Proc. Nail. Acad. Sci. USAVol. 88, pp. 4104-4108, May 1991Biochemistry

Functional domains of the poliovirus receptor(truncated receptors/mouse L-cell transformants/virus binding site/cytoplasmic domain/monoclonal antibodies against receptor)

SATOSHI KoIKE*, IKU ISE, AND AKIo NOMOTODepartment of Microbiology, The Tokyo Metropolitan Institute of Medical Science, Honkomagome, Bunkyo-ku, Tokyo 113, Japan

Communicated by Robert M. Chanock, February 19, 1991

ABSTRACT A number of mutant cDNAs of the humanpoliovirus receptor were constructed to identify essential re-gions of the molecule as the receptor. All mutant cDNAscarrying the sequence coding for the entire N-terminal immu-noglobulin-like domain (domain I) confer permissiveness forpoliovirus to mouse L cells, but a mutant cDNA lacking thesequence for domain I does not. The transformants permissivefor poliovirus were able to bind the virus and were alsorecognized by monoclonal antibody D171, which competes withpoliovirus for the cellular receptor. These results stronglysuggest that the poliovirus binding site resides in domain I ofthe receptor. Mutant cDNAs for the sequence encoding theintracellular peptide were also constructed and expressed inmouse L cells. Susceptibility of these cells to poliovirus revealedthat the entire putative cytoplasmic domain is not essential forvirus infection. Thus, the cytoplasmic domain of the moleculeappears not to play a role in the penetration of poliovirus.

Virus receptors are considered to have important roles in theearly steps of viral infection such as binding to the cellsurface, penetration, and uncoating of the virus. Therefore,elucidation offunctional regions ofthe virus receptors in eachinfection step is essential to an understanding of the molec-ular mechanisms of the interaction between cells and virusesin early infection. Indeed DNAs encoding several virusreceptors have been isolated (1) and extensive studies havebeen performed to identify and characterize regions of thereceptors required for infection, particularly those of thehuman immunodeficiency virus (2-8) and the major grouphuman rhinovirus (9, 10).

Poliovirus, the causative agent of poliomyelitis, infectsprimates and invades the target tissues including the centralnervous system (11). The characteristic species specificityand tissue tropism of poliovirus are considered to be primar-ily determined by a unique cell surface receptor (12, 13).Although crystallographic studies on poliovirus revealed theprecise three-dimensional structure of the virion and pro-posed a putative attachment site for the poliovirus receptor(PVR) that is a depression (called a "canyon") near thefivefold axis of the virion particle (14, 15), little is knownabout structure of the counterpart, the PVRs. To understandthe specific interaction between poliovirus and PVRs thatleads to the establishment of the infection, several monoclo-nal antibodies (mAbs) that block poliovirus infection havebeen isolated (16-19). Furthermore, the genomic and com-plementary DNAs for human PVRs were isolated fromgenomic and complementary DNA libraries prepared fromHeLa S3 cells (19, 20). Mouse L cell transformants carryingthese DNAs acquired susceptibility to poliovirus and wererecognized by mAbs against PVRs. Transgenic mice carryingthe human PVR gene are permissive for all three poliovirusserotypes (21, 22). Thus, it is clear that the human PVR gene

confers permissiveness for poliovirus to mice in vivo as wellas mouse cells in vitro.

Nucleotide sequence analysis of cDNAs for human PVRsrevealed that a functional PVR contains a putative signalpeptide, three immunoglobulin-like domains, a transmem-brane domain, and a cytoplasmic domain and that there areat least four mRNA isoforms (mRNAs for PVRa, PVR,8,PVRy, and PVR8) generated by alternative splicing from theprimary transcript (19). PVRa and PVR8 are integral mem-brane proteins that function as the virus receptor, althoughthese two PVRs differ in amino acid sequence in the cyto-plasmic domain. PVRP and PVRy lack the transmembranedomain that has an essential function in anchoring the PVRmolecules to the surface membrane of the cells. In fact, it hasbeen proved that PVR,3 is secreted from the cells and is,therefore, not a functional PVR. Thus the transmembranedomain is an essential region of a functional PVR molecule(19). However, it is not clear whether the three immunoglo-bulin-like domains and the cytoplasmic domain play essentialroles in the establishment of poliovirus infection.

Herein we describe the construction of a number of mutanthuman PVR cDNAs and examine the susceptibility to polio-virus of mouse L cells carrying these mutant cDNAs. Theresults indicate that the binding site of poliovirus resides inthe N-terminal immunoglobulin-like domain (domain I) andthat the entire cytoplasmic domain is not necessary for virusinfection.

MATERIALS AND METHODSConstruction of the Mutant Receptor cDNAs. The mutant

receptor cDNAs with internal deletions were constructed bythe following procedure. PVRa cDNA was progressivelydigested with nuclease BAL-31 from the HindIII site or BglII site ofpSV2PVRa (19) to make a series of 5' deletions and3' deletions, respectively. Digested DNA was repaired withthe Klenow fragment ofDNA polymerase I and ligated withsynthetic Sal I linkers (5'-GGTCGACC-3'). PVR cDNAfragments thus obtained were subcloned in pUC118 and thenthe nucleotide sequences were determined. Several pairs ofDNA fragments (HindIlI-Sal I fragments with 3' deletionsand Sal I-Bgl II fragments with 5' deletions) suitable forconstructing cDNAs encoding the mutant receptors wereselected. Pairs of DNA fragments encoding amino acids1-146 and 245-417, amino acids 1-262 and 344-417, aminoacids 1-164 and 344-417, and amino acids 1-146 and 116-417were used for the construction of pSV2PVRd2, -d3, -d4, and-d5, respectively. These fragments were inserted into pSV2vector that had been digested with HindIII and Bgl II (Fig. 1).Plasmid pSV2PVRdl was constructed by ligating a HindIII-Nar I fragment (Nar I site was treated with Klenow fragmentto fill-in the protruding end) encoding amino acids 1-33 of

Abbreviations: PVR, poliovirus receptor; mAb, monoclonal anti-body; pfu, plaque-forming unit(s).*To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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PVR a

PVR dl

PVR d2

PVR d3

PVR d4

PVR d5

PVR d6

PVR d7

domain I domain 11 domain III27 143 242 331V rs Sl Y rs is Y r-s s V

TM Cyt

33 137

146 245RST I /

262 344GRP

164 344L:-1 GRP

1461::::1 F--lRST

116 -383

:AGE356

::~GE

PVR d8 I::344IGE

FIG. 1. Structures ofthe mutant receptors. The structure of the PVRa is shown at the top. The arrowheads with amino acid positions indicatethe boundary of the domains where the introns are present in the PVR gene. The signal-peptide domain, transmembrane domain (TM), andcytoplasmic domain (Cyt) are indicated by stippled, solid, and hatched boxes, respectively. The constructs that have mutations in theextracellular domain (PVRdl-PVRd5) or the cytoplasmic domain (PVRd6 and PVRd7) or that lack the entire transmembrane and cytoplasmicdomains (PVRd8) are shown. Numbers above each construct indicate the amino acid positions of the boundaries. Lines between boxes representdeleted amino acid sequences. The letters between lines or behind the boxes are the amino acid sequences that are generated by the ligationwith the synthetic linker DNAs. Amino acids are shown in single-letter code. Names of individual PVRs are indicated at the left of the figure.

PVRa with a larger HindIII-EcoRV fragment encodingamino acids 137-417 ofPVRa from the plasmid pSV2PVRa.The mutant receptor cDNAs lacking parts ofthe nucleotide

sequence encoding C-terminal proximal peptides were con-structed as follows. PVRa cDNA was digested with BAL-31as described above or digested at the Xho I site (correspond-ing to amino acid 383) and then the DNAs were repaired withKlenow fragment. Modified PVR cDNA fragments thusobtained were digested with HindIII and then inserted intothe HindIII-BgI II site of vector pSV2 with the synthetictermination adaptor

5'-GGTGAGTGACTGAA-3'3'-CCACTCACTGACTTCTAG-5'.

Amino acids that are generated by the synthetic DNAs inmutant PVRs are shown in Fig. 1. Nucleotide sequences ofall junctions of the constructs were determined again for theconfirmation.Transformant Cells Producing the Mutant Receptors.

Mouse L-cell transformants Ldl-Ld8 producing the mutantreceptors PVRdl-PVRd8, respectively, were established asdescribed (19). The RNAs oftransformant cells were isolatedas described by Okayama et al. (23). Total RNA (10 ,ug) oftransformants was separated by agarose gel electrophoresisand the mRNAs for mutant receptors were analyzed byNorthern blot hybridization (24) using the entire codingregion of PVRa cDNA as probe.

Infection with Poliovirus. Transformant cells (106 cells)were seeded in a 6-cm plastic dish in duplicate. The cells wereinfected with 107 plaque-forming units (pfu) of light-sensitivepoliovirus type 1 Mahoney strain (19). After a 30-min ad-sorption period and a 30-min penetration period at roomtemperature and 370C, respectively, in the dark, the cellswere washed twice with 5 ml of Eagle's minimum essentialmedium (MEM). Dishes were frozen immediately or after theincubation at 370C for 24 h. Cells were disrupted by threecycles of freezing and thawing under a fluorescent lamp (19),and then the virus titers at 0 h and 24 h after infection weredetermined by the plaque assay using African green monkeykidney cells (25).

Calculation of Infection Efficiency of Transformant Cells.Transformants in 6-cm plastic dishes were infected with 5 x102 or 5 x 104 pfu of poliovirus as described above. Theinfected cultures were incubated at 370C under agar overlay.The numbers of plaques displayed on transformant cells werecounted and compared with the number on HeLa S3 cells.Binding of Poliovirus and Antibodies to Transformant Cells.

Poliovirus Mahoney strain was metabolically labeled with[35S]-methionine as described (26). 35S-labeled virus waspurified by chromatography on DEAE-Sepharose CL-6B (27)followed by sucrose density gradient centrifugation as de-scribed (28). Specific activity of the labeled virus was 0.5cpm/pfu. Transformant cells (5 x 105 cells per well) wereseeded in 24-well culture plates. Poliovirus (5 x 105 cpm) wasadded to the medium with or without a 1000-fold excess ofunlabeled poliovirus and incubated at 40C for 12 h. Unat-tached virus was removed by washing three times with 2 mlof MEM. Virus bound to cells was recovered with 250 gi of0.5% SDS and radioactivity was measured in a liquid scin-tillation counter. The radioactivity of virus that specificallybound to cells was calculated by subtracting the cpm ob-tained in the presence of unlabeled virus from that in theabsence of unlabeled virus.Anti-PVR mAb P44 (19) was purified by affinity chroma-

tography on protein A-Affi-Gel (Bio-Rad). Anti-PVR mAbD171 (17) was kindly provided by Peter Nobis (UniversitatHamburg) through Eckard Wimmer (State University ofNewYork at Stony Brook). Antibodies were labeled with 1251I byusing the Bolton-Hunter reagent (29). Specific activities oflabeled mAbs P44 and D171 were 8 x 106 and 1.6 x 106cpm/pug, respectively. mAbs (5 x 105 cpm) were added to theculture medium and incubated at 40C for 2 h. Radioactivityassociated with transformants was measured in a y-counter.

RESULTSEffect of Deletions in Extracellular Domain. We constructed

cDNAs encoding a series of mutant PVRs (PVRdl-d4) thatlacked various regions of the extracellular peptide to identifythe domain that contained the binding sites of poliovirus andmAbs against PVRs (Fig. 1). Transformant cell lines, Ldl-

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1 2 3 4 5 6 7 8 9 10 Table 2. Binding of poliovirus and anti-PVR mAbs to cells

28Sv

18S v ~*mwgW

FIG. 2. Detection of the mRNAs for the mutant receptors. TotalRNA (10 ,tg) of the transformants was analyzed by Northern blothybridization. Total RNA of La, Ldl-Ld8, and Ltk- cells wasanalyzed in lanes 1-10, respectively. The entire coding region of the32P-labeled PVRa cDNA fragment was used as a probe. Positions of28S and 18S rRNA markers are shown with arrowheads.

Cells Virus bound, cpm

HeLaLtk-LaLdlLd2Ld3Ld4Ld5Ld6Ld7Ld8

27,5320

37,7040

1766,6621,422

31,52826,64918,462

0

Antibodiesbound, cpm

D171 P44

31,7800

34,6880

97031,26829,44154,31353,88534,568

0

3890

22200000

14451241408

0For antibodies bound, values after the subtraction of backgrounds

are shown (backgrounds are radioactivities associated with Ltk-cells and are 98 cpm and 81 cpm for D171 and P44, respectively).

Ld4, carrying cDNAs encoding the mutant receptors wereestablished. The expression of the mutant cDNAs in the cellswas examined by Northern blot hybridization (Fig. 2, lanes2-5). RNA transcripts of the expected size were detected inindividual transformants.

Susceptibility to the poliovirus infection of these cells wasexamined. Among the transformants (Ldl-Ld3) producingmutant receptors that lack one of the three immunoglobulin-like domains, transformants Ld2 and Ld3 with receptorscarrying domain I showed cytopathic effects and producedinfectious particles (Table 1). Transformant Ld4 that lacksdomain III and most of domain II was also susceptible topoliovirus infection (Table 1). These results indicate thatdomain I is the extracellular region that is essential for thepoliovirus infection. Transformant Ld2 produced a relativelylower amount of infectious particles by 24 h after infection ascompared with other transformants (Table 1). This maysuggest that part of domain II acts to maintain the structureof domain I that is required for effective virus infection. Tochange the distance or configuration of domains I and II,transformant Ld5 whose receptor had an internal duplicationbetween these two domains was constructed and examinedfor its susceptibility to poliovirus. This transformant wasfully susceptible to poliovirus (Table 1). Details of the inter-action of domain I with other immunoglobulin-like domainsremain to be elucidated.

Binding of Poliovirus and Anti-PVR mAbs to the Trans-formant Cells. Establishment of virus infection must require

Table 1. Susceptibility of transformant cells topoliovirus infection

Virus titer 24 hafter infection,

Cells log1o (pfu/ml)HeLaLtk-LaLdlLd2Ld3Ld4Ld5Ld6Ld7Ld8

7.6ND6.8ND5.27.17.37.47.27.3ND

Cytopathic effect

+

+

+

several steps of specific virus-cell interaction, and conse-quently, virus binding to cells does not always result in theinfection. Accordingly, we measured the amount of virusbound to transformants Ldl-Ld5 whose PVRs contain mu-tations in the extracellular moiety (Table 2). Virus bound onlyto the cells susceptible to poliovirus infection, although theamount of the bound virus varied from cell line to cell line.These results indicate that the binding site for poliovirus islocated only in domain I. All transformant cells that aresusceptible to type 1 poliovirus are also susceptible to type 2and type 3 polioviruses (data not shown). This stronglysuggests that all three poliovirus serotypes bind to domain I.We also analyzed mAbs that compete with the virus for the

PVR binding site. mAb D171 has proved to satisfy thiscriterion (17). mAb P44 also competes with poliovirus andD171 for the binding sites (data not shown). As shown inTable 2, D171 binds only to the cells transformed withcDNAs containing the nucleotide sequence encoding domainI. This observation supports the conclusion that poliovirusbinds to domain I of the receptor. Relative amounts ofantibody bound to individual transformants were similar tothe amounts ofbound virus. In contrast to D171, mAb P44 didnot bind most of the transformants carrying mutant receptorswith an altered extracellular domain. Thus, we could notidentify the location of the epitope recognized by mAb P44 inthis experiment but it is clear that it is not identical to theepitope recognized by mAb D171.

Since a difference in the amount of virus bound to cells mayalter the efficiency of infection, we examined the efficiencyof poliovirus infection of individual cells by comparing thenumber of plaques that developed on the transformants withthe number of plaques that developed on HeLa S3 cells.Plaque numbers are presented as a percentage of the controlplaque number on HeLa S3 cells (Table 3). The number ofplaques on HeLa S3 cells was twice that observed on Lacells, although the amount of virus bound to La cells was

Table 3. Efficiency of the infection of the transformants cells

Plaque number,Cells % of control

HeLa (control) 100La 49.3Ld2 0.3Ld3 26.3Ld4 8.5Ld5 19.5Ld6 35.9Ld7 23.3

Virus titer was measured at 0 and 24 h after infection. At 0 l afterinfection, the virus titer was not detectable in any cell culture. +,Effect detected; -, no effect detected.

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higher than that bound to HeLa S3 cells. Thus, HeLa S3 cellsshowed the highest efficiency of infection among cell linestested. This observation suggests that the efficiency of theinfection is due not only to amount of virus bound to the cellsurface but also to other factors affecting virus replication.Among L-cell transformants, the efficiency of plaque forma-tion appears to vary in accordance with the amount of virusbound to the cells. Thus, the efficiency of infection seems toreflect the amount of virus bound if other conditions of thecells are the same.

Effect of Deletions from the C-Terminal End of the PVR.The penetration of poliovirus has been suggested to occurthrough receptor-mediated endocytosis (30-32). There aremany ligands including viruses that enter cells through re-ceptor-mediated endocytosis. In some cases, the cytoplasmicdomain of the receptor (33-36) or phosphorylation of thisdomain (7, 8) plays an essential role in endocytosis of theligand-receptor complex. Although functional PVRs, PVRaand PVR8, differ from each other in amino acid sequence inthe cytoplasmic domain and do not contain important signalsfor internalization (37-39), the amino acid sequences of thesePVRs contain many serine residues that might be substratesfor phosphorylation. We, therefore, tested whether this do-main of the PVR also plays an important role in poliovirusinfection. We constructed cDNAs encoding three mutantreceptors (PVRd6-PVRd8) that differed in the amino acidsequence of the C-terminal region (Fig. 1) and transformantcells, Ld6-Ld8, carrying these cDNAs, respectively, wereestablished. Mutant mRNAs were detected in the transform-ant cells by Northern blot hybridization (Fig. 2, lanes 7-9).Mutant receptors on the surface of transformants Ld6 andLd7 were also confirmed by the binding of mAb D171 (Table2). As expected from the results of experiments involvingPVRJ3, a secreted form of PVR (19), PVRd8, that lacks thetransmembrane domain was not detected on the cell surface,and transformant Ld8 was not permissive for poliovirusinfection. This observation confirms our previous conclusionthat PVRs lacking this region are secreted forms of PVRmolecules (19). Susceptibility of these transformants to po-liovirus infection was examined. As shown in Table 1,transformants Ld6 and Ld7 showed cytopathic effects andproduced a high titer of virus similar to transformant La(Table 1). However, a slight-to-modest reduction in theamount of virus bound was observed (Table 2) but theefficiency of infection was not significantly lowered. Theseresults indicate that the cytoplasmic domain is not essentialfor virus infection.

DISCUSSIONWe show that the poliovirus binding site is located in domainI of the PVR molecule. This conclusion is reasonable con-sidering the size of the canyon to which only one immuno-globulin-like domain is accessible, as suggested by the resultof an experiment using the major group human rhinovirusesand its cellular receptor, intercellular adhesion molecule 1(ICAM-1) (10). The virus binding sites for other virus recep-tors have been identified using deletion mutants or chimericreceptors (2-6, 9, 10). CD4, the receptor for human immu-nodeficiency virus, and ICAM-1 contain three and five im-munoglobulin-like domains, respectively. The amino acidresidues critical for virus binding have also been identified.These two receptors have their primary virus binding site indomain I (2-6, 10). In both cases, deletion or amino acidchange in other domains affects virus binding of the receptor(2-4, 10). Similar phenomena may occur with regard todomain I of PVR, since virus receptor activity appears to beinfluenced by the deletion of domain(s) II and/or III (Tables1-3). It is possible that the deletion of domain(s) II and/or IIIaffects to some extent the three-dimensional structure of

domain I and that minor conformational changes caused bythe deletion of other domain(s) result in reduction of theamount of the virus or D171 bound to the cell surface. Thedistance of domain I from the cell surface may also affect itsaccessibility to virus. The reason why mAb P44 does not bindto most of the mutant PVRs is not clear at present. It ispossible that P44 recognizes a structure in domain I that iseasily destroyed by deletions introduced in any part of thePVR.

Poliovirus releases capsid protein VP4 as a consequence ofbinding to its receptor on the cell surface, and the S value ofthe particle changes from 160 S to 135 S (40). Recently, it wasdemonstrated that the solubilized membrane fraction of cellsexpressing the PVR causes a similar alternation of virionstructure (41). These observations suggest that the PVRtriggers the uncoating of poliovirus. Indeed, our preliminaryresults indicate that a partially purified peptide containingonly the extracellular domains of PVR is able to convert the160S poliovirus particle into 135S or 80S particles that areconsidered to be the important intermediates in the viraluncoating process (unpublished result). By taking the factthat Ld4 is susceptible to poliovirus infection into consider-ation, this observation suggests that the domain I ofPVR hasan important role in the uncoating process of the virus as wellas in recognition and binding by virus.

Several stages of poliovirus infection are still unclear. Theuncoating model proposed by Madshus et al. (30) is ques-tioned by Gromeier and Wetz (42). Deletion of the entirecytoplasmic domain does not significantly affect PVR func-tion in virus infection. This is of particular interest becausethe cytoplasmic domain of other receptors appears to beimportant for internalization ofreceptors through coated pits.Therefore, the internalization of the PVR may not be neces-sary during poliovirus infection, although we have not testedyet whether the PVR has the ability to be internalized by thecell. It is entirely possible that uncoating of poliovirus leadingto initiation of virus replication occurs on the cell surface aswell as in endosomes (30-32). Alternatively, it is possible thatthe internalization of PVR might occur by a totally differentmechanism from other receptors. For example, other mole-cules associated with PVRs may contribute to virus penetra-tion. Thus the mechanisms for the penetration of poliovirusremain to be elucidated. The transformant cells established inthis study will be useful tools to investigate these mechanismsin poliovirus infection.

We thank Akira Oinuma for help in the preparation of the illus-trations. We also thank Peter Nobis and Eckard Wimmer for thegenerous gift of mAb D171. This work was supported in part byresearch grants from The Ministry of Education, Science, andCulture of Japan, The Ministry of Health and Welfare of Japan, TheNaito Foundation, and The Tokyo Biochemical Research Founda-tion.

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