vicinity of acidic residues located downstream from the phosphorylat-able amino acid. The CK2 phosphorylation sites are conserved across the mamma-lian species (Meggio and Pinna., 2003). Both kinases CK1 and CK2 have an unusual expression profile, similar to that of housekeeping proteins. High numbers of proteins in signaling cascades, metabolic enzymes, and protein phosphatases interact with casein kinase II (CK2). An increasing number of essential proteins for the virus life cycle are phosphorylated by constitutively active CK2. Intensive use of CK2 as a

17

phosphorylating agent for viral proteins makes it an interesting target for antiviral drugs (Meggio and Pinna, 2003; McShan and Wilson., 1997; Mitchell et al., 1997; Morrison et al., 1998, Schwemmle et al., 1997).

1.12 Protein kinase II (CK2)Protein kinase CK2, widely studied in animals, adjusts global cellular processes. As discussed, a specific stimulus regulates the expression and activation of most protein kinases. Regulatory mechanism independence and constitutive activity are a special-ity of the CK2 (Meggio et al., 2003; Montenarch and Faust, 2000). Usually mammali-an CK2 kinase exists as α2 β2 tetramers, where α subunits are catalytically active, and β subunits play regulatory role adjusting α towards certain substrates (Garankowski et al., 1991; Pinna, 2002). Oligomeric activity of CK2 was isolated from a variety of plants such as maize, Arabidopsis, broccoli, and pea (Dobrowolska et al., 1989; Do-browolska et al., 1991; Dobrowolska et al., 1999; Klimczak et al., 1992 and 1995; Li et al., 1992; Mizoguchi et al., 1993). The only CK2 crystallized subunit is maize-CK2 (mCK2), and other CK2 structures are its sequence comparison derivatives (Niefind et al., 1998). Unusual features of CK2 are its insensitivity to the universal kinase in-hibitor staurosporine, and its utilization of both ATP and GTP as phosphoryl-donors (Niefind et al., 1999).

1.13 Function and comparison of plant CK2 –subunitsDue to the high conservation of CK2α subunits, different species are able to share their main regulatory functions. Structurally, these active CK2 subunits consist of two regulatory subunits, CK2β1 and β2, and two catalytically active subunits CK2α1, -α2 generating the heterotetramer of α2β2, α´2β2 or αα´β2 (Espunya and Martinez, 1997). Molecular masses of these subunits range between 37 and 44 kDa. These catalytic subunits form stable structures, making it possible to purify catalytic subunits and a tetrameric α2 β2 form (Espunya and Martinez, 1997). Riera et al., (2001) postulate plant CK2α as belonging to a multigene family consists of three members. Most of the amino acid differences are located at the C-terminal region. The C-terminal region of maize CK2α (mCK2α) is 60 amino acids shorter than the human, making mCK2 stability higher, but still capable of forming a stable and functional heterotetramer with human CK2β (Niefind et al., 1998). It is speculated that plant CK2α kinase is not so dependent on CK2β-subunit regulation (Riera et al., 2001). Phylogenetic analysis shows that identity of CK2β-subunit similarity between plant, yeast, and human is lower than in the case of CK2α. A.thaliana genome encodes two subunits, CK2β1 and CK2β2, and presumably a third CK2β3 member (Sugano et al., 1998). Maize contains three regulatory subunits with very similar properties to those described in other organisms (Riera et al., 2001). Structurally, plant CK2β share con-served features present in other organisms (Krehan et al., 1996).Similar to human CK2, plant CK2 has been shown to phosphorylate increasing num-bers of substrate proteins, in the literature, human CK2 (hsCK2) phosphorylates 307 substrates (Meggio and Pinna, 2003). Somewhat enigmatic is that the catalytic prop-erties or function of the target protein is usually not influenced by phosphorylation by CK2. CK2 does not act like a transient messenger; rather it participates like a univer-sal maintenance activator (Hardie, 1999; Meggio and Pinna, 2003).

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2.0 AIMS OF THE STUDY

The role of different phosphorylation events in the regulation of Potyvirus VPg and CP function has remained unclear. The purpose of the present study was:

To examine in vitro phosphorylation events of two movement related proteins, CP and VPg.

To study the tobacco and S.commersonii kinase catalyzed in vitro phosphorylation of VPg, and second, to examine the virus particle-localized VPg function.

To study CK2 phosphorylation sites in PVA CP and examine the role of phosphoryla-tion in the regulation of RNA -binding in vitro and in vivo.

To map the VPg nucleotide binding domain, and examine its role in the NIb-catalyzed uridylylation reaction.

19

3.0 MATERIALS AND METHODS

Detailed description of the materials and methods are found in the original publica-tions.

Materials and methods Original publication

1. CD spectroscopy III, IV.2. Chemical cross-linking experiment with glutaraldehyde. IV.3. 2D-analysis of nucleotidylated VPg IV.4. Electron microscopy I, III.5. Fusion protein purification and expression I, II, III, IV.6. Gel retardation assay (electrophoretic mobility shift assay) III.7. Identification of the amino acid involved in formation of the VPg-nucleotide linkage. IV.8. In vitro enzymatic dephosphorylation assays I.9. In vitro kinase assays I, II, III.10. In vitro nucleotidylation competition assay IV.11. In vivo 33P-orthophosphate labelling and PVA CP immunoprecipitation I, III.12. In vivo phosphorylation I, II.13. In vitro synthesis of RNA I.14. Immunogold labelling and IEM. II.15. Immunoprecipitation of virions with anti-VPg antibodies. II.16. Immunofluorescence staining III.17. Liquid chromatography–tandem mass spectrometry III.18. NTP-binding assay IV.19. Plants and viruses. II, III.20. Plant material and preparation of plant protein extracts I.21. Plasmid construction I, II, III, IV,.22. Phosphoamino-acid analysis. I. III. 23. Purification of the PVA CP kinase from tobacco III.24. Reverse-phase chromatography and mass spectrometry III.25. RNA-protein blotting II.26. RNA-protein binding assays using metal chelate magnetic beads I.27. SDS-PAGE and immunoblotting. I, II, III, IV.28. Tryptic phosphopeptide mapping I, II, III, IV.29. UV Cross-linking/label-transfer assay III.

20

4.0 RESULTS

4.1 N.tabacum protein extracts phosphorylates PVA CP and VPgIn this study, we investigated the role of phosphorylation in regulating the PVA VPg and CP, and the consequences for viral infection. We used first in vitro phosphoryla-tion reactions with recombinant proteins and plant protein extracts. Later we verified the putative phosphorylation sites by using site directed mutations in the in vitro phos-phorylated proteins VPg and CP. The role of CK 2 in the regulation of PVA cell-to-cell movement was examined in whole plants using GFP tagged icDNA. We studied in preliminary experiments the role of divalent metal cations required in the activation of unknown plant protein kinases phosphorylating PVA MPRs VPg, and CP (I, II,). We compared the phosphorylation of PVA CP, VPg and TMV MP, and results revealed that the unknown N.tabacum kinase activity phosphorylates each of the tested proteins (Fig. 4B in I and Fig. 1 B in II). Our results also showed that PVA CP and TMV MP may be phosphorylated by the same protein kinase. These results confirm that the phosphorylation of PVA CP and TMV MP is substrate-specific and that PVA VPg is a substrate for another kinase (Fig. 6 in I).To determine the in vivo phosphorylation status of the PVA CP, the PVA-infected leaves were incubated with 32P-labeled orthophosphate to replace part of their cellu-lar phosphate reserves. Infected and mock-inoculated orthophosphate-labeled plant material was used for anti-PVA CP antibody immunoprecipitation. SDS-PAGE analy-sis of immunoprecipitated proteins followed by autoradiography revealed that labeled bands with an electrophoretic mobility similar to that of CP were observable only from plants infected with PVA. To further support the in vivo phosphorylation results, PVA CP phosphorylation was inhibited with a concentration of 1 μM staurosporine (Fig. 2a, and b, in I).

4.2 Identification of CK2 activity phosphorylating PVA CPCK2 is shown to interact with ToMV in the genus Tobamovirus (Matsushita et al., 2000). ToMV is closely related to TMV, and our results revealed that TMV MP and PVA CP are substrates for the same Ser/Thr tobacco kinase. To investigate the pos-sible role of CK2 in the phosphorylation of PVA CP and TMV MP, we first carried out an in vitro kinase assay. Phosphorylation of PVA CP was tested first in the presence of ATP, GTP, and of the CK2-specific kinase inhibitors heparin and 5,6-dicloro-1-(β-D-ribofuranosyl)benzimidazole (DRB). To address the effect of these two inhibi-tors, an α-catalytic subunit of maize CK2 sharing 90% identity at the protein level to tobacco CK2 was used in the kinase reactions. Our results revealed that rmCK2 phosphorylates PVA CP and TMV MP in the presence of Mn2+. Heparin inhibited PVA CP phosphorylation with ATP and GTP as phosphorylation donors (Fig. 1A in III). The second inhibitor 5,6-dicloro-1-(β-D-ribofuranosyl)benzimidazole (DRB), is known as a cell-permeable inhibitor that can also be used to inhibit CK2 in vivo. The addition of DRB to in vivo phosphorylation experiments strikingly reduced the phosphorylation of PVA CP. The in vivo phosphorylation results with infected tobacco leaf suggest that PVA CP is phosphorylated by CK2 during the infection cycle (Fig. 1C in III). Next, CK2 was purified almost to homogeneity by one chromatographic step with

21

heparin sepharose (Fig. 2 in III). Proteins from the active fractions were subjected to an in-gel kinase assay to determine the molecular mass of the kinase and its sensitivity to specific inhibitors (Fig. 1 in III). The catalytically active fractions were further separated by SDS-PAGE and subjected to LC-MS/MS. Results of LC-MS/MS sequence tag analysis revealed the catalytic domains of the tobacco CK2 peptide as having a molecular mass of 39 kDa, which is identical to the α-catalytic subunit of CK2 from tobacco (tCK2).

4.3 Identification of CK2 phosphorylation in vivoTo further investigate the relevance of the CK2 activity, the recombinant tCK2 was used to produce a polyclonal antiserum. We then used tCK2 and PVA CP antibodies to localize proteins in infected protoplasts by confocal microscopy (Fig. 4 in III). We could confirm the results of the previous CK2 localization experiments (Faust and Montenarh, 2000) showing that CK2 is localized to the cytoplasm and nucleus. The observed co-localization of tCK2 and PVA CP in the cytoplasmic patches usually near the nucleus support in vitro and in vivo findings that PVA CP is a target for CK2 kinase activity.

To study the sites of phosphorylation in more detail, we separated CK2-phosphor-ylated CP peptides by two-dimensional (2D) tryptic phosphopeptide mapping. We observed five radioactive spots when using total protein extract from tobacco, but only one with similar mobility as in total protein extract, when using isolated rmCK2 to phosphorylate CP in tryptic peptide mapping. Radioactive peptides were isolated from cellulose plates and further analyzed by combination of radioactive phosphate-release sequencing and phosphoamino-acid analysis. Results revealed that all the peptides contained only phosphothreonine, and phosphate-release sequencing of the major spot indicated that radioactive amino acid was released after 14 cycles. Peptides analyzed with these two methods contain a threonine residue after 14 se-quencing cycles, which matches with peptide 229-NSNTNMFGLDGNVTTSEED-TER-250 (Fig. 5b in III). CK2 phosphorylated CP residues were also determined by a matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF). MALDI-TOF analysis characterizes one peptide from liquid chromatography fractions further mea-sured to be radioactive. This single peptide was identified as 229-NSNTNMFGLDGN-VTTSEEDTER-250 with a calculated monoisotopic mass of 2430 kDa. It contained a CK2 phosphorylation consensus site motif (S/TXXD/E), 242-TTSEED-247, which was the main phosphorylation site for tobacco CK2 (Fig. 6 in III).

The role of phosphorylation in the regulation of CP function during viral infection was further investigated by use of infective 35S-PVA-GFPNIb/CP and particle bombardment to introduce different 35S-PVA-GFPNIb/CP mutants to N. benthamiana plants. GFP was introduced to the infectious 35S-PVA genome at the junction of NIb and CP genes using short NIa protease cleavage sites to excise the GFP during polyprotein pro-cessing. First, we introduced into the CP triple mutations of Thr- 242, Thr-243, and Ser-244, to alanine. Then triple Asp and Tyr mutants were introduced at the same sites to mimic both electrostatic and steric effects of phosphorylated Thr (Dean and

22

Koshland, 1990). We showed that phosphorylation of these sites was blocked and produced movement-deficient viruses without affecting replication, since viruses pro-duced reversion mutations (Fig 7C and E in III). To further investigate the relevance of substituted amino acids in CK2 phosphorylation, we constructed a mutant virus with altered triple CK2 consensus sequences, where acidic residues following the phosphorylation site were replaced with basic Arg residues. In these mutants, Thr-242, Thr-243, and Ser-244 were left intact. Again, 7 days after inoculation, only single bombarded cells were visible. Results revealed that the CK2 target Thr-242 was not phosphorylated because of CK2 consensus sequence alteration. We could confirm the results from these mutational experiments showing that interference with CK2 phosphorylation of PVA CP Thr-242 significantly and strongly impaired PVA infec-tion.

4.4 Phosphorylation of PVA CP regulates its RNA-binding activityThe role of phosphorylation in the regulation of RNA binding was further investigated by using biochemical analysis of phosphorylated and nonphosphorylated PVA CP proteins. We showed that PVA CP bound RNA in a sequence-independent manner, and that plant Ser/Thr kinase phosphorylation inhibited binding capacity in vitro (Fig. 8 C and D in I). Data from gel retardation and UV cross-linking techniques suggests that non-phosphorylated PVA CP exhibits higher affinity towards RNA than does the CK2 phosphorylated protein (Figs. 9 A and B in III). Additionally, our hypothesis is further supported by results demonstrating that virion-derived LiCl extract was effec-tively phosphorylated by the plant protein kinases but intact purified virions were not targets for plant kinase activity.

4.5 Phosphorylation of PVA VPgOur results show that an unknown tobacco kinase phosphorylated PVA VPg in vitro (Fig. 1 D. in I). We therefore wanted to analyze substrate specificity of these unknown tobacco kinases. Several amino acid substitutions in chimeric PVA viruses influence virus accumulation or phloem loading in leaves of S.commersonii. Among these virus strains B11-M differs from B11 within VPg. One of the mutations, at the C- terminus, affects PVA accumulation in infected cells (Rajamäki et al. 2002). These two strains, B11 and B11-M, have point mutations in the VPg region (Val116Met, Tyr118His, and Leu185Ser), and the 6K2 region (Met5Val) (Fig. 6A in II). Phosphorylation patterns of E.coli expressed chimeric virus VPg isoforms were compared using N.tabacum and S.commersonii were sources of kinase activity. Kinase activity from tobacco resulted in a phosphorylation pattern similar for all recombinant VPg proteins, when identified by tryptic phosphopeptide mapping. S.commersonii kinase activity produced two dif-ferent phosphorylation patterns, indicating that it is able to distinguish between these different chimeric virus-derived VPg molecules. (Figs. 6 and 7 in II). Our immunoprecipitation experiment from infected tobacco with VPg antibody sug-gests that VPg-antibodies could be used to immunoprecipitate viruses (Fig. 3. A in II). Our further Immunoelectron microscopy (IEM) results demonstrate that VPg was located in an exposed position at one end of the particles and suggest the possibility of a protein-protein interaction (Fig. 3 in II). PVA virions were also used as substrates

23

for tobacco kinase phosphorylation experiments. Phosphoimager analysis of virion extracted VPg showed that tobacco kinase was able to interact and further phos-phorylate in vitro the PVA VPg in the virion.

4.6 Nucleotidylation of PVA VPg To examine the putative role of PVA VPg working as a primer in genome replication, we set up experiments using recombinant PVA NIb and VPg, which were based on a modified in vitro assay used by Paul et al. (1998). A reaction mixture, mimicking a highly simplified PVA replication complex, contained only a poly (A) template, VPg, purified RNA polymerase, labeled UTP, and the divalent cations manganese or mag-nesium. In the presence of manganese, the NIb RNA polymerase catalyzes covalent linkage between UTP and VPg tyrosine. As a control for the in vitro reaction, removal of NIb from the reaction mixture abolished the covalent linkage between UTP and VPg, indicating that VPg itself does not catalyze the reaction. PVA NIb RNA polymerase was further studied using an in vitro reaction with a poly(A) template. Products from in vitro reactions indicated that the NIb RNA polymerase is active and capable of producing high molecular weight-labeled poly(A) polymers (Fig. 1 in IV). The presence of synthesized high molecular-weight polymers was de-pendent on addition of the poly (A) template. The yield of labeled product increased with increasing amounts of NIb RNA polymerase. The homopolymeric products were analyzed further by immunological methods, and the results indicated that the high molecular weight RNA products did not contain VPg. This in turn indicated that syn-thesized RNA is not primed by VPg. The presence of high molecular-weight RNA products is thought to be a result of unspecific RdRp template-independent polymer-ization or terminal transferase activity of the NIb -polymerase. We also examined the divalent metal cation specificity of the polymerase-catalyzing VPg-nucleotidylation reaction. We could confirm the results from previous studies (Caldentey et al., 1992; Crotty et al., 2003) showing that Mg2+ -dependent DNA polymerase of the bacterio-phage PRD1 and poliovirus RNA-dependent RNA polymerase have a similar broad concentration range for the activating metal cation.Replication of picornaviruses requires a cis-acting RNA element (cre-element), acting independently of the other RNA signals in the genomic RNA (Yin et al. 2003). This RNA hairpin structure stimulates uridylylation efficiency of PV polymerase tenfold when compared to a reaction with poly (A). Our in vitro reaction indicates that nucleo-tidylation of VPg, catalyzed by NIb RNA polymerase, is template-independent. Either the presence or absence of artificial poly (A) or in vitro-produced infectious PVA RNA had no effect on the nucleotidylation efficiency of VPg (Fig. 2C in IV).

We tested the involvement of RNA template and VPg in selection of the incoming nucleotide was tested. Since selection of the nucleotide in the NIb-catalyzed reac-tion was independent of the template RNA, we assume that it is not dependent on the polymerase reactive pocket. The reactions were carried out in the presence of a constant amount of 32P-labeled UTP, and the amounts of each cold ribonucleotide ATP, CTP, GTP, and UTP was varied. The results indicated that the presence or absence of poly(A) template did not change the reaction specificity towards UTP. In

24

addition, concentrations of cold competing nucleotide above 0.75 µM abolished the weak nucleotide specificity of the reaction. The competition assay revealed a weak selection for UMP in the nucleotidylation reaction, in the absence of the artificial RNA template, suggesting an RNA-independent selection for UTP by either VPg or NIb RNA polymerase. Crawford and Baltimore (1983) found in infected cells that free po-liovirus VPg carries short polymerized UMPs. Our finding that NIb-polymerase cata-lyzed the template-independent nucleotidylation reaction raises the question whether polymerase may elongate the chain from one added nucleotide. 2D gels were used to analyze differences between VPg uridylylated with or without a poly (A) template. However, the low resolution of 2D-gels and the minor heterogene-ity in the protein samples made it impossible to determine the number of nucleotides within VPg (Fig. 3 in IV). We used phosphopeptide mapping to separate and visualize 32P–labeled nucleotidylated VPg peptides. Phosphopeptide mapping resulted in two unidentified peptides carrying labeled UMP. We were unable to determine whether the separate peptides represented either VPg -pU or VPg -pUpU (Fig. 4. in IV). An al-ternative explanation may be that separate tyrosine amino acids in different peptides were nucleotidylated. The low yield of nucleotidylation reaction impeded MALDI-TOF analysis of trypsinated VPg, but according to amino acid analysis, only tyrosine resi-dues formed covalent linkage with UTP in the PVA RdRp-catalyzed reaction (Fig. 5. in IV). PVA VPg has a tendency to form dimers (Oruetxebarria et al. 2001). We studied whether VPg oligomerization plays a role in a NIb-catalyzed nucleotidylation reaction by using chemical cross-linking with glutaraldehyde during an in vitro nucleotidylation reaction. Only a minor amount of dimerized VPg was found to be labelled with UMP, indicating that polymerase may interact in such a way that dimers and higher form oligomers are not substrates for the protein nucleotidylation reaction.

4.7 Identification of nucleotide binding site in PVA VPgWe used lysine-specific chemical-cross linking of oxidized nucleotides in the pres-ence of NaCNBH3 to study PVA VPg nucleotide-binding capacity as in Richards and Ehrenfeld (1997). The results from periodate-oxidized UTP-binding studies revealed that PVA VPg had strong nucleotide-binding capacity in the presence of the diva-lent metal cations Mg2+ and Mn2+. Divalent metal cations have been shown to be important in activating polymerases of several viruses (Crotty et al., 2003). This re-sult indicates a coordinative role for divalent metal ions when incoming nucleotides bind to a VPg nucleotide-binding domain. Computer aided analysis of the VPg amino acid sequence (http://us.expasy.org/prosite) predicted the presence of a function-ally distinct nucleotide-binding domain. The putative nucleotide-binding domain had the (AG)X(4)GK(ST) sequence motif and the corresponding VPg amino motif was located at (38AYTKKGK44). Truncated VPg ∆38-44, having a deleted NTP-binding domain, had a strongly reduced nucleotide-binding capacity (Fig. 7A in IV).The nucleotide binding domain participated also in the NIb-catalyzed nucleotidylation reaction, since truncated VPg ∆38-44 reduced nucleotidylation efficiency by more than 70% compared to wt VPg (Fig. 7B in IV). Circular dichroism (CD) spectroscopy allows estimation of the secondary structure of purified His-tagged VPg (Fig. 3). CD-data for wt VPg presented in fig. 3 show double minima ellipticity values at 222 and 208 nm, and the maximum at 195 nm. Interpretation from these values may show that

25

mostly α-helical structures present at wt VPg, whereas a high proportion of β sheets led to spectra with a broader minimum around 215 nm and a maximum at 200 nm (Kelly and Price, 1997; Schmid 1997; Vila et al., 2001). From these measurements it follows that PVA wt VPg was highly ordered, giving 57% beta sheet, only (4%) helix, and (36%) random orientation

Fig. 3. CD spectrum of PVA VPg. The spectrum of recombinant his-tagged protein (2 mg/ml) was meas-ured in 0.05 mM HEPES buffer, pH 7.5, in a 1-mm cuvette at room temperature. Saw-toothed line rep-resents VPg protein CD absorbance. Upper fine line loosely following the CD(mdeg) 0-line represents calculated differences between measured and ideal protein absorbencies.

5.0 DISCUSSION

5.1 Phosphorylation of the VPg proteinThe plant virus life cycle is regulated at several levels. One of the levels is the regu-lation of movement protein functions, the mechanism of which has remained poorly understood. We have investigated the role of potyvirus VPg and CP phosphorylation in regulating potyvirus movement and replication initiation. Involvement of VPg in multiple functions during the virus life cycle has been recently discovered: it is crucial for replication, translation, and phloem loading, as well as in avirulence determination (Reviewed in Rajamäki et al., 2004). Due to its multiple functions, VPg is presumably targeted to interact with viral and host components during the infection process. Only a minor part of VPg becomes covalently linked to RNA. Most of the VPg is thought to accumulate in the nucleus (Carrington et al., 1993). The covalent VPg-RNA link is not disrupted during virion assembly, and the VPg remains as a part of the virion structure (Murphy et al., 1990; Oruetxebarria et al., 2001; II).Previous reports have identified eIF(iso)4E and eIF4E interaction with turnip mosaic virus (TuMV) and tobacco etch virus (TEV) VPg. Several roles for this interaction should be considered. Binding of VPg to eIF(iso)4E may act to suppress the host cell functions; interaction may suppress one or more defence responses that specifically limit long-distance movement (Wittmann et al., 1997; Schaad et al., 2000). Amino acid substitution studies indicate that the central domain of VPg is important for inter-action with HC-Pro. Interestingly, the central region of the HC-Pro is also required for

-16

9

-10

0

185 250200 220 240

CD[mdeg]

Wavelength [nm]

Accumulation: 20

Cell Length: 0.1 cm

Concentration: 0.02 (w/v)%

Solvent: HEPES

Temperature: Room Temperature

Fraction Ratio

Helix: 0.0 4.1%

Beta: 0.1 57.5%

Turn: 0.0 2.1%

Random:0.1 36.2%

Total: 0.2 100.0%

RMS: 17.861

26

long-distance movement; evidently HC-Pro interaction with VPg is connected with vi-ral movement functions (Schaad et al., 1996b; Yambao et al. 2003). VPg may also be connected with RNA-induced silencing (RNAi) via interaction with HC-Pro (Marathe et al. 2000). Site-directed mutations have demonstrated with TVMV-S that minor differences in VPg manage to overcome va–resistance. The va-gene blocks cell-to-cell movement of TVMV, but has no influence on replication (Masuta et al., 1998; Nicolas et al., 1997). The virulence phenotype studies of Rajamäki and Valkonen (1999 and 2002) have shown that different domains and certain amino acids within VPg are crucial for systemic infection in N. physailoides and S.commersonii. The mutational analysis results indicate that movement function and replication may be located at different VPg domains (Fig. 4) (Nicholas et al. 1997; Schaad et al. 1997 (A)

Fig 4. Diagrammatic representation of the PVA VPg protein. The amino acid sequence between positions 36 and 58 contains six ba-sic residues: five Lys and one Arg. The nu-clear localization signal (amino acids 36-58) overlaps with NTP-binding domain 38-44. The potyvirus VPg NLS signal shows a gen-eral similarity to the Simian virus 40 large-T antigen-type NLS (Raikhel 1992). Amino acid positions within a central domain of the VPg are significant for systemic infection in potyvi-rus-host combinations. There are several con-served amino acids within the central domain of the VPg of different potyviruses.

Our results indicate that unknown kinase activity is involved in regulating PVA VPg function. The kinase activity from S. commersonii and tobacco produced different tryp-tic phosphorylation patterns, when recombinant VPg from different PVA virus isolates was used. We found that the capability of VPg to support accumulation and transport in S. commersonii was correlated with differences in the phosphorylation patterns. Infection and phloem loading accelerated in S.commersonii but not in N.physailoides when the strain B11 VPg central part had Tyr instead of His at position 118. The B11 VPg mutation is Val 116 Met, which reduces virus accumulation only in S. commerso-nii (Rajamäki and Valkonen, 2002). In addition, virus accumulation is reduced when 185 Leu is substituted for the C-terminal amino acid Ser-185 with no effect on virus titers in tobacco (Rajamäki and Valkonen, 2002). The differences observed in phos-phorylation patterns of PVA strains B11 and B11-M VPg in S. commersonii may be the result of a defence response which down regulate or inactivates various different VPg functions. Results agree that the VPg may influence the virus host range (II). Similar host range selection is observed with tobacco vein-mottling virus (TVMV) VPg in which four critical amino acids are necessary for resistance-breaking determinance (Nicolas et al., 1997).

Tobacco and wild potato may be differently susceptible to different virus strains, and our results suggest that phosphorylation of VPg plays a functional role in certain host interactions. Substitution of one or more amino acids or phosphorylation-induced con-formational changes may influence the recognition of putative host factors involved

PVA VPg 188 aa 21 kDa

38-A Y T G T-44

NTP -binding site

K K K

NLS Tyr -63

Central domain

27

in VPg function. Direct demonstration of the involvement of the VPg in a movement complex is still lacking. Phosphorylated VPg may assume a conformation which in-teracts with viral proteins and host factors facilitating movement. We failed to prove that improper movement is a result of phosphorylation, or is needed for engaging host factors supporting movement. Phosphorylation regulates functions of several plant virus proteins. Interaction of TMV 30 K MP with RNA down regulates transla-tion, but phosphorylation of MP reduces its RNA binding and releases it for transla-tion (Karpova et al., 1997). Furthermore, mutation of the C-terminal phosphorylation site of MP has been shown to change its affinity towards regulation of plasmodesma (Karpova et al., 1999; Waigmann et al. 2000). Alternatively, phosphorylation of VPg might control viral genome passage through plasmodesmata. TEV VPg is known to facilitate long-distance movement in a genome replication-in-dependent manner, and it is necessary for both long-distance and cell-to-cell move-ment (Schaad et al., 1997). VPg mutations studied here are distant from the domain interacting with eIF(iso)4E, from the nuclear localization signal, from TMV VPg amino acids overcoming va –resistance, or from tyrosine linking the VPg to the RNA (Mur-phy et al., 1991; Nicholas et al., 1997; Leonard et al., 2000; Schaad et al., 1996).

5.2 Polar localization of VPg Covalently RNA-linked VPg remained encapsidated in a virion. Considering the size of VPg (22-23 kDa), it may not fit into the helical structure of repeating CP. Identifica-tion of VPg linked to RNA as a part of the virion structure was one significant finding of this work. Polar particle structure is demonstrated to be similar to that of beet yellow virus (BYV) in the family Closteroviridae. This particle structure is thought to involve in an inter-action with both host and virus proteins (Peremyslov et al., 2004). PVA VPg was located at one end of the virion in such a way that makes protein-to-protein interac-tion possible. The exposed position of VPg in the particle of PVA may play a role in transport of virion through plasmodesma or phloem loading (Nicholas et al., 1997; Rajamäki and Valkonen, 2002). Moreover, VPg-coordinated particle stabilization is possible, since large PVA particles needs to be protected from degradation by the host machinery. Particle disassembly on arrival into adjacent cells should be tightly regulated and coupled to primary translation. VPg phosphorylation may also trigger virion disassembly or regulate translation initiation of the virus genome by changing VPg affinity towards CP. Alternatively, phosphorylation may regulate VPg affinity with eIF(iso)4E (Wittmann et al., 1997). The eIF(iso)4E is a translation initiation factor, and its interaction with VPg indicates its role in translation initiation (Ruud et al.,1998; Sachs et al., 1997). VPg interaction with eIF(iso)4E may involve functions required for intracellular localization and transport of potyviral RNA (Lellis et al., 2002). Arabidopsis eIF(iso)4E interacts with eIF(iso)4G protein. The linkage to the plant cell microtubule network may occur via eIF(iso)4G which is shown in reconstitution assays to interact directly with Arabi-dopsis microtubules and wheat microtubules (Bokros et al. 1995). The evidence sug-gests that exposed VPg may mediate transport by either providing stability through eIF4(iso)E, or interact with microtubules through a VPg-eIF(iso)4E-eIF(iso)4G (Schwartz et al., 2000; Lellis et al 2002).

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5.3 Nucleotidylation of the VPg The role of VPg in the replication initiation remains poorly understood, and polymerases of (+) RNA viruses use a different priming mechanism to initiate RNA synthesis. Many RNA polymerases have the capacity to initiate RNA synthesis de novo, but picorna-like viruses, like potyvirus, are thought to be primer-dependent enzymes (Agol et al., 1999). In picornaviruses, nucleotidylation of VPg is the first step in replication initiation. RdRp then catalyzes RNA synthesis when VPg-pU or VPg-pUpU serves as a primer. PVA VPg is uridylylated by NIb, the virally encoded RdRp. A mutational study revealed that the point mutation Tyr-63 to Phe-63 of the individual nucleotidylation site in the potyvirus VPg abolishes replication of TVMV and TEV (Murphy et al. 1991; Murphy et al. 1996; Schaad et al., 1996). Interestingly, in the light of the current model of potyvirus replica-tion initiation, the location of tyrosine linking the PVA RNA to VPg remains unknown (Kekäräinen et al., 2002).

Replication is a complex process, and host factors are involved in initiation of both (+) and (-) strand synthesis. Most if not all potyviral proteins are essential for virus replication (Kekäräinen et al., 2002). The replication complex is anchored via 6K2 to host membranes (Martin et al., 1995, Restrepo-Hartwig and Carrington, 1994). Several reasons for association with membranes have been suggested. Local concentrations of RNA and polymerases are elevated, which may favours viral RNA selection. The isolated compartment protects viral RNA from host dsRNA suppression system. In the poliovirus replication initiation, the cre (2C) RNA sequence was found to be the pri-mary template for PV VPg nucleotidylation. Mutation of the PV cre sequence reduced nucleotidylation reaction efficiency (Rieder et al., 2000). The potyvirus RdRp failed to elongate uridylylated VPg-pU in our study, and it should be noted that poliovirus RdRp elongates VPg-pU in a template-independent manner (Paul et al., 1998). VPg in an in vitro competition assay showed template-independent preference for UTP over the three nucleotides. Explanation for such a result suggests VPg-derived nucleotide selec-tion. Results may reflect the fact that the VPg first binds the incoming NTP. The VPg-NIb interaction is conceivably required to catalyze the linkage of the initially bound NTP to a tyrosine residue of VPg.

We confirmed that the in vitro nucleotidylation reaction of PVA NIb is stimulated by the simultaneous presence of the divalent metal cations Mn2+ and Mg2+. The PVA RNA polymerase may require Mn2+ for polymerase activity, which coordinates and stabilizes the incoming NTP in the polymerase-binding pocket. Alternatively, Mg2+ may enhance polymerase function during the conceivable structural rearrangement of polymerase when polymerase changes activity from protein nucleotidylation to RNA elongation. The structural rearrangement of T7 RNA polymerase has been determined during the transi-tion from initiation to elongation (Yin and Steitz, 2002). With PRD DNA-dependent DNA polymerase, the use of Mn2+ stimulate protein –primed initiation, but has no effect in acti-vating phage genome replication (Caldentey et al., 1992). In addition, an x-ray structural study of φ6 RdRp has revealed a second cation-binding site near the expected catalytic pocket (Butcher et al., 2001). It is conceivable that plant virus polymerases have an additional requirement for Mn2+ rather than Mg2+. It is known that plants contain a signifi-cantly higher amount of Mn2+ stores than of Mg2+ (Crotty et al., 2003).

29

5.4 The NTP-binding domain of VPgOur intriguing finding that VPg bound nucleotides in the absence of the RdRp and RNA template, led us to suggest that it may have an NTP-binding domain. The results from nucleotide cross-linking experiments showed that VPg has lysines close enough to react with incoming oxidized nucleotides. The reaction used here was lysine spe-cific (Richards et al., 1995). We also showed that Mn2+ strengthened the NTP-binding efficiency of VPg, possibly by coordinating or stabilizing the incoming nucleotide. Deletion of VPg domain ∆38-44 debilitated nucleotide binding-efficiency, and VPg ∆38-44 mutant in vitro uridylylation activity was strongly reduced, compared to wt VPg. This led us to predict that the positively charged lysines, Lys-41, Lys-42, and Lys-44 may play a role in NTP binding. Interestingly, this region of PVA VPg over-lapped with the nuclear localization signal of TEV VPg. Mutational analysis of TEV VPg with Lys-42 and Lys-44 resulted in a tenfold reduction in infection in experiments with protoplasts as well as in nuclear translocation efficiency (Schaad et al., 1996b). We found sequence homology between PV VPg and the nucleotide-binding domain of PVA VPg; moreover deletion of either one or both of the adjacent lysines resulted in nonviable phenotype and debilitated in vitro the uridylylation of PV VPg (Paul et al., 2003). PV VPg amino acids (Tyr-3, Gly-5, Lys-9, Lys-10, and Arg-17), are essential for nucleotide binding, uridylylation, and viral growth in studies of Paul et al. (2003). It should be mentioned here that the exact roles of these PV VPg amino acids remains unclear.

5.5 Phosphorylation of the CP moleculeSurprisingly little is known of how phosphorylation of PVA movement-related proteins is regulated. Our working model proposes that CP phosphorylation negatively regu-lates its interaction with RNA early in the assembly pathway. Later, dephosphoryla-tion of the CP is required before RNA binding and efficient movement intermediate can arise. The PVA CP is phosphorylated on threonine, and CP and TMV MP are both targets for the same in vitro tobacco Ser/Thr kinase activity (I). Our results indicated that TMV MP and PVA CP phosphorylation is activated by Mn2+. In our study, the metal cation concentration used was higher than the concentration necessary to saturate ATP present in the phosphorylation reaction suggesting that plant kinases may have more than one metal-binding site for regulating enzyme activation (Sun and Budde, 1997; 1999). The isolated PVA virions themselves are not deficient targets for in vitro tobacco kinase phosphorylation, suggesting that the CP phosphorylation sites are located in the particle core. Interestingly, it was shown that other potyviruses, PPV virions, interact with phosphoserine, and phosphothreonine-specific antibod-ies. These results suggested that virion surface-located sites were phosphorylated by another unknown Ser/Thr-specific kinases. Additionally, PPV CP contains other modification, O-linked N-acetylglycosamine (O-GlcNAc). This O-GlcNAc modification was as abundant as phosphorylation events. These modifications are reciprocal with phosphorylation, occurring at the same or at adjacent hydroxyl moieties. O-GlcNAc modifications are involved in regulation of very diverse cellular functions (Comer and Hart, 2000; Fernandez-Fernandez et al., 2002).

30

5.6 RNA-binding function of CP is regulated by phosphorylationIt is plausible that the RNA-binding activity of CP is regulated. The nature of the poty-virus movement complex is largely unknown. There are two alternative hypotheses: If potyviruses use virions for cell-to-cell translocation, to form phosphorylation may control movement by regulating the amount of CP available for interaction with viral RNA. Alternatively, viral RNA interacts with the CP in replication complexes, forming ribonucleoprotein complexes, and this movement intermediate is directed to plas-modesmata. The movement intermediate is then transported through plasmodes-mata to neighboring cells, where certain protein kinases may phosphorylate CP mol-ecules. Phosphorylation reduces CP affinity towards RNA, and viral RNA is released for translation in the uninfected cell (Fig. 5). A similar movement-protein phosphoryla-tion-regulated strategy has been suggested for intermediate viral ribonucleoprotein movement complexes (Mas and Beachy, 1999; Karpova et al., 1997, 1999).

Fig 5. General model for phosphorylation events regulating coat protein functions during virus life cycle. During virus protein synthesis, the capsid proteins are phosphorylated. Phosphorylation of capsid proteins prevents interaction with cellular and viral RNA, and thus prevents formation of particles. Later in the repli-cation cycle, the amount of genomic RNA increases and particle formation is favored by dephosphorylation of capsid proteins. The capsid protein is dephosphorylated by an unknown protein phosphatase resulting in increased affinity towards RNA. Particle dephosphorylation by phosphatases may activate genomic RNA release from the particle during translocation through plasmodesmata.

Many RNA-binding proteins share a similar structural domain orientation. These do-mains may be structurally flexible and thus promote RNA binding (Baratova et al., 2001; Forsell et al. 1995). Structural data on PVA CP was obtained by tritium bom-bardment and secondary structure prediction, a method recently used successfully to probe several domains of complex proteins (Baratova et al., 2001). The CP C-ter-minal region domains are organized in a βαββα order. This organized order is found in several RNA binding proteins (Baratova et al., 2001). Proteins having this domain order have a structure called an abCd-unit (Efimov. 1995). The secondary structure length and distribution of PVA CP is closely related to TMV CP structure orientation (Agafonov et al., 1997; Baratova et al., 2000). We hypothesize that the PVA CP C-terminal part may regulate the RNA binding activity. CP C-terminal CK2 phosphoryla-tion site Thr-242 is located in a flexible loop, inside in the particle. The CP β7-strand may change affinity towards RNA after the charge or structure of this region changes.

VPg

RNA

-- - -

--

--

-

- -

VPg

RNA

-- - -

--

--

-

- -

VPg

RNA

-- - -

--

--

-

- -

CP

CPCP

CP

CP

P PPPPP

P

Early assembly

(on membraneous

environment)

X XPhosphatases

Late assembly

on membraneous

environment

Kinases+

CK2

Nucleocapsid uncoating

Host

RNA

Host

RNA

31

This potyviral C-terminal CP region is conserved in multiple sequence alignments amongst 28 members of the potyvirus genus ( Shukla et al., 1994).

The negative charge of phosphate is thought to prevent electrostatic interactions with RNA. Alternatively, phosphorylation may also trigger conformational changes in CP and thus prevent RNA binding. Ribonucleoprotein complex assembly and formation may trigger conformational changes (Williamson, 2000). For example, phosphoryla-tion of duck hepatitis virus CP triggers a conformational effect on a structural level (Yu and Summers, 1994). Phosphorylation of a viral structural protein may change its affinity towards RNA, but this depends on the virus studied. In several cases, phos-phorylation is shown to reduce the RNA-binding affinity of a given protein (Gazina et al., 2003; I and III; Kann and Gerlich, 1994; Lan et al., 1999; Wu et al. 2002). Phosphorylation of virus coat protein during early replication may inhibit non-spe-cific complex formation with cellular RNA, since the level of viral RNA is low. Our model proposes that CP phosphorylation regulates RNA binding in a negative way. Phosphorylation of the RNA-binding domain of PVA CP is an ideal way to regulate the amount of genomic RNA available for replication on the membranous replication complexes (Schaad et al., 1997).

5.7 PVA CP is phosphorylated by CK2CK2 activity is involved in cell proliferation, cell-cycle progression, transcriptional control, light-regulated gene expression, and plant growth (Espunya et al., 1999, Landesman et al., 2001; Plana et al., 1991). We confirmed and investigated in detail PVA CP Thr-242 as a major in vitro CK2 phosphorylation site. We found also that the Thr-242 phosphorylation site is located in the interconnecting loop inside the struc-ture, as often found in RNA-binding proteins. Additionally, Kekäräinen et al. (2002) showed, with an in vitro DNA transposition based genome insertion mutant library, that CP domain does not tolerate five amino acid insertions near Glu-254, which may disrupt the CK2 recognition domain. We examined the role of CP phosphorylation by constructing a full-length icDNA with mutations that affect the CK2 consensus sequence. Mutations that affect the highly conserved domain 242-TTSEED-247 in CP produced cell-to-cell and long-distance movement-deficient phenotypes. The phosphoacceptor site was further mutated with a substitution of acidic residues for basic Arg residues downstream. This mutant also produced a movement deficient virus. This mutation study shows that CK2 plays a critical role in regulating PVA movement. Our results propose that a delicate balance between protein kinase and still unknown phosphatase activity is crucial for PVA infectivity.The article of Zhang and Klessig (2001) focuses on plant MAPKs in stress responses, which is functionally analogous to mammalian c-Jun kinase (JNK) and p38 MAPKs. Because plant MAPKs are probably derived from the ancient MAPK that also gave rise to the mammalian anti-extracellular signal-regulated kinase (ERK) subfamily of MAPKs, their functions in plant stress responses probably evolved independently from mammalian JNKs and p38s. The implication of CK2 in mediation of stress sig-nalling downstream of p38 MAP kinase is a surprising finding Fig. 6 (Sayed et al., 2000). Virus infection induced defence reactions, which in turn activate plant MAPK

32

cascade and may result in upregulation of plant CK2. Plant CK2 is likely regulated through the stress-activated MAPK signalling cascade. It is plausible to think that the PVA may benefit from pathogen activated stress response that modifies CK2 kinase activity.

Fig. 6. Schematic rep-resentation of plant MAPK-signalling cas-cade. Arrows indicate up-regulation of downstream kinases. A. Overall repre-sentation of MAPK-sign-aling cascade. B. Stress-induced activation of CK2 through p38 MAPK-sign-aling cascade (Sayed et al., 2000). C. Hypothetic upregulation of plant CK2 through p38 signaling cascade after pathogen induced defence reac-tion. Upregulation of the CK2, the major kinase phosphorylating PVA CP, may be beneficial for the virus.

In addition to CK2 phosphorylation at Thr-242, PVA CP is phosphorylated by a still unidentified kinase/s. The broad-range kinase inhibitor staurosporine inhibits in vitro phosphorylation of the CP only partially. Staurosporine inhibition against CK2 is re-markably low. However its use should result in a complete block of other kinases (Da-vies et al., 2000). Localization of protein kinases is one way to modulate the activity of these enzymes (Hubbard et al., 1993; Mochly-Rosen, 1995). Differential localization of kinases can be regulated by interaction through specifically targeted anchoring proteins (Huang et al., 1999), and many kinases involved in signaling cascades are dynamically regulated at post-translational-level (Murillo et al., 2001).

Diverse viruses localize their RNA replication complexes on different membrane subsets. As an example, alphaviruses use endosomal and lysosomal membranes (Kääriäinen and Ahola, 2002). Brome mosaic virus (BMV) ER membranes and PV replication complexes are formed from budding transport vesicles (Restrepo-Hartwig and Ahlquist, 1996; Rust et al., 2001). The present study showed that ER-associated CK2 activity may phosphorylate CP. Localization of CK2 activity to ER is notable, since this observation is in agreement with earlier results showing that there are also other CK2 substrates, tightly associated with the ER or with Golgi complexes. To be precise, CK2 subunits are located at the cytosolic side of the ER (Faust et al 2001).

Conseivable CK2 activation by MAPK kinase cascade

STIMULUS

Sensor or

reseptor

G-protein

or kinase?

MAPKKK

MAPKK

MAPK

Substrate

other components

RESPONSE

TNF-R, other

reseptors

STRESS

MEKK-x

MKK3

p38 MAPK

CK2

upregulation

PVA infection

R-protein, or

alternative stress

induced proteins?

MEKK1

MEK1

MPK4

Plant p38 MAPK

homolog

CK2

upregulation

SUBSTRATE

PVA CP

A B C

33

This is logical, since viral RNA replication complexes of potyviruses are known to as-sociate to the ER derived membranes (Schaad et al., 1997). A similar result is shown with Tomato mosaic virus MP phosphorylated with CK2α (Matsushita et al., 2000; Matsushita et al., 2003).

Phosphorylation of the structural protein suggests a ubiquitous regulation mecha-nism for genome encapsidation. Indeed, phosphorylation of virus structural proteins seems to have different effects depending upon the virus. Mutations affecting CP phosphorylation sites have a negative effect on replication with Cauliflower mosaic virus and Potato virus X (Atabekov et al., 2001; Leclerc et al., 1999). Site-directed mutation of HIV Cap24 protein suggests that phosphorylation may be implicated in disassembly of the viral capsid (Cartier et al., 1999). Phosphorylation of the core pro-tein of the hepatitis B virus has a dual role in encapsidation, creating conformational changes, which enables the protein structure to participate in encapsidation (Gazina et al., 2000). Rabies viruses regulate transcription and replication by phosphorylation (Wu et al., 2002). In closing, regulating virus capsid proteins may be important not only for virus movement but also for other types of virus host interactions.

34

6.0 CONCLUDING REMARKS

The results of the present work demonstrate that MPRs of PVA, VPg, and CP are regulated by host cell kinase phosphorylation. We also suggest that phosphorylation of VPg plays a role in virus movement. Different host kinases from tobacco and wild potato recognized VPg, and differences in phosphorylation patterns correlated with the phenotypic differences. We also found that virion-bound VPg could interact with host proteins. Second, we have found that CK2 phosphorylation of CP downregulated RNA binding. Affinity towards RNA may regulate premature particle association during early repli-cation. Analysis of CK2 phosphorylation site-deficient mutant viruses in vivo showed that they were defective in cell-to-cell and long distance movement. Third, we have investigated the role of VPg serving as a primer for genome replica-tion. We show that VPg could be uridylylated in vitro by NIb RNA-dependent RNA polymerase. Additionally, nucleotide-binding domain deletion diminished the nucleo-tide-binding capacity of VPg and decreased the uridylylation reaction.

35

ACKNOWLEDGEMENTS

This study has been carried out at the Institute of Biotechnology and Department of Applied Biology, University of Helsinki, under the supervision of Docent Kristiina Mäkinen during the years 1999 – 2004. The Academy of Finlad, National Technology Agency of Finland, the Ella and Georg Ehrnrooths Foundation, Finnish Cultural Foun-dation, Finnish Virus Disease Research Foundation (VITUS), and Swedish Cultural Foundation have financially supported the workI want to thank Professor Mart Saarma for excellent scientific facilities, and stimulat-ing research environment at the Institute of Biotechnology. I want to thank Kristiina Mäkinen for providing great facilities for this work, and for encouragement over these years. I am also grateful for their continuous interest in and critical reading of the manuscript to Kristiina, and reviewers Lesley Torrance, and Tero Ahola. My special thanks go to Kostya Ivanov, for excellent training during intense periods in the labo-ratory. I am proud to have been able to spend these four years with such a person always ready to share excellent scientific guidance. Special thanks to Katri, without her magic eye, most of the desperately needed, and sought reagents, and apparatus would still be missing. Most of the time I spend in the lab with Katri and Kostya; spe-cial thanks to both of you, for supportive friendship when the moments were boring, and frustrating. Also thanks to my friends for the helpful discussions in Biokeskus, Mikko F, Anne S, Mikael B, Susanna F, Leena V, Andres M. Thanks to everyone else, working in our former virus/biochemistry lab.Experimental science is a hard work, but even harder is sometimes to shut off the system that makes our knowledge possible. Warmest thanks for helping to clarify me out thoughts from science, and the academic environment goes to friends actually constructed to carry something a bit heavier than paper piles, Jari-Pekka M., Petri M., Marko S., and Jaro K. Thanks to plant science collaborators; Petri S and Tony W.My special thanks go to my parents: Maria, Matti, and sisters, Milena, and Miro for being there, and last but not least, to Virpi, for continuous love, support, and encour-agement.

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