Salt-stable association of simian virus 40 capsid with simian virus 40 DNA

In 8 M CsCl, a fraction of the wild-type previrions and tsB228 nucleoprotein complexes lose their core histones but retain their capsid. These histone-depleted complexes appear in the electron microscope as a protein shell attached to supercoiled DNA. Consistent with this result, we find that in 1 M NaCl, the wild-type previrions dissociate into two populations of nucleoprotein complexes. One population sediments between 50 and 140 S and morphologically resembles the shell-DNA complexes isolated in CsCl gradients. The other population is comprised primarily of nucleoproteins which sediment at 40 S.     

Analysis of a nuclear localization signal of simian virus 40 major capsid protein Vp1.

The nuclear localization signal of the major structural protein, Vp1, of simian virus 40 was further defined by mutagenesis. The targeting activity was examined in cells microinjected with SV-Vp1 variant viral DNAs bearing either an initiation codon mutation of the agnoprotein or mutations in the Vp1 coding sequence or microinjected with pSG5-Vp1 and pSG5-Vp1 mutant DNAs in which Vp1 or mutant Vp1 is expressed from simian virus 40 early promoter. The Vp1 nuclear localization signal functioned autonomously without agno-protein once the Vp1 protein was synthesized in the cytoplasm. The targeting activity was localized to the amino-terminal 19 residues. While replacement of cysteine 10 with glycine, alanine, or serine did not affect the activity, replacement of arginine 6 with glycine caused the cytoplasmic phenotype. When multiple mutations were introduced among residue 5, 6, 7, 16, 17, or 19, the targeting activity was found to reside in two clusters of basic residues, a cluster of lysine 5, arginine 6, and lysine 7 and a cluster of lysine 16, lysine 17, and lysine 19. The clusters are independently important for nuclear localization activity.     

The VP2/VP3 minor capsid protein of simian virus 40 promotes the in vitro assembly of the major capsid protein VP1 into particles

The SV40 capsid is composed primarily of 72 pentamers of the VP1 major capsid protein. Although the capsid also contains the minor capsid protein VP2 and its amino-terminally truncated form VP3, their roles in capsid assembly remain unknown. An in vitro assembly system was used to investigate the role of VP2 in the assembly of recombinant VP1 pentamers. Under physiological salt and pH conditions, VP1 alone remained dissociated, and at pH 5.0, it assembled into tubular structures. A stoichiometric amount of VP2 allowed the assembly of VP1 pentamers into spherical particles in a pH range of 7.0 to 4.0. Electron microscopy observation, sucrose gradient sedimentation analysis, and antibody accessibility tests showed that VP2 is incorporated into VP1 particles. The functional domains of VP2 important for VP1 binding and for enhancing VP1 assembly were further explored with a series of VP2 deletion mutants. VP3 also enhanced VP1 assembly, and a region common to VP2 and VP3 (amino acids 119-272) was required to promote VP1 pentamer assembly. These results are relevant for controlling recombinant capsid formation in vitro, which is potentially useful for the in vitro development of SV40 virus vectors.     

DNA-binding properties of the major structural protein of simian virus 40.

We investigated whether the VP1 protein of simian virus 40 binds to DNA. In vitro DNA-binding experiments clearly indicate that VP1 bound strongly to double-stranded and single-stranded DNA, with a higher affinity for the latter; additional experiments show that VP1 did not bind to a specific sequence of simian virus 40 DNA.     

Resolution of simian virus 40 proteins in whole cell extracts by two-dimensional electrophoresis: heterogeneity of the major capsid protein.

The major capsid protein (VP1) of simian virus 40 (SV40) has been analyzed by two-dimensional electrophoresis. This system separates protein according to isoelectric point by isoelectric-focusing, and according to molecular weight by sodium dodecylsulphate electrophoresis (O'Farrell, 1975). VP1 synthesis in infected CV-1 cells can be monitored directly by analysis of unfractionated whole cell extracts; the resolution of VP1 from cellular proteins allows its detection as early as 13 hr after infection. The two-dimensional separation of VP1 reveals that it is heterogeneous, consisting of one major protein (molecular weight 47,000 daltons and isoelectric point of approximately pH 6.8) and five minor protein components. The minor forms of VP1 are 10% of the total VP1 and differ from the major form of VP1 both in molecular weight (by approximately 500 daltons) and isoelectric point (ranging from approximately pH 6.7 to pH 6.9). Evidence is presented to show that two of the minor forms are phosphorylated derivatives of VP1, and it is further suggested that all the different forms of VP1 are the result of modifications of the primary product of translation. A temperature-sensitive mutant of the BC complementation group (BC11) of SV40 results in the synthesis of VP1 with an altered electrophoretic mobility; both the major form of VP1 and the minor forms are shifted in their isoelectric points. In addition to the specific case of SV40, two aspects of these studies should be generally significant to investigators studying eucaryotic gene expression by two-dimensional gel electrophoresis: first, the genetic origin of a protein can be determined by a temperature-sensitive mutation which causes a charge change in the resultant protein; and second, two or more protein spots on a two-dimensional separation may be the products of a single gene.     

High cooperativity of the SV40 major capsid protein VP1 in virus assembly

SV40 is a small, non enveloped DNA virus with an icosahedral capsid of 45 nm. The outer shell is composed of pentamers of the major capsid protein, VP1, linked via their flexible carboxy-terminal arms. Its morphogenesis occurs by assembly of capsomers around the viral minichromosome. However the steps leading to the formation of mature virus are poorly understood. Intermediates of the assembly reaction could not be isolated from cells infected with wt SV40. Here we have used recombinant VP1 produced in insect cells for in vitro assembly studies around supercoiled heterologous plasmid DNA carrying a reporter gene. This strategy yields infective nanoparticles, affording a simple quantitative transduction assay. We show that VP1 assembles under physiological conditions into uniform nanoparticles of the same shape, size and CsCl density as the wild type virus. The stoichiometry is one DNA molecule per capsid. VP1 deleted in the C-arm, which is unable to assemble but can bind DNA, was inactive indicating genuine assembly rather than non-specific DNA-binding. The reaction requires host enzymatic activities, consistent with the participation of chaperones, as recently shown. Our results demonstrate dramatic cooperativity of VP1, with a Hill coefficient of approximately 6. These findings suggest that assembly may be a concerted reaction. We propose that concerted assembly is facilitated by simultaneous binding of multiple capsomers to a single DNA molecule, as we have recently reported, thus increasing their local concentration. Emerging principles of SV40 assembly may help understanding assembly of other complex systems. In addition, the SV40-based nanoparticles described here are potential gene therapy vectors that combine efficient gene delivery with safety and flexibility.     

A simian virus 40-encoded protein of Mr 74,000 daltons is structurally related to the capsid proteins of the virus

We have demonstrated the synthesis of a 74,000-dalton protein (74K protein) in African green monkey kidney cells infected with simian virus (SV)40. The 74K protein was detected late during the lytic cycle. Its synthesis was inhibited by arabinosyl cytosine as was the synthesis of the capsid proteins. Monospecific antibodies raised against VP1 and VP3 precipitated the structural proteins and the 74K protein. The 74K protein was not found in purified virions. Tryptic peptide analysis demonstrated that the 74K protein shares methionine- and serine-containing peptides with VP1 and VP3 and thus is structurally related to the capsid proteins.     

Simian virus 40 agnoprotein facilitates perinuclear-nuclear localization of VP1, the major capsid protein.

The agnoprotein of simian virus 40 (SV40) is a 61-amino-acid protein encoded in the leader of some late mRNAs. In indirect immunofluorescence studies with antisera against SV40 capsid proteins, we show that mutants which make no agnoprotein display abnormal perinuclear-nuclear localization of VP1, the major capsid protein, but not VP2 or VP3, the minor capsid proteins. In wild-type (WT) SV40-infected CV-1P cells, VP1 was found predominantly in the cytoplasm until 36 h postinfection (p.i.), approximately the time that high levels of agnoprotein became detectable under our infection conditions. Thereafter, VP1 localized rapidly to the perinuclear region and to the nucleus. In contrast, in agnoprotein-minus mutant-infected CV-1P cells, perinuclear-nuclear accumulation of VP1 occurred much less efficiently; a significantly greater fraction of cells with predominantly cytoplasmic fluorescence was observed up to 48 h p.i. At 48 and 60 h p.i., more cells with largely perinuclear and little nuclear staining were seen than in WT-infected controls. In similar analyses with stably transfected cell lines constitutively expressing the agnoprotein, VP1 localized to the nucleus before 30 h p.i., regardless of the infecting virus. Delayed nuclear entry of VP1 in a mutant which makes no agnoprotein was also overcome in a revertant which has a second site point mutation in VP1. This suggests that an alteration of VP1 can partially overcome the defect of the agnogene mutation by enhancement of the rate of its own nuclear localization. Taken together, these results indicate that at least one function of the agnoprotein is to enhance the efficiency of perinuclear-nuclear localization of VP1.     

Identification of simian virus 40 protein A.

A large simian virus 40 (SV40)-specific protein can be efficiently immunoprecipitated from infected cell extracts with antisera obtained from hamsters bearing SV40-induced tumors. The protein has an apparent molecular weight of 88,000 to 100,000 with respect to markers with known molecular weights, but behaves anomalously on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Cell lines infected by two different strains of SV40 synthesize immunoreactive proteins that differ slightly in mobility during SDS-polyacrylamide gel electrophoresis, evidence that the protein is coded for by the virus. These differences in protein size correlate with differences in the electrophoretic mobility of viral DNA fragments obtained by digestion with HindII and III restriction enzymes. The size of the viral capsid proteins VP2 and VP3 also varies with the strain of virus. dl-1001, a constructed deletion mutant that lacks part of the SV40A gene, directs the synthesis of a 33,000-dalton polypeptide that is not detected in cells infected with wild-type virus. The deletion fragment, like the larger protein, is phosphorylated. Maps of tryptic peptides from the 88,000- to 100,000-dalton protein and the 33,000-dalton fragment show common peptides and provide strong direct evidence that the proteins are products of the SV40 A gene. The deletion fragment reacts with antitumor sera and binds to double-stranded DNA in the presence of the complete A protein.     

Modification of simian virus 40 protein A.

The A protein of simian virus 40 is phosphorylated in both productive and transforming infection. The phosphorylated amino acid has been identified as serine and has been localized in a single tryptic peptide of the protein. Because the A protein synthesized in infection by A mutants is phosphorylated to the same extent and in the same peptide as in infection by wild-type virus, the functional defect of the A mutants is apparently unrelated to phosphorylation. At least three distinct forms of the A protein with apparent molecular weights of 85,000, 88,000, and 100,000 can be identified in extracts of cells infected by wild-type virus. After exposure of cells to Nonidet P-40, the 85,000- and 88,000-dalton proteins were found in varying amounts in extracts of permissive cells but not in extracts of transformed cells. This finding raised the question of the possible functional importance of the smaller proteins in productive infection. However, the virtual absence of the 85,000- and 88,000-dalton proteins in some extracts of the fully permissive CV-1 cell line indicates that a conversion of the larger to the smaller forms of the A protein is not required in significant quantity for productive infection. Furthermore, a study of extraction conditions shows that the smaller proteins are easily generated during extraction and provides an explanation for the appearance of these proteins in some cells after extraction under unfavorable conditions.     

Two separable functional domains of simian virus 40 large T antigen: carboxyl-terminal region of simian virus 40 large T antigen is required for efficient capsid protein synthesis.

The carboxyl-terminal portion of simian virus 40 large T antigen is essential for productive infection of CV-1 and CV-1p green monkey kidney cells. Mutant dlA2459, lacking 14 base pairs at 0.193 map units, was positive for viral DNA replication, but unable to form plaques in CV-1p cells (J. Tornow and C.N. Cole, J. Virol. 47:487-494, 1983). In this report, the defect of dlA2459 is further defined. Simian virus 40 late mRNAs were transcribed, polyadenylated, spliced, and transported in dlA2459-infected cells, but the level of capsid proteins produced in infected CV-1 green monkey kidney cells was extremely low. dlA2459 large T antigen lacks those residues known to be required for adenovirus helper function, and the block to productive infection by dlA2459 occurs at the same stage of infection as the block to productive adenovirus infection of CV-1 cells. These results suggest that the adenovirus helper function is required for productive infection by simian virus 40. Mutant dlA2459 was able to grow on the Vero and BSC-1 lines of African green monkey kidney cells. Additional mutants affecting the carboxyl-terminal portion of large T were prepared. Mutant inv2408 contains an inversion of the DNA between the BamHI and BclI sites (0.144 to 0.189 map units). This inversion causes transposition of the carboxyl-terminal 26 amino acids of large T antigen and the carboxyl-terminal 18 amino acids of VP1. This mutant was viable, even though the essential information absent from dlA2459 large T antigen has been transferred to the carboxyl terminus of VP1 of inv2408. The VP1 polypeptide carrying this carboxyl-terminal portion of large T could overcome the defect of dlA2459. This indicates that the carboxyl terminus of large T antigen is a separate and separable functional domain.     

Cell-free translation of simian virus 40 16S and 19S L-strand-specific mRNA classes to simian virus 40 major VP-1 and minor VP-2 and VP-3 capsid proteins.

Simian virus 40 capsid proteins VP-1, VP-2, and VP-3 have been synthesized in wheat germ and reticulocyte cell-free systems in response to either poly(A)-containing mRNA from the cytoplasm of infected cells or viral RNA purified by hybridization to simian virus 40 DNA linked to Sepharose. All three viral polypeptides synthesized in vitro are specifically immunoprecipitated with anti-simian virus 40 capsid serum. VP-2 and VP-3 are related by tryptic peptide mapping to each other but not to VP-1. The most abundant class of L-strand-specific viral mRNA, the 16S species, codes for the major capsid protein. The relatively minor 19S class directs the cell-free synthesis of VP-1, VP-2, and VP-3. Whether the 19S RNA represents more than one distinct species of mRNA is not yet clear. VP-1 mRNA can be isolated from the cytoplasm, detergent-washed nuclei, and the nuclear wash fraction. The mRNA from the nuclear wash fraction is enriched for VP-2 mRNA when compared to other viral or cellular polypeptides.     

Nuclease-sensitive sites in the two major intracellular simian virus 40 nucleoproteins

Simian virus 40 nucleoprotein isolated from the nuclei of infected cells contains a nuclease-sensitive site adjacent to the viral origin of replication (between 0.66 and 0.73 map unit). Nuclear extracts were subfractionated by sucrose gradient centrifugation to yield provirions (200S) and simian virus 40 chromatin (80S). The 80S fraction was cleaved either by DNase I or by an endonuclease endogenous to BSC-1 cells with high preference for the 0.66 to 0.73 region. The 200S fraction was treated to release core particles that were sensitive to nuclease cleavage; however, DNase I showed little or no preference for the 0.66 to 0.73 region of the provirion core nucleoprotein.     

Assemblages of simian virus 40 capsid proteins and viral DNA visualized by electron microscopy

SV40 assembles in the nucleus by addition of capsid proteins to the minichromosome. The VP15VP2/3 capsomer is composed of a pentamer of the major protein VP1 complexed with a monomer of a minor protein, VP2 or VP3. In the capsid, the capsomers are bound together via their flexible carboxy-terminal arms. Our previous studies suggested that the capsomers are recruited to the packaging signal ses via avid interaction with Sp1. During assembly Sp1 is displaced, allowing chromatin compaction. Here we investigated the interactions in vitro of VP1(5)VP2/3 capsomers with the entire SV40 genome, using mutant VP1 deleted in the carboxy-arm that cannot assemble, but retains DNA-binding capacity. EM revealed that VP1(5)VP2/3 complexes bind non-specifically at random locations around the DNA. Sp1 was absent from mature virions. The findings suggest that multiple capsomers attach simultaneously to the viral genome, increasing their local concentration, facilitating rapid, concerted assembly reaction and removal of Sp1.     

Charge microheterogeneity of the major capsid protein of polyoma virus.

The behavior in isoelectric focusing of the major capsid polypeptide VPI of several strains of polyoma virus was studied. Two previously recognized phenomena were reexamined, namely, (i) the separation of the VP1 polypeptide into multiple subspecies differing only slightly from each other in apparent isoelectric point and (ii) strain differences in the overall apparent net charge of the family of VP1 subspecies. It was found that the pattern of subspecies was reproducible when focusing was initiated from either the basic or acidic region of the gel, keeping the ampholyte mixture constant. However, individual subspecies were unstable, and labeled polypeptide could be shifted dramatically by either refocusing of separated subspecies or by altering the concentration of ampholytes. These findings suggest that protein-protein and protein-ampholyte interactions play an important role in the generation of this charge heterogeneity. The basis for the overall charge difference between the VP1 of 3049 virus and several other strains (lpD, lpS, ts59, and A2) was studied, using recombinant viruses constructed of specific sequences derived from 3049 and lpD genomes. The portion of the VP1 polypeptide carrying the altered charge could be mapped to the body of the molecule 3' to the HindIII site at 45.0 map units (3,918 base pairs). This clearly segregates the VP1 charge phenotype from the cyc phenotype of 3049 in which capsid proteins are overproduced and accumulate in the cytoplasm of infected cells.     

Expression of Semliki Forest virus capsid protein from SV40 recombinant virus

Here, the proteolytic processing of the Semliki Forest virus (SFV) capsid protein was studied in the absence of other viral functions. Two different fragments of the SFV messenger cDNA, coding for capsid protein and 174 and 38 extra amino acids from the envelope proteins, respectively, were cloned in the late region of the SV40 viral DNA. Cells infected with the SV40 recombinant virus stocks were analyzed for the production of SFV capsid mRNA and polypeptide. Immunofluorescence staining of the infected cells indicated that the produced SFV capsid protein accumulated mainly in the nucleus. Polyacrylamide gel electrophoresis of the immunoprecipitated SFV capsid proteins showed that both recombinants yielded a labelled band equivalent in size to the SFV capsid protein. Thus the proteolytic processing takes place even under conditions where the capsid protein is the only virus-specified protein synthesized.     

Specific association of simian virus 40 tumor antigen with simian virus 40 chromatin

Simian virus 40 tumor antigen (SV40 T antigen) was bound to both replicating and fully replicated SV40 chromatin extracted with a low-salt buffer from the nuclei of infected cells, and at least a part of the association was tight specific. T antigen cosedimented on sucrose gradients with SV40 chromatin, and T antigen-chromatin complexes could be precipitated from the nuclear extract specifically with anti-T serum. From 10 to 20% of viral DNA labeled to steady state with [3H]thymidine for 12 h late in infection or 40 to 50% of replicating viral DNA pulse-labeled for 5 min was associated with T antigen in such immunoprecipitates. After reaction with antibody, most of the T antigen-chromatin complex was stable to washing with 0.5 M NaCl, but only about 20% of the DNA label remained in the precipitate after washing with 0.5 M NaCl-0.4% Sarkosyl. This tightly bound class of T antigen was associated preferentially with a subfraction of pulse-labeled replicating DNA which comigrated with an SV40 form I marker. A tight binding site for T antigen was identified tentatively by removing the histones with dextran sulfate and heparin from immunoprecipitated chromatin labeled with [32P]phosphate to steady state and then digesting the DNA with restriction endonucleases HinfI and HpaII. The site was within the fragment spanning the origin of replication, 0.641 to 0.725 on the SV40 map.     

Stimulation of non-histone chromosomal protein synthesis in simian virus 40-infected simian cells.

The pattern of synthesis of non-histone chromosomal proteins in simian virus (SV) 40-infected African green monkey kidney cells was analyzed by polyacryl-amide gel electrophoresis to see whether the changes in chromosomal protein metabolism are involved in the viral-induced synthesis of cellular DNA and mRNA. During the prereplicative phase of infection, the rate of histone synthesis was decreased until 15 h postinfection, whereas that of non-histone protein synthesis was increased after 5 h postinfection and reached a maximum at 10 to 15 h postinfection when viral-induced synthesis of cellular DNA and mRNA began to be observed. Stimulation of non-histone protein synthesis was also observed in the infected cells treated with cytosine arabinoside and was dependent on the multiplicity of infection. Stimulation occurred in almost all species of non-histone proteins. These results suggest that the stimulation of non-histone protein synthesis is caused by an early SV40 function and occurs prior to the viral-induced synthesis of cellular DNA and mRNA. During the replicative phase of infection, a marked increase in the rate of synthesis was observed in the non-histone proteins with molecular weights of about 48,000, 35,000, and 23,000, which were subsequently found to be SV40 capsid proteins.