Importance of Vp1 Calcium-Binding Residues in Assembly, Cell

JOURNAL OF VIROLOGY, July 2003, p. 7527–7538 Vol. 77, No. 130022-538X/03/$08.00�0 DOI: 10.1128/JVI.77.13.7527–7538.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Importance of Vp1 Calcium-Binding Residues in Assembly, Cell Entry,and Nuclear Entry of Simian Virus 40

Peggy P. Li,1 Akira Naknanishi,1 Mary A. Tran,1 Ken-Ichiro Ishizu,2 Masaaki Kawano,2Martin Phillips,3 Hiroshi Handa,4 Robert C. Liddington,5 and Harumi Kasamatsu1*

Department of Molecular, Cell and Developmental Biology and Molecular Biology Institute1 and Department of Chemistry andBiochemistry,3 University of California at Los Angeles, Los Angeles, California 90095; Frontier Collaborative Research

Center4 and Faculty of Bioscience and Biotechnology,2 Tokyo Institute of Technology, Midori-ku, Yokohama226-8501, Japan; and The Burnham Institute, La Jolla, California 920375

Received 8 January 2003/Accepted 4 April 2003

For polyomaviruses, calcium ions are known to be essential for virion integrity and for the assembly of capsidstructures. To define the role of calcium ions in the life cycle of the virus, we analyzed simian virus 40 (SV40)mutants in which structurally deduced calcium-binding amino acids of Vp1 were mutated singly and incombination. Our study provides evidence that calcium ions mediate not only virion assembly but also theinitial infection processes of cell entry and nuclear entry. Mutations at Glu48, Glu157, Glu160, Glu216, and/orGlu330 are correlated with different extents of packaging defects. The low packaging ability of mutant E216Rsuggests the need to position the Glu216 side chain for proper virion formation. All other mutants selected forfurther analysis produced virus-like particles (VLPs) but were poorly infectious. The VLPs of mutant E330Kcould not attach to or enter the cell, and mutant E157A-E160A and E216K VLPs entered the cell but failed toenter the nucleus, apparently as a result of premature VLP dissociation. Our results show that five of the sevenacidic side chains at the two calcium-binding sites—Glu48 and Glu330 (site 1), Glu157 and Glu160 (site 2), andGlu216 (both sites)—are important for SV40 infection. We propose that calcium coordination imparts not onlystability but also structural flexibility to the virion, allowing the acquisition or loss of the ion at the two sitesto control virion formation in the nucleus, as well as virion structural alterations at the cell surface and in thecytoplasm early during infection.

The capsid of simian virus 40 (SV40), like those of othersmall DNA viruses in the polyomavirus family, is composed of72 pentamers of the major capsid protein Vp1 arranged on aT�7d icosahedral lattice (2, 19, 26). The architecture of theSV40 capsid, resolved at atomic resolution (19), provides cluesto aspects of capsid assembly and stability. Each Vp1 monomercomprises a core �-barrel structure with a jelly-roll topology,an amino-terminal extension, and a long carboxy-terminal arm.The five monomers in a pentamer are intimately associated viainterlocking secondary structures. Interaction between pen-tamers (in six different modes, �, ��, ��, �, ��, and �) is madethrough the insertion of carboxy-terminal arms into the coresof neighboring pentamers. Several lines of evidence have sug-gested that this interpentamer interaction is strengthened bycalcium ion chelation and disulfide bonding (4, 6, 19, 32).Structural refinement on SV40, in which divalent calcium ions(Ca2�) were replaced with trivalent gadolinium ions (Gd3�),has identified two probable sites of calcium ion coordinationper Vp1 monomer on the capsid (32) (Fig. 1). Site 1 consists ofthe Glu216 side chain and Ser213 carbonyl oxygen of onemonomer, the Glu46 and Glu48 side chains of a second mono-mer from the same pentamer, and the Glu330 side chain (C-terminal arm) of a third monomer from a neighboring pen-tamer. Site 2 consists of the Glu157, Glu160, and Glu216 side

chains and Lys214 carbonyl oxygen of the first monomer andthe Asp345 side chain (C-terminal arm) of the third monomer.Thus, each pair of calcium ions is expected to tie together twodifferent pentamers by interacting with mostly acidic aminoacid residues contributed by three Vp1 chains. All but one(Glu46) of the seven acidic residues are conserved in the poly-omavirus family (25). It is suspected that besides contributingto capsid integrity, calcium ion-mediated interactions play arole in various processes of virus dissemination, including cellentry, intracellular trafficking, disassembly, and uncoating, aswell as in capsid assembly. We have previously shown thatmutating Cys254, a residue located on a conserved loop nearthe calcium sites, can severely reduce the formation of infec-tious particles. That this defect is observed for substitution intocertain side chains (leucine, glycine, serine, and arginine) butnot into alanine is consistent with the local structure of Cys254,including the putative calcium-binding residues, being impor-tant for particle assembly and infectivity (17). Mutating certainacidic residues of the calcium-binding sites also affects theshape or stability of virus-like particles (VLPs) formed in Vp1-expressing insect cells (12).

In this study, we tested whether the seven putative calcium-binding acidic residues play a role in the viral life cycle. Wereport that five acidic side chains—Glu48 and Glu330 of site 1,Glu157 and Glu160 of site 2, and Glu216, shared by bothsites—contribute to the formation of infectious virions. Ouranalysis included mutation of the residues into lysine and ar-ginine as well as into alanine. We expected that the substitutedbasic side chains would either perturb the local structure of thesites and disrupt assembly or lead to the formation of poorly

* Corresponding author. Mailing address: Molecular Biology Insti-tute, 456 Boyer Hall, University of California at Los Angeles, 611 E.Charles E. Young Dr., Box 951570, Los Angeles, CA 90095-1570.Phone: (310) 825-3048. Fax: (310) 206-7286. E-mail: [email protected].

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infectious particles with new salt bridges in place of calciumatoms. Both categories of defects were observed. MutantE216R was ineffective in packaging into VLPs. Mutant E330Kassembled VLPs that cannot bind or enter the cell. MutantsE216K and E157A-E160A made VLPs that appear to prema-turely dissociate following cell entry, thus failing to target theviral DNA to the nucleus. Our findings support the idea thatcalcium coordination at both of the calcium sites is importantnot only for virion assembly but also for initiation of infectionat the stages of cell entry and nuclear entry of the viral genome.A model for calcium ion-dependent, sequential structural al-terations that are involved in the entry of the virion into thecell and into the cell nucleus is presented.

MATERIALS AND METHODS

Plasmid construction. Oligonucleotides for PCR, for linkers, and for sequenc-ing were synthesized by Genosys (The Woodlands, Tex.). All mutations createdwere confirmed by dideoxynucleotide sequencing analysis. DNA nucleotide se-quences are given in capital letters except for mutated SV40 nucleotides, whichare highlighted in lowercase. Relevant restriction sites are underlined.

Mutagenesis of the Vp1 calcium-binding acidic residues was performed withinpBS-based plasmids. pBS-Vp1-E46A, pBS-Vp1-E48A, and pBS-Vp1-E46A-E48A were made by inserting into pBS-Vp1 (17) an AccI-to-AflII linker in whichthe Glu46 codon, the Glu48 codon, or both codons, respectively, were replacedwith gct. pBS-Vp1-E157A, pBS-Vp1-E160A, and pBS-Vp1-E157A-E160A wereconstructed by inserting into pBS-Vp1 the XbaI-to-PstI PCR fragment generatedusing pBS-Vp1 as a template, the sense primer 5�-GGAGTAGCTCTAGAATGAAGATG-3�, and the antisense primer 5�-AACACACCCTGCAGwxCCAAAGGyzCCCCACCAACAGCAAAAAATG-3�, where wx and yz are CT and ag(E157A), ag and TT (E160A), or ag and ag (E157A- E160A), respectively.pBS-Vp1-E216A, pBS-Vp1-E216K, and pBS-Vp1-E216R were made by insert-ing into pBS-Vp1 the BstBI-to-MluI PCR fragment generated using the antisenseprimer 5�-TGCCCATCCACGCGTTGTGTtCTgCgGTTAATcAGGTCACTTAACAAAAAGGA-3� and the sense primer 5�-GGGTTCCTGATCCTTCGAAAAATxyzAACACTAGATATTTTGGAACCTACACAG-3�, where xyz repre-

sents Gcg (Ala), aAg (Lys), or cgc (Arg), respectively. pBS-Vp1-E329A, pBS-Vp1-E330A, pBS-Vp1-E330K, pBS-Vp1-E330R, pBS-Vp1-E329A-E330A, pBS-Vp1-E329A-E330K, and pBS-Vp1-E329A-E330R were made by inserting intopBS-WT (modified pBluescriptII containing the whole wild-type Vp1 codingregion) (12) respective PstI-to-XbaI fragments, which were generated via twoconsecutive rounds of PCR using pBS-Vp1 as the initial PCR template. The firstround of PCR used the following sense and antisense primer pairs: 5�-CCTCTCAAGTAGcGGAGGTTAGGGTTTATGAGGACACAG-3� and 5�-CTAACCTTACAGGAGAGTTCATCgCCTCCAATCC-3� (E329A); 5�-CCTCTCAAGTAGAGuvwGTTAGGGTTTATG-3� and 5�-CATAAACCCTAACxyzCTCTACTTGAGAGG-3� (E330A, E330K, and E330R), where uvw and xyz represent Gctand agC, aAG and CTt, or aga and tct, respectively; and 5�-CCTCTCAAGTAGcGuvwGTTAGGGTTTATGAGGACACAG-3� and 5�-CCTAACxyzCgCTACTTGAGAGGACATTCCAATC-3� (E329A-E330A, E329A-E330K, and E329A-E330R), where uvw and xyz represent the same codons as for the correspondingE330 single mutants. The second round of PCRs used the primer pair E160Q-Sense (12) and WT3�-XbaI (5�-CCGGTCTAGATCACTGCATTCTAGTTGTGGTTTG-3�). pBS-D345A, pBS-D345K, pBS-D345R, and pBS-D345N weremade by inserting into pBluescriptII the BamHI-to-PstI PCR fragment gener-ated using pUCVP1 (15) as a template, the anitsense primer M13 Reverse, andthe sense primers 5�-CTGGGGATCCAgccATGATAAGATAC-3� (D345A), 5�-CTGGGGATCCAaagATGATAAGATAC-3� (D345K), 5�-CTGGGGATCCAcgcATGATAAGATAC-3� (D345R), and D345N-Sense (12), respectively.

To make nonoverlapping SV40 plasmids (NO-pSV40) containing mutations inGlu46, Glu48, Glu157, Glu160, Glu216, Glue329, and Glu330, suitable restric-tion fragments of the Vp1 coding region from respective mutant pBS-Vp1s wereinserted into NO-pSV40-BSM. To make NO-pSV40-D345A, NO-pSV40-D345K, NO-pSV40-D345R, or NO-pSV40-D345N, pBS-D345A, pBS-D345K,pBS-D345R, or pBS-D345N was sequentially subjected to insertion with thefollowing: the PstI-to-KpnI fragment of NO-pSV40-BSM containing the SV40 oriand the N-terminal coding region for the large T antigen, the KpnI-to-EcoRIfragment of NO-pSV40-BSM encoding Vp2/3 and the N-terminal portion ofVp1, and the EcoRI-to-EcoRI fragment of pBS-WT encoding the rest of Vp1.NO-pSV40-E157Q-E160Q-D345N and NO-pSV40-E329Q-E330Q-D345N weremade by inserting the EcoRI-to-EcoRI Vp1-encoding fragments from pBS-mtEand pBS-mtF (12), respectively, into NO-SV40-D345N. All NO-SV40 viral ge-nomes were prepared from their respective NO-pSV40 plasmids by digestion

FIG. 1. Calcium-binding side chains of Vp1. At left is a ribbon diagram of an SV40 Vp1 pentamer, with one of the Vp1 monomers highlightedin white. An invading arm from a neighboring pentamer is shown in pink. Two blue circles represent the twin Ca2� sites. At right, an enlarged viewof the Ca2�-binding sites shows the coordination of the two Ca2� ions by glutamate and aspartate side chains from three Vp1 monomers on twodifferent pentamers. Glu157, Glu160, and Glu216 are from the highlighted monomer. Glu46 and Glu48 (E46 and E48; indicated by *) are froma neighboring monomer in the same pentamer. Glu329, Glu330, and Asp345 (E329, E330, and D345; indicated by **) are from a second pentamer,and the helix �C** is from a third pentamer. The left-hand image is adapted from reference 32 with permission of the publisher.

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with BamHI and recircularization with T4 DNA ligase as described previously(11).

Antibodies. Polyclonal rabbit anti-Vp1 serum (14) and affinity-purified poly-clonal rabbit anti-Vp3 immunoglobulin G (IgG) (21) have been described pre-viously. Monoclonal mouse anti-importin-� (anti-Rch-1) and anti-importin-�(anti-karyopherin-�) antibodies were obtained from Transduction Laboratories.Protein A-Sepharose beads were obtained from Amersham-Pharmacia.

Assays for viability, replication, and Vp1 production. Viability was determinedby plaque formation assays using serial dilutions of lysates prepared by freeze-thawing NO-SV40 DNA-transfected CV-1 monkey kidney cells at 72 h posttrans-fection, as described previously (17, 18). For select NO-SV40 mutants, plaqueswere lifted from the culture dish, resuspended in 0.1 to 1.0 ml of TD buffer, andlysed by freeze-thawing. A 5-�l aliquot of the plaque lysate was incubated with5 �l of trypsin-EDTA (Gibco-BRL) at 37°C for 15 min and at 99.9°C for 10 minto release viral DNA from virions in the lysate. A 1-�l volume of the trypsindigest was serially diluted over a 100-fold range with 10 mM Tris-HCl (pH 8.0),and 1 �l of each dilution was subjected to semiquantitative PCR (22) followed byagarose gel electrophoresis and ethidium bromide staining to determine theconcentration of the viral DNA. The amount of DNA in individual plaquesranged from 200 to 400 pg. Aliquots of the original plaque lysates containingequal amounts of viral DNA were then serially diluted and reassayed for plaqueformation. Throughout this assay, mutants such as E157A were found to retainthe characteristic plaque number and size relative to the wild type.

The extent of viral DNA replication was determined by gel quantitation ofDpnI-resistant viral DNA extracted from 1 106 transfected cells, and Vp1production was assessed by anti-Vp1 Western blot analysis of 5 104 transfectedcells, as described previously (17, 18).

DNase I treatment and packaging assay. The methods used for the DNase Itreatment and packaging assay are similar to those described previously (17),with minor modifications. CV-1 cells harvested at 72 h after transfection witheach NO-SV40 DNA were lysed by freeze-thawing in serum-free culture mediumor by sonication in hypotonic buffer at a concentration of 107 cells per ml. ForDNase I treatment, an aliquot of the lysate was cleared of cellular debris anddigested with 500 U of DNase I per ml at 37°C for 30 min. To determine theextent of viral DNA packaging, 20 �l of transfected lysate (corresponding to 2 105 cells) was treated with DNase I and the DNase I-resistant viral DNA wasextracted, digested with BamHI, and quantitated by Southern blot analysis fol-lowed by phosphorimaging. The value obtained was expressed relative to thatderived from a non-DNase I-treated but otherwise identically processed lysatealiquot, which was taken to be 100%.

VLP analysis and preparation by sucrose sedimentation. Sonicated lysatesprepared from 5 106 to 2 107 transfected cells were treated with DNase I andfractionated in a 5 to 32% continuous sucrose gradient, as described previously(17). For each of the 17 fractions, half was subjected to Southern blot analysis ofviral DNA content and 1/50 was subjected to Western blot analysis of Vp1content. For infection assays, the two or three peak VLP fractions (based on viralDNA content, usually between fractions 4 and 8 from the bottom) were pooledand used as a source of VLPs. The VLP concentration was determined byextracting viral DNA from an aliquot of these preparations and analyzing it bySouthern blot analysis alongside known amounts of viral DNA.

For electron microscopy analysis, 5 ml of sonicated lysate, prepared from 4 108 cells transfected with NO-SV40-E330K, was centrifuged at 10,000 g at 4°Cfor 10 min. The resulting supernatant was pelleted through a 20% sucrosecushion in 10 mM HEPES (pH 7.5) at 35,000 rpm at 4°C in an SW50 rotor. Thepellet was resuspended in 0.2 ml of fetal bovine serum supplemented with aprotease inhibitor cocktail (1 �g of aprotinin/ml, 1 �g of leupeptin/ml, 10 �g ofpepstatin/ml, 1 mM phenylmethylsulfonyl fluoride), further sedimented througha 5 to 32% continuous sucrose gradient as above, and separated into 30 fractions.The peak VLP fractions, determined from anti-Vp1 Western blot analysis of 2 �lfrom each fraction alongside known amounts of Vp1, were pooled and pelletedthrough a 20% sucrose cushion again. The pellet was resuspended in 0.1 ml of 10mM HEPES (pH 7.5)–10 mM NaCl–protease inhibitor cocktail. The VLP con-centration in this purified preparation was again quantitated by anti-Vp1 West-ern blot analysis.

Cell entry assay. The cell entry assay method was similar to that describedpreviously (22), with minor modifications. TC7 monkey kidney cells on 100-mmdishes were infected with approximately 1,000 VLPs per cell, incubated at 4°C for1 h and then at 37°C for 4 h, and harvested by either scraping or trypsintreatment. An aliquot of harvested cells was extracted for viral DNA, which wasdigested with BamHI and quantitated by Southern blot analysis followed byphosphorimaging. Another aliquot of the cells was assayed for Vp1 by anti-Vp1Western blot analysis.

Analysis of internalized VLPs by immunoprecipitation. The immunoprecipi-tation procedure was carried out essentially as described previously (22). Briefly,TC7 cells were infected with approximately 1,000 VLPs per cell, incubated at 4°Cfor 1 h and then at 37°C for 6 h, and harvested by trypsin treatment. An aliquotof the cytoplasmic fraction, prepared as the supernatant from the homogeniza-tion followed by low-speed centrifugation of 5 105 harvested cells, was reactedwith each specified antibody, and the immune complexes were collected byreaction with protein A beads alone or with protein A beads bound with rabbitanti-mouse IgG. After extensive washing, the immunoprecipitates were mixedwith NO-pSV40NcoI control DNA before the collective DNA was extracted,amplified by semiquantitative PCR, and quantitated by Southern blot analysis.

Electron microscopy. A 10-�l volume of purified virions (4 1010 particles/ml) or VLP preparation (2 1010 particles/ml) was allowed to adhere for 60 s oncarbon-coated copper grids that had been freshly glow-discharged (to make thegrid hydrophilic), and excess sample was removed by touching it with the edge ofa Whatman no. 4 filter paper wedge. While still wet, the grids were washed threeor four times successively with a drop of 1% aqueous uranyl acetate each time,and the final drop was left on the grids for 40 to 45 s. Excess stain was removedby the filter paper wedge, and the grids were allowed to air dry and stored underdesiccation until used for electron microscopy observation. The samples wereviewed under a Hitachi H6000 electron microscope, operating at 75,000 eV witha condenser aperture of 200 �m and an objective aperture of 50 �m, at anapparent magnification of 30,000.

RESULTS

Viability of single alanine substitution mutants of site 1 andsite 2 acidic residues. To test if the seven Vp1 acidic residuesof presumed calcium-binding sites 1 and 2 (Glu46, Glu48,Glu157, Glu160, Glu216, Glu330, and Asp345) play a role inthe viral infection cycle, these residues were individually mu-tated in the nonoverlapping, infectious viral genome, NO-SV40. Glu329 is not among the inferred metal-coordinatingresidues but was included in the analysis because of its possibleinfluence on the adjacent Glu330. The viability of the mutantswas determined by plaque formation assays using lysates ofmutant viral DNA-transfected cells. As shown in Table 1, sin-gle alanine substitutions had a range of effects on plaque for-mation efficiency. Mutants E46A, E216A, E329A, and D345Awere comparable to the wild type in terms of the infectioustiter (PFU per unit lysate) and the average size of the plaques.Mutants E157A, E160A, and E330A gave 50- to 80-fold-re-duced titers with concomitant reductions in plaque sizes. Mu-tant E48A showed a 70,000-fold reduction in PFU and a sim-ilar small-plaque phenotype. Thus, of all acidic side chainsmapped to the calcium-binding sites, that of Glu48 at site 1 isthe most critical to viability.

Effect on viability of basic side chain substitution at Glu216and Glu330 or Asp345. The minimal to moderate effect on theviability of alanine substitution at Glu216, Glu330, or Asp345(Table 1) suggests that the small, neutral alanine side chainsare largely compatible with the structural environment of theoriginal glutamic acids. On the other hand, a bulky, basic sidechain of lysine or arginine placed at residue 216, 330, or 345could exert a much greater effect on viability for two reasons.One is that the alignment of the calcium-binding residueswould be perturbed, compromising calcium binding and dimin-ishing the yield of particles. The other is that the basic sidechain might form salt bridges with other acidic side chains,thereby producing particles that do not bind calcium. Suchparticles may lack the potential to undergo the appropriateconformational changes or dissociation during subsequent in-fection.

Basic side chain substitutions at Glu216 and Glu330 proved

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highly detrimental to viability. Mutants E216K, E216R, andE330R had substantially (�300,000-fold) reduced PFUs and asmall-plaque phenotype, whereas mutant E330K failed to pro-duce any measurable infectious units (Table 1). In contrast,mutants D345K and D345R were as viable as their alanine-substituted counterpart. These results suggest that the Glu330side chain at site 1 and the Glu216 side chain which coordi-nates calcium ions at both sites 1 and 2 can influence the localstructure of the calcium-binding sites. The Asp345 side chainat site 2 is dispensable for productive infection.

Viability of double and triple mutants of site 1 and site 2acidic residues. The substantial viability of most single alaninemutants (Table 1) could indicate that their original acidic sidechains collectively coordinate the twin calcium ions and thatlosing individual side chains does not significantly compromisethe calcium coordination. We therefore combined alanine mu-tations for pairs of residues that are close in amino acid se-quence and structure (glutamic acids 46 and 48, 157 and 160,and 329 and 330). As seen in Table 1, each double mutantpreserved the small-plaque phenotype of the single-mutantmember with the smaller plaque size. For mutant E46A-E48A,the PFU value approximated that of the less viable single-mutant member, E48A. For mutants E157A-E160A andE329A-E330A, the PFU values reflected a multiplicative ac-cumulation of PFU reductions sustained by the single-mutantcounterparts. Thus, the reductions in infectious titer were atleast 200,000-fold for the three double mutants. Adding theE329A mutation to the nonviable or nearly nonviable basic-residue-substituted mutants, E330K and E330R, yielded twononviable double mutants, as might be expected (Table 1).

We also tested the viabilities of two triple glutamine-aspar-

agine substituted mutants, E157Q-E160Q-D345N and E329Q-E330Q-D345N, whose mutant Vp1s have been previously ex-pressed and found to form VLPs in insect cells (12). Therelative unimportance of the side chain identity at residue 345makes it reasonable to compare the viability of the triple mu-tants with that of their alanine-substituted double-mutantcounterparts, E157A-E160A and E329A-E330A. Whereas mu-tant E329Q-E330Q-D345N had a similar titer to mutantE329A-E330A, mutant E157Q-E160Q-D345N had a 3-log-unit-higher titer than did mutant E157A-E160A (Table 1).This difference could be due to the smaller disruptive effect ofreplacing the more structurally similar glutamines, rather thanalanines, by glutamic acids in mutant E157Q-E160Q-D345Nand is consistent with the greater-than-wild-type resistance ofE157Q-E160Q-D345N and E329Q-E330Q-D345N VLPs tocalcium-chelating agents (12).

The collective results show that five of the seven putativecalcium-binding residues of Vp1—Glu48 and Glu330 from site1, Glu157 and Glu160 from site 2, and Glu216 from bothsites—contribute to viral viability, presumably by jointly coor-dinating calcium ions at respective sites. Glu46 and Asp345, onthe other hand, are nonessential. Glu329 can apparently sub-stitute for Glu330 in the absence of the latter residue. The lowviability of basic-residue-substituted Glu216 and Glu330 mu-tants may be due to either the disruption of the calcium-binding site structure or the displacement of site 1 calcium.

Viral DNA replication and Vp1 production by mutants. Weproceeded to dissect the effects of calcium-binding-site muta-tions on various processes of the infection cycle. We began byexamining viral DNA replication and Vp1 production. Theamount of replicated viral DNA extracted from transfectedcells was similar for the wild type and all mutants, as judged bythe intensity of DpnI-resistant viral DNA bands following aga-rose gel electrophoresis and ethidium bromide staining (datanot shown). The amount of Vp1 in transfected cell lysates, asdetected by Western blot analysis, was also comparable for thewild type and the mutants, with the exception of mutantE157Q-E160Q-D345N (Fig. 2, lane T1). The low steady-statelevel of this mutant Vp1 was reproducible and was not inves-tigated further.

Packaging efficiency of mutants: DNase I resistance assay.We further tested whether the mutants could package viralDNA into particles, using a DNase I resistance assay. The

FIG. 2. Vp1 production by mutants. Cells transfected with wild-type (lanes W) or mutant (lanes a through T2 as designated in Table1) NO-SV40 DNA were subjected to anti-Vp1 Western blot analysis asdescribed in Materials and Methods.

TABLE 1. Viability of Vp1 calcium-binding site mutants

Label NO-SV40 Titer (PFU)a Plaque diam(mm)

Wild type (1.8 � 1.1) 108 6.6 � 2.2a E46A 2.0 108 5.5 � 0.7b E48A 2.5 103 1.9 � 1.2c E157A 2.3 106 2.0 � 0.3d E160A 3.6 106 1.6 � 0.4e E216A 2.8 107 5.8 � 1.3f E329A 5.0 107 4.8 � 1.3g E330A 2.4 106 1.2 � 0.4h D345A 3.5 107 6.4 � 2.3i E46A–E48A 6.2 102 1.7 � 0.9j E157A–E160A 1.0 103 1.2 � 0.3k E329A–E330A 6.6 102 1.7 � 0.5L E216K 3.1 102 2.0 � 0.9m E216R 5.0 102 1.7 � 0.8n E330K 0b

o E330R 7.5 1.0 � 0.0p D345K 4.0 107 6.5 � 2.0q D345R 4.0 107 5.9 � 2.2r E329A-E330K 0b

s E329A-E330R 0b

T1 E157Q-E160Q–D345N 1.2 106 2.1 � 0.7T2 E329Q-E330Q–D345N 3.5 102 2.2 � 0.6

a PFU contained in the lysate of one 60-mm dish of cells that were transfectedwith the respective NO-SV40 DNA. The value represents the average from fiveexperiments for the wild-type sample and the average from two experiments formutants a through T2.

b No plaques detected in one-eighth of the lysate harvested from one dish oftransfected cells.



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fraction of intracellular viral DNA that was packaged intosome protective structure should remain after the nucleasedigestion of the transfected lysate and was quantitated bySouthern blot analysis. As seen in Fig. 3, the percentages ofDNase I resistance for the mutants range from 59 to 5%. Aplot of these percentages against PFU (Table 1) in Fig. 4 showsdistinct clustering of the mutants into five groups, which welabel as wild-type-like, I, II, III, and IV.

The wild-type-like group had wild-type-like PFUs andplaque sizes and packaged viral DNA either at a wild-type levelof 60% � 9% (D345A and D345K) or in a somewhat lowerrange of 47 to 30% (E46A, E216A, E329A, and D345R) (Fig.3 and 4). Group I mutants also packaged in the 47 to 30%range, except that they had 50- to 150-fold-reduced PFUs anda small-plaque phenotype (E157A, E160A, E330A, andE157Q-E160Q-D345N) (Table 1; Fig. 3 and 4). Thus, group Imutants appeared to form reasonable quantities of particlesthat had lower infectivities than wild-type particles.

Group II mutants had substantially (70,000- to 600,000-fold)lower PFUs than did the wild type, along with small-plaquephenotypes, and packaged from 36 to 21% (E48A, E46A-E48A, E157A-E160A, E216K, E329A-E330A, and E329Q-E330Q-D345N) (Table 1; Fig. 3 and 4). The group III mutantE216R is in the same PFU range as group II, except that its

packaging level was extremely low, about 5% (Fig. 3 and 4).Thus, group II mutants appeared to make particles that wereeven more poorly infectious than group I particles whereas themutant E216R was significantly defective in packaging viralDNA into a nuclease-resistant structure.

Group IV mutants were nonviable (E330K, E329A-E330K,and E329A-E330R) or nearly so (E330R) but were surprisinglycapable of packaging into a DNase I-resistant form (24 to40%) (Fig. 3 and 4). Thus, group IV mutants are expected toassemble almost exclusively noninfectious particles.

The above results show that mutants of the calcium-bindingsite residues are, in general, defective to various extents in theassembly of virus particles. For mutants of the wild-type-likegroup and groups I and II, there is a reasonable correlationbetween packaging efficiency and viability. The group III mu-tant E216R has the largest defect in packaging. It indicates thatthe positioning of the Glu216 side chain relative to that of its�-carbon is important for packaging and virion assembly andsuggests that the local structure of Glu216 is susceptible todisturbance by the insertion of a bulky, positively charged sidechain. The packaging-competent group IV mutants are ex-pected to be defective in initiating new infections.

VLP formation by mutants. To confirm that the nuclease-protected fraction for the mutants represented VLPs, theDNase I-resistant materials of select mutants from groups II,III, and IV were analyzed by sedimentation in sucrose gradi-ents. As seen in Fig. 5A, the wild-type-transfected sample hada peak of viral DNA (in the form of nicked open circular,linear, and covalently closed circular species) in fractions 4 and5, coinciding with the sedimentation position for purified viri-ons, and Vp1 was found mostly in the same fractions.

For group II mutants E48A, E157A-E160A, and E216K, themutant DNA, along with a notable amount of the mutant Vp1,

FIG. 3. Extent of packaging by mutants. An aliquot of wild-type ormutant NO-SV40-transfected lysates was treated with DNase I, andthe remaining, nuclease-resistant DNA was quantitated and expressedas a percentage of total viral DNA in the aliquot, as described inMaterials and Methods. Each bar, with standard deviation marked,represents the average value from two to four experiments, as indi-cated on the right of the graph.

FIG. 4. DNase I-resistant packaging as a function of the number ofPFU of the mutants. Percent DNase I-resistant viral DNA from Fig. 3is plotted against PFU from Table 1 for NO-SV40 wild type (Wt) andmutants. Clusters of mutants are boxed and designated as one of thefollowing groups: Wt-like, I, II, III, and IV. The names of the mutantsare listed near each boxed group in descending order of DNase Iresistance. Site 1 mutants are plotted as dots; site 2 mutants are plottedas dots surrounded by a single circle; and mutants containing both site1 and site 2 mutations are plotted as dots surrounded by a doublecircle.



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sedimented mostly in fractions 4 through 6, consistent with theformation of VLPs (Fig. 5B). The viral DNA found in fractions1 through 3 (near the bottom of the gradient), together withsome Vp1, may represent particles of somewhat differentshape or size from wild-type virions or could be aggregatedVLPs. The substantial amount of Vp1 that was found in frac-tions 7 through 9 without much cosedimented viral DNA mayrepresent mostly capsid protein aggregates or Vp1 assemblyintermediates.

The group III mutant E216R had a strikingly different sed-imentation profile (Fig. 5C). Viral DNA was nearly absent infractions 2 through 11, whereas Vp1 was distributed broadlythroughout the gradient. This pattern suggests that mutantE216R formed capsid protein aggregates, packaging interme-diates, and even VLPs, but most of the viral DNAs in thesecomplexes was susceptible to DNase I, consistent with a lowDNase I resistance level of only 5% (Fig. 3). The elevatedamount of mutant DNA in fraction 17 was most probably dueto the overloading of the mutant sample onto the sucrosegradient relative to other samples, but it could also signify thatsome of the DNA that was protected during the nucleasedigestion was released from the unstable structures duringsedimentation.

For group IV mutants E330K and E330R, the cofraction-

ation of a majority of the viral DNA with Vp1 between frac-tions 3 and 9 suggests the formation of VLPs (Fig. 5D). Thedistribution of viral DNA and Vp1 is shifted toward the right(top of the gradient) relative to that of the wild type (Fig. 5A)and may indicate a difference in the size or shape of mutantE330K and E330R VLPs compared to wild-type virions. How-ever, electron microscopy revealed the E330K VLP to be al-most identical in appearance and diameter (47.9 � 3.4 nm [Fig.6A]) to wild-type SV40 particles (47.8 � 2.7 nm [Fig. 6B]).

The collective results showed that at least some of theDNase I-resistant materials for group II and IV mutants rep-resented VLPs, supporting the idea that the poor plaque-form-ing ability of these mutants is due to a block in reinfectionprocesses.

Cell attachment and entry by mutant E157A-E160A, E216K,and E330K VLPs. We further analyzed two group II mutants,E157A-E160A and E216K, and one group IV mutant, E330K,to test the ability of their VLPs to initiate new infections. Twosources of VLPs were assayed: peak VLP fractions from thesucrose gradients above (Fig. 7A and B) and crude transfectedcell lysates (Fig. 7C). The former type of VLP preparations formutants E157A-E160A, E216K, and E330K contained allthree capsid proteins, Vp1, Vp2, and Vp3, at similar ratios tothose observed for wild-type particles (Fig. 7D). Therefore, allthree mutant VLPs had a wild-type-like capsid protein com-position.

We first asked whether the VLPs could attach to cells andbecome internalized following infection. To distinguish cellsurface-bound and internalized VLPs, cells were harvested at4 h postinfection by two different methods: by scraping, forquantitating the VLPs that had become cell associated eitherat the surface or in the interior, and by trypsin treatment, forquantitating only the VLPs that had been internalized. Whenpeak VLP fractions were used for the infections, mutantE157A-E160A and E216K VLPs attached to cells just as wellas wild-type particles did, as judged by the amount of cell-associated viral DNA (Fig. 7A) and Vp1 (Fig. 7B). Theamounts of their internalized viral DNAs were also compara-ble to that of the wild-type sample (Fig. 7A and B). Infectionwith the crude lysates for mutants E157A-E160A and E216Kgave essentially the same results (Fig. 7C). In contrast, little orno viral DNA or Vp1 became cell associated or internalizedfollowing infection with mutant E330K VLPs (Fig. 7A and B).However, cell-associated mutant E330K DNA was detectedwhen crude mutant E330K lysate was used for infection (Fig.

FIG. 5. VLP formation by mutants. DNase I-treated, transfectedcell lysates for wild-type (A) and mutant E48A (B), E216R (C), andE330K (D) NO-SV40s were sedimented through 5 to 32% sucrosegradients, and the 17 fractions collected from the bottom of the gra-dients were analyzed for viral DNA (top half of each panel) or for Vp1(bottom half of each panel), as described in Materials and Methods.The amount of lysates sedimented was equivalent to 1 107 (A), 2 107 (B), 4 107 (C), and 1.5 107 (D) transfected cells. A solidarrowhead above the junction of fractions 4 and 5 indicates the posi-tion for purified virions sedimented in a parallel gradient. Three barsto the right of each viral DNA half panel mark the positions (from topto bottom) of the open circular, linear, and closed circular forms of theviral DNA. The profiles of mutants E157A-E160A and E216K resem-ble that of mutant E48A (B), and the profile of mutant E330R resem-bles that of mutant E330K (D).

FIG. 6. Electron micrograph of mutant E330K VLPs. MutantE330K VLPs (A) and wild-type SV40 virions (B) were visualized byelectron microscopy together with polystyrene spheres (diameter, 91nm) as an internal particle standard. Bars, 10 nm. The diameter of theSV40 virion is 47.8 � 2.7 nm (n � 337), and that of the E330K VLP is47.9 � 3.4 nm (n � 257).



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7C), suggesting that non-particle-derived viral DNA could en-ter cells together with viral proteins in the lysate. These resultsindicate that mutant E157A-E160A and E216K particles arecapable of cell attachment and entry whereas mutant E330Kparticles are defective in cell attachment.

T-antigen expression following infection of mutant E157A-E160A, E216K, and E330K VLPs. Since mutant E157A-E160Aand E216K VLPs could enter the cell, we then asked if theycould deliver their viral DNA to the nucleus and initiate viralgene expression. At 20 h postinfection, 46.2% � 11.7% of thewild-type-particle-infected cells became positive for the large Tantigen (Fig. 7E). However, only a small portion of E157A-E160A (0.43% � 0.05%) or E216K (0.065% � 0.02%) VLP-infected cells was T-antigen positive (Fig. 7E). Not surpris-ingly, no T-antigen-positive cells resulted from E330K VLPinfection (Fig. 7E), consistent with the nonviability and defectin cell attachment of this mutant. These results show thatinternalized mutant E157A-E160A and E216K VLPs areblocked at some event preceding T-antigen expression, such asdelivery of the viral genome to the nucleus.

State of internalized mutant E157A-E160A and E216KVLPs in the cytoplasm. We have previously shown that onceinternalized in the cytoplasm, the SV40 particle undergoessome alteration in capsid structure such that the nuclear local-

ization signals (NLSs) of the virion, the internal Vp3 NLSs,become exposed for recognition by cellular importins (22).Thus, the defect of mutant E157A-E160A and E216K VLPscould be either due to an inability to undergo this criticalalteration or due to a premature disassembly of the VLPs.These two possibilities can be distinguished by analyzing thecoimmunoprecipitation of internalized, VLP-associated viralDNA with anti-capsid protein antibodies. Poor viral DNA co-precipitation with anti-Vp3 but normal coprecipitation withanti-Vp1 would suggest failure to undergo a structural alter-ation, whereas reduced coprecipitation with both anti-Vp1 andanti-Vp3 would suggest premature disassembly. In addition,immunoprecipitation with anti-importin antibody would revealwhether the internalized VLPs could be recognized by theimportins.

Cytoplasmic lysates prepared from wild-type-particle- ormutant VLP-infected cells were reacted with individual anti-bodies, and coimmunoprecipitated viral DNA was detected viasemiquantitative PCR. Control antibody immunoprecipitationyielded no detectable viral DNA, as expected (Fig. 8, lane 2).Although similar amounts of viral DNA were present in wild-type and mutant VLP-infected cytoplasms (lane 1), much lessof the mutant viral DNA than wild-type viral DNA was coim-munoprecipitated by either anti-Vp1 or anti-Vp3 (lanes 3 and

FIG. 7. VLP composition and infection processes for mutants E157A-E160A, E216K, and E330K. (A to C) Cell attachment and internalizationby mutant VLPs. Cells grown on 100-mm dishes were infected with 1,000 particles per cell for 4 h, with peak sucrose fractions containing wild-typeparticles or mutant E157A-E160A (E157/160A), E216K, or E330K VLPs. Cells in one dish were harvested by scraping (representing “Cellassociated” viral DNA or Vp1), those in another dish were harvested by trypsin treatment (“Internalized” viral DNA or Vp1). (A) Viral DNA wasextracted from one-eighth of the input particles (“Input”) or from half of the respectively harvested infected cells, linearized, and detected bySouthern blot analysis. (B) One-hundredth of the input particles (“Input”) and one-twentieth of each type of harvested cells were analyzed for Vp1Western blot analysis. (C) Cells were infected with transfected cell lysates instead of peak VLP fractions and processed for viral DNA detectionby Southern blot analysis, as above. (D) Capsid protein composition of mutant VLPs. Peak sucrose fractions corresponding to wild-type particlesor various mutant VLPs were analyzed for viral proteins by Western blotting using anti-Vp1 (top panel) or anti-Vp3 (bottom panel) antibody.Bands corresponding to Vp1, Vp2, and Vp3 are marked. (E) T-antigen expression in mutant VLP-infected cells. Cells grown on coverslips wereinfected for 20 h with peak sucrose fractions corresponding to wild-type particles or various mutant VLPs. The number of T-antigen-positive cellswas determined by immunofluorescence microscopy. Each bar, with marked standard deviation, represents the average from three sets ofexperiments, in each of which approximately 2,000 cells were counted.



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4). For example, whereas 52.4% of the internalized wild-typeDNA was detected in association with Vp1, only 2.8 and 14.6%of the internalized mutant E157A-E160A and E216K viralDNAs, respectively, were detected. Not surprisingly, the mu-tant DNAs were also poorly coimmunoprecipitated by mixedanti-importin-� and -� antibodies (lane 5). These results areconsistent with the interpretation that mutant E157A-E160Aand E216K particles fail to sustain adequate structural integ-rity in the infected cytoplasm and hence cannot mediate thenuclear transport of the viral DNAs via interaction with theimportins.

DISCUSSION

In the present study, we analyzed SV40 mutants in which theVp1 side chains mapped by crystallographic studies to the twocalcium-binding sites (Fig. 1) (19, 32) were mutated. A DNAtransfection system and assays that probe for particle forma-tion and the effect of particle infection in the next host haveallowed us to dissect the defects of the mutants at variousstages of the SV40 life cycle (e.g., particle formation and par-ticle entry into the cell and nucleus). We show that a subset ofthose seven acidic side chains—Glu48 and Glu330 of site 1,Glu157 and Glu160 of site 2, and Glu216, which is a part ofboth sites—are important for SV40 infection. Our in vivo studyprovides the first evidence for the involvement of calcium ion-mediated interactions not only in virion assembly but also incell entry and delivery of the viral genome into the nucleus.

Our data suggest that Glu48, Glu330, Glu157, Glu160, and

Glu216 are the primary side chains that coordinate calcium.We have also found Glu329, which is not among the predictedcalcium-binding residues, to be important in the absence of theadjacent Glu330. All six residues are well conserved in thepolyomavirus family. As for the two acidic residues that wefound to be unimportant for SV40 infection, Glu46 is not wellconserved and Asp345 does not assume an ordered structurefor �-type Vp1 monomers on the capsid (32). Thus, Glu330 ofsite 1 could be the only residue on the invading C-terminal armthat normally contacts calcium ions. This C-terminal contribu-tion, however, is essential for calcium coordination, as sug-gested by our analysis below. It concurs with the predictionthat calcium would not bind to both sites 1 and 2 unless theinvading arm is present in the pentamer core (32).

For SV40 and the closely related murine polyomavirus andhuman JC virus, the importance of calcium binding in virionstability and assembly is well documented. Experimental evi-dence includes the in vitro dissociation and/or reassemblyproperties of virions (4, 5, 6), of VLPs from bacterially ex-pressed Vp1 (10, 29), and of VLPs formed in insect cells (12)and yeast cells (8). The observation that VLPs unexpectedlyform in the cytoplasm when polyomavirus Vp1-expressing in-sect cells are treated with a calcium ionophore (20) impliesthat VLP formation is controlled by the availability of calciumions in specific intracellular compartments. These studies,however, do not address the possible role of the metal ions inthe earlier phase of infection, during virion entry into the celland into the nucleus.

The SV40 virion is presumed to be a carrier of calcium ions.The metal ions, jointly coordinated by mostly acidic Vp1 res-idues, map near the base of Vp1 pentamers at a stoichiometryof two ions per Vp1 chain (Fig. 9A and B). If all availablebinding sites on the capsid are filled, the total 720 metal ionswould amount to 5 to 10 �M, or 100 times the estimatedaverage level of free intracellular calcium, 0.1 �M. It is notknown whether all available binding sites are occupied in SV40or how the metal ions can be extensively accumulated duringparticle formation in the nucleus. Later, such as during theinfection of a new host cell, release of the calcium ions fromthe particle may occur at different stages in a controlledmannner. Although the number of calcium ions bound pervirion or mutant VLP is not known, the results presented heresuggest that the calcium ions carried by an SV40 virion partic-ipate in multiple steps of the infection cycle.

Model. Calcium coordination links the C-terminal arms ofneighboring pentamers with the core of each pentamer,thereby helping to stabilize the virion particle by reinforcinginterpentamer interactions (19, 32). Our working model for therole of calcium binding in SV40 infection is as follows. Thecapsid structure held together by calcium ions has the potentialto change after loss or gain of the ions at some locations of thecapsid. Consequently, appropriate structural alterations canoccur during different steps of the infection cycle. Duringvirion assembly in the nucleus, calcium ion addition drives theformation of the capsid structure from Vp1 pentamers, to-gether with Vp2 and Vp3 and the viral minichromosome, andstabilizes the particle structure. During cell attachment, thevirion undergoes a conformational change, perhaps via calciumion release, to achieve receptor binding. Such a structuralalteration can also underlie internalization via the caveolar

FIG. 8. Immunoprecipitation analysis of internalized particles. Thecytoplasmic fraction was prepared from cells infected with wild-typeparticles (top panel) mutant E157A-E160A particles (middle panel),or mutant E216K particles (bottom panel) as described in the legendto Fig. 7A. Aliquots of the cytoplasmic fractions were reacted withanti-mouse IgG (lane 2, Cont), anti-Vp1 (lane 3, Vp1), anti-Vp3 (lane4, Vp3), and mixed anti-importin-� and anti-importin-� antibodies(lane 5, Imps), as described in Materials and Methods. The coimmu-noprecipitated (IP) viral DNA was purified from the immune com-plexes in the presence of a fixed amount of control DNA and detectedvia semiquantitative PCR. The expected amplification product of theNO-SV40 genome is 2.2 kbp (arrow, viral DNA), and that of thestandard DNA is 1.7 kbp (arrowhead, cont. DNA). For Input (lane 1),one-fifth as much cytoplasmic lysate as the amount used for eachimmunoprecipitation was purified for viral DNA in the presence of thecontrol DNA and detected by PCR.



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pathway (1, 24). Furthermore, nuclear entry of the viral DNAmay depend on a calcium-mediated structural alteration of thevirion, without dissociating the capsid proteins from the DNA,so that the NLSs of the virion, the Vp3 NLSs, are exposed andrecognized by the importins (22). In short, calcium ion coor-dination can contribute to different processes of the SV40infection cycle by imparting stability to the capsid structurewhile allowing for the flexibility of that structure to alter oncue. That is, biologically significant conformational change ap-pears to occur at early stages of infection. This model is con-sistent with the results we report here.

Regulation of virion conformation by calcium ions, alsothought to occur in the early steps of infection, has beendocumented for several T�3 icosahedral plant viruses. Forexample, when divalent cations are removed and the pH israised above neutrality, tomato bushy stunt virus (16, 27) andcowpea chlorotic mottle virus (3, 30) undergo swelling, a re-versible change in capsid structure that results from the loss ofthe positively charged metal chelated at interfaces of capsidprotein subunits. We envision that controlled or local releaseof calcium ions from the virion under certain extra- or intra-cellular conditions would also trigger a defined conformationchange in SV40, unlike the complete disruption of virions orVLPs after treatment with divalent cation-chelating and -re-ducing agents in vitro (4, 6, 29).

Viability of mutants. Viability, measured as the number ofPFU detected in each viral DNA-transfected cell lysate and thesize of plaques, allows us to rate the impact of the calcium-binding site mutations on the overall ability to make infectiousparticles. The rating reveals that five of the seven putativecalcium-binding residues (Glu48, Glu157, Glu160, Glu216, andGlu330), plus Glu329, are important, because mutating themsingly or in combination produced the lowest infectious titersobserved in this study, along with a small-plaque phenotype(Table 1 and Fig. 4, group II mutants E48A, E157A-E160A,E329A-E330A, and E216K and group III mutant E216R), orproduced no detectable infectious titer at all (group IV mu-tants E330K and E330R). The defects of these mutants lie inpackaging and particle formation and/or in stages of reinfec-tion (see below).

Glu46 and Asp345, which can be mutated while maintaininga wild-type-like viability, are likely to play only a minor roleduring infection. It may be that Glu46 and Asp345 do notactually contact calcium ions, contrary to structural prediction(32). The equivalent viability of mutants D345A, D345K, andD345R suggests that the Asp345 side chain does not contributeto the local structure of the capsid; i.e., the region of theC-terminal arm encompassing residue 345 may be somewhatflexible. An alternative possibility, that the infectious D345K orD345R particles contain rigid salt bridges between the basic

FIG. 9. Location and function of calcium-binding sites. (A and B) Positions of Glu330 and Glu160 in SV40. The �-carbon positions for Glu330(site 1) and Glu160 (site 2) are marked in red and yellow, respectively, in a cross section that includes parts of pentamers (A) as viewed from thetop, with a cluster of seven pentamers (a hexavalent one surrounded by six others) highlighted by enclosure in a white circle (B). (C) Proposedfunctions of Vp1 calcium-binding amino acids in infection: cell attachment through viral DNA nuclear import. The model shows an SV40 virionundergoing cell attachment through MHC class I (“Y”), internalization via caveolae (crisscrossing double lines) on the plasma membrane (straightdouble lines), structural alteration in the cytoplasm to expose the Vp3 NLS (Vp3-NLS), recognition by importin-� and importin-�, and nuclearentry through a nuclear pore complex embedded in the nuclear envelope. Glu330 is important for attachment to the cell. The Glu157-Glu160 pairand Glu216 function in the controlled and selective structural alteration to achieve a nuclear import-competent state. Panel C modified fromreferences 13 and 23, with permission of the publishers.



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side chain at residue 345 and the acidic side chain at residue157, 160, or 216, is unlikely in the light of the poor infectivityof the analogous mutants E330K and E330R (Table 1).

Packaging and VLP assembly. The extent of packaging intoDNase I-resistant structures in transfected cells gave us a mea-sure of the ability of the mutants to assemble into VLPs. Thatthe wild-type-like mutant D345R packaged 31% of the DNA,or roughly half the wild-type level (Fig. 3 and 4), suggests thatviability is not greatly affected by this level of packaging reduc-tion per se. In fact, only 6 of 21 mutants analyzed packagedbelow 30% (E48A, E329A-E330A, E157A-E160A, E216K,E216R, and E330R). Significantly, two of the six are doublymutated and three contain a basic-residue substitution. Con-ceivably, losing one calcium-binding side chain may not greatlyaffect the calcium coordination affinity at a particular site be-cause of the presence of other side chains, but losing two couldhave a significant effect. If an acidic residue were replaced witharginine, the basic, bulky side chain could disrupt the structureof one or both calcium sites, hence disrupting calcium ionbinding and possibly other interactions normally made by thecalcium site residues with other residues. These observationssupport the idea that calcium coordination at two sites is adriving force in virion assembly.

Among the mutants that packaged least efficiently, all butmutant E216R made particles that sedimented in similar po-sitions to wild-type particles, although group II mutants appearto form some particle aggregates or assembly intermediatesbesides VLPs (Fig. 5). The VLPs of the group IV mutantE330K are indistinguishable from wild-type virions (Fig. 6).Mutant E157A-E160A, E216K, and E330K VLPs also hadnormal capsid protein compositions (Fig. 7D). Therefore, theprimary reason for the low viabilities of group II and IV mu-tants is the low infectivity of the VLPs, which are blocked atsome stage of reinfection.

The single group III member, mutant E216R, packaged viralDNA poorly and was poorly viable. Its sucrose gradient profilesuggests that it assembled the mostly capsid protein aggregatesor nucleoprotein complexes whose viral DNA was accessible toDNase I (Fig. 5C). The detrimental effect on assembly of thearginine substitution at residue 216 may stem from the uniquelocation of Glu216, which links calcium ions at both sites 1 and2 and might have an influence on the local capsid structure.The bulky basic side chain of arginine can perturb both sites 1and 2, possibly abolishing the coordination of any calcium ions.Although the Glu216 side chain may normally make contactswith both ions, substitution by the small, neutral side-chainalanine (as in the wild-type-like mutant E216A) is well toler-ated (Table 1). Therefore, the positioning of the Glu216 sidechain relative to that of the �-carbon may influence minichro-mosome packaging and virion assembly. The phenotype ofmutant E216R is reminiscent of the poorly packaging andpoorly viable Vp1 cysteine mutant, C254L (17). Cys254, resid-ing on the short, highly conserved GH loop near the calcium-binding sites, also appears to be in a highly structurally sensi-tive local environment that is perturbed by certain substitutedside chains at that residue (17).

Virus attachment to cells, Glu330. The phenotype of groupIV mutants, including E330K, E330R, E329A-E330K, andE329A-E330R, is intriguing: VLPs with the same morphologyand composition as virions were effectively made but were largely

noninfectious (Fig. 3, 5, and 6A). In the mutant E330K particle,the substituted lysine side chain at residue 330 may be positionedsuch that it actually fits in the space normally occupied by theGlu330 side chain and the calcium ion. The lysine side chainprobably makes salt bridges with other acidic side chains of site 1,Glu48 and Glu216, thus permanently excluding calcium from site1. This new arrangement would allow E330K VLPs to assembleeffectively while chelating only half of the calcium ions chelated bywild-type particles. Future in vitro assays could test whether theseVLPs are more stable than wild-type virions in the presence ofcalcium-chelating agents.

Mutant E330K VLPs turned out to be defective in adsorbingto cells (Fig. 7A and B). Conceivably, the salt bridges in placeof calcium ion at site 1 could have prevented the E330K VLPfrom undergoing the calcium-dependent structural shift nec-essary for binding to the cell receptor, the major histocompat-ibility complex (MHC) class I molecule (7, 31), leading to theadsorption defect. If the structurally unresponsive mutant par-ticle failed to expose the myristylated N terminus of Vp2,which is thought to insert into the plasma membrane to facil-itate virion entry (28, 33), this could contribute to the adsorp-tion defect. Glu330 residues, marked in red in Fig. 9A and B,lie on the C-terminal arms of neighboring pentamers and aresituated near the base of the pentamers. The results obtainedwith mutant E330K suggest a role of the calcium ions in sup-porting the dynamic alteration of capsid structure that takesplace at the pentamer base during cell attachment (Fig. 9C).For example, the virion may release site 1 calciums to achievethe conformation for the MHC class I interaction. This sce-nario is part of our working model that cell attachment re-quires a particle structural flexibility conferred by the coordi-nation of calcium ions. Analyzing the structural differencebetween the mutant E330K VLP and the wild-type virion couldhelp delineate the virus site or epitope responsible for attach-ment to MHC class I.

Nuclear import of infecting virus, calcium site 2. For the twogroup II mutants, E157A-E160A and E216K, the particlescould attach to and enter cells (Fig. 7A and B) but wereineffective at delivering their viral genomes into the nucleus, asseen by the low incidence of T-antigen expression (Fig. 7E).Little internalized DNA of the mutants was in complex withthe capsid proteins or with the importins (Fig. 8). It is strikingthat not only Vp1, but also Vp2 and Vp3, have dissociatedfrom the DNAs. Since the structural proteins promote thenuclear entry of SV40 DNA (21) and since the Vp3 NLS isnecessary for virion DNA entry into the nucleus (22), it is notsurprising that mutant E157A-E160A and E216K DNAs, withfew associated structural proteins, were not recognized by theimportins or targeted to the nucleus. Thus, the Glu157- Glu160and Glu216 mutations appear to weaken the affinity of calciumbinding at site 2 sufficiently to cause a premature dissociationof the mutant particles once internalized into the cell, thoughthe VLPs still assembled in the nucleus of the mutant DNA-transfected cells (Fig. 3 and 5). It is not known what conditionsin the caveolae or the cytoplasm or which aspect of the attach-ment and entry process might have compromised the integrityof the mutant VLPs. The infectivity of mutant E157A-E160Aand E216K particles is more than 1,000-fold lower than that ofVp3 NLS-null mutant particles, also blocked in viral genomenuclear targeting (22). This difference suggests that the pre-



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mature dissociation of particles is an even greater obstacle toviral DNA nuclear entry than is the loss of functional Vp3NLSs in the particles.

Our study thus shows that effective nuclear targeting of theviral DNA cannot be achieved when the particle dissociates.We propose that a selective series of conformational changesin the particle is necessary for the exposure of Vp3 NLSs tocytoplasmic importins (Fig. 9C). Such a model is consistentwith the result of a preliminary analysis in which the SV40-infected cytoplasm was fractionated by sucrose gradient sedi-mentation and then reacted with anti-importin antibodies.Whereas a majority of the antibody-precipitable viral DNAsedimented near the viral chromatin position, some was foundin virion fractions (A. Nakanishi, unpublished data). Still un-known is the extent of the structural alteration or the compo-sition of the internalized particle. We seek clues to the possiblelocation of a structural alteration by building an atomic modelof the SV40 Vp1-Vp3 complex based on the structure of thehomologous mouse polyomavirus Vp1-Vp2 complex (9). Fromthis model, the distance between the Vp3 Leu178 side chain,which is within the defined Vp1-contacting structure, and thecalcium-binding Vp1 Glu48 side chain, is 13 A. The Vp3 NLS,21 residues downstream from Leu178, is inferred to be nearthe pentamer base. Since Glu157, Glu160, and Glu216 are allsituated in calcium site 2 near the base of pentamers, a struc-tural change in the wild-type particle could be brought aboutthrough a depletion of calcium ions from site 2 to permit theexposure of the Vp3 NLSs. Such a particle would then becompetent for importin recognition (Fig. 9C). Potential calci-um-binding-site mutants that have a permanent bond in placeof site 2 calcium, analogous to the site 1 mutant E330K, wouldhelp test this scenario. Again, calcium coordination by theSV40 particle is likely to be a key to the structural flexibilitynecessary for the processes involved in initiating a new infec-tion cycle.

In summary, we have shown that certain acidic side chainsfrom the two structurally deduced calcium-binding sites con-tribute to the formation of infectious SV40. The potential tochelate calcium ions at both sites is important for virion as-sembly in the nucleus. The cell attachment defect of the site 1mutant, E330K, and the DNA nuclear import defect of the site2 mutants, E157A-E160A and E216K, due to premature par-ticle dissociation in the cytoplasm, imply that local structuralalterations occurring at the base of Vp1 pentamers are impor-tant in the initiation of an infection cycle. Thus, the properformation of the virion structure and the ability of the virion toundergo structural alterations on cue at the cell surface or inthe cytoplasm are essential for infectivity and are probablyregulated by calcium coordination by residues at the two cal-cium sites (Fig. 9C). These structural alterations set the stagefor the nuclear entry of the infecting SV40 through interactionwith importin complex and for launching the viral gene expres-sion program.

ACKNOWLEDGMENTS

P.P.L., A.N, and M.A.T. contributed equally to this work.We thank Akiko Nakamura for assistance in performing plaque

assays and Walter Eckhart for critical reading of the manuscript.This work was supported by awards to H.K. from the National

Institutes of Health (Public Health Service grant CA50574) and theUniversity of California, Los Angeles (UCLA) Academic Senate.

P.P.L. was supported in part by an award from the UCLA JonssonComprehensive Cancer Center. The sabbatical stay of H.K. at TokyoInstitute of Technology, Yokohama, Japan, was supported in part byan award to H.H. from New Energy and Industrial Technology De-velopment Organization (NEDO).

REFERENCES

1. Anderson, H. A., Y. Chen, and L. C. Norkin. 1996. Bound simian virus 40translocates to caveolin-enriched membrane domains, and its entry is inhib-ited by drugs that selectively disrupt caveolae. Mol. Biol. Cell 7:1825–1834.

2. Baker, T. S., J. Drak, and M. Bina. 1988. Reconstruction of the three-dimensional structure of simian virus 40 and visualization of the chromatincore. Proc. Natl. Acad. Sci. USA 85:422–426.

3. Bancroft, J. B., G. J. Hills, and R. Markman. 1967. A study of the selfassembly process in a small spherical virus formation of organized structurefrom protein subunits in vitro. Virology 31:354–379.

4. Brady, J. N., V. D. Winston, and R. A. Consigli. 1977. Dissociation ofpolyoma virus by the chelation of calcium ions found associated with purifiedvirions. J. Virol. 23:717–724.

5. Brady, J. N., J. D. Kendall, and R. A. Consigli. 1979. In vitro reassembly ofinfectious polyma virions. J. Virol. 32:640–647.

6. Brady, J. N., C. Lavialle, and N. P. Salzman. 1980. Efficient transcription ofa compact nucleoprotein complex isolated from purified simian virus 40virions. J. Virol. 35:371–381.

7. Breau, W. C., W. J. Atwood, and L. C. Norkin. 1992. Class I major histo-compatibility proteins are an essential component of the simian virus 40receptor. J. Virol. 66:2037–2045.

8. Chen, P.-L., M. Wang, W.-C. Ou, C.-K. Lii, L.-S. Chen, and D. Chang. 2001.Disulfide bonds stabilize JC virus capsid-like structure by protecting calciumions from chelation. FEBS Lett. 500:109–113.

9. Chen, X. S., T. Stehle, and S. C. Harrison. 1998. Interaction of polyomavirusinternal protein VP2 with the major capsid protein VP1 and implications forparticipation of VP2 in viral entry. EMBO J. 17:3233–3240.

10. Garcea, R. L., D. M. Salunke, and D. L. Caspar. 1987. Site-directed mutationaffecting polyomavirus capsid self-assembly in vitro. Nature 329:86–87.

11. Ishii, N., A. Nakanishi, M. Yamada, M. H. Macalalad, and H. Kasamatsu.1994. Functional complementation of nuclear targeting-defective mutants ofsimian virus 40 structural proteins. J. Virol. 68:8209–8216.

12. Ishizu, K.-I., J. Watanabe, S.-I. Han, S.-N. Kanesashi, M. Hoque, H. Yajima,K. Kataoka, and H. Handa. 2001. Roles of disulfide linkage and calciumion-mediated interactions in assembly and disassembly of virus-like particlescomposed of simian virus 40 Vp1 capsid protein. J. Virol. 75:61–72.

13. Kasamatsu, H., and A. Nakanishi. 1998. How do animal DNA viruses get tothe nucleus? Annu. Rev. Microbiol. 52:627–686.

14. Kasamatsu, H., and A. Nehorayan. 1979. Intracellular localization of viralpolypeptides during simian virus 40 infection. J. Virol. 32:648–660.

15. Kosukegawa, A., F. Arisaka, M. Takayama, H. Yajima, A. Kaidow, and H.Handa. 1996. Purification and characterization of virus-like particles andpentamers produced by the expression of SV40 capsid proteins in insectcells. Biochim. Biophys. Acta 1290:37–45.

16. Kruse, J., K. M. Kruse, J. Witz, C. Chauvin, B. Jacrot, and A. Tardieu. 1982.Divalent ion-dependent reversible swelling of tomato bushy stunt virus andorganization of the expanded virion. J. Mol. Biol. 162:393–414.

17. Li, P. P., A. Nakanishi, M. A. Tran, A. M. Salazar, R. C. Liddington, and H.Kasamatsu. 2000. Role of simian virus 40 Vp1 cysteines in virion infectivity.J. Virol. 74:11388–11393.

18. Li, P. P., A. Nakanishi, D. Shum, P. C. Sun, A. M. Salazar, C. F. Fernandez,S. W. Chan, and H. Kasamatsu. 2001. Simian virus 40 Vp1 DNA-bindingdomain is functionally separable from the overlapping nuclear localizationsignal and is required for effective virion formation and full viability. J. Virol.75:7321–7329.

19. Liddington, R. C., Y. Yan, J. Moulai, R. Sahli, T. L. Benjamin, and S. C.Harrison. 1991. Structure of simian virus 40 at 3.8-A resolution. Nature(London) 354:278–284.

20. Montross, L., S. Watkins, R. B. Moreland, H. Mamon, D. L. D. Caspar, andR. Garcea. 1991. Nuclealr assembly of polyomavirus capsids in insect cellsexpressing the major capsid protein Vp1. J. Virol. 65:4991–4998.

21. Nakanishi, A., J. Clever, M. Yamada, P. P. Li, and H. Kasamatsu. 1996.Association with capsid proteins promotes nuclear targeting of simian virus40 DNA. Proc. Natl. Acad. Sci. USA 93:96–100.

22. Nakanishi, A., D. Shum, H. Morioka, E. Otsuka, and H. Kasamatsu. 2002.Interaction of the Vp3 nuclear localization signal with importin �2/� het-erodimer directs nuclear entry of infecting simian virus 40. J. Virol. 76:9368–9377.

23. Pante, N., and U. Aebi. 1995. Toward a molecular understanding of thestructure and function of the nuclear pore complex. Int. Rev. Cytol. 162B:225–255.

24. Pelkmans, L., D. Puntener, and A. Helenius. Local actin polymerization anddynamin recruitment in SV40-induced internalization of caveolae. 2002.Science 296:535–539.



http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

25. Pipas, J. M. 1992. Common and unique features of T antigens encoded bythe polymavirus group. J. Virol. 66:3979–3985.

26. Rayment, I., T. S. Baker, D. L. Caspar, and W. T. Murakami. 1982. Polyomavirus capsid structure at 22.5 A resolution. Nature 295:110–115.

27. Robinson, I. K., and S. C. Harrison. 1982. Structure of the expanded state oftomato bushy stunt virus. Nature 297:563–568.

28. Sahli, R., R. Freund, T. Dubensky, R. Garcea, R. Bronson, and T. Benjamin.1993. Defect in entry and altered pathogenicity of a polyoma virus mutantblocked in VP2 myristylation. Virology 192:142–153.

29. Salunke, D. M., L. D. Caspar, and R. L. Garcea. 1986. Self-assembly ofpurified polyomavirus capsid protein Vp1. Cell 46:895–904.

30. Speir, J. A., S. Munshi, G. J. Wang, T. S. Baker, and J. E. Johnson. 1995.Structures of the native and swollen forms of cowpea chlorotic mottle virusdetermined by X-ray crystallography and cryoelectron microscopy. Structure3:63–78.

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

32. Stehle, T., S. J. Gamblin, Y. Yan, and S. C. Harrison. 1996. The structure ofsimian virus 40 refined at 3.1 A resolution. Structure 4:165–182.

33. Streuli, C. H., and B. E. Griffin. 1987. Myristic acid is coupled to a structuralprotein of polyoma virus and SV40. Nature 326:619–622.



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Dow

nloaded from

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Documents

Importance of Vp1 Calcium-Binding Residues in Assembly, Cell