Structural Polypeptides of Simian Virus 40

To determine the number and molecular weights of the structural polypeptides of simian virus 40, we have analyzed purified virus by electrophoresis on 14% polyacrylamide gels containing sodium dodecyl sulfate. Full virus purified by several different methods showed six distinct bands with molecular weights of approximately 43,000 (VP1, containing 70% of virion protein), 32,000 (VP2, 9%), 23,000 (VP3, 10%), 14,000 (VP4, 6%), 12,500 (VP5, 4%), and 11,000 (VP6, 3%) both by analysis of radioactively labeled virions and by visualization of the polypeptide bands after staining. “Empty” virions contain decreased amounts of VP4, 5, and 6. The approximate molecular ratios of the polypeptides were 6.0, 1.0, 1.5, 1.5, 1.1, and 1.0. When virus degraded in an alkaline buffer was analyzed by velocity centrifugation in sucrose gradients, the two larger polypeptides (VP1 and VP2) remained at the top of the gradient, whereas the three smallest polypeptides (VP4, 5, and 6) sedimented as a complex with the viral deoxyribonucleic acid. VP3 was found in association with either VP1 and 2 or VP4, 5, and 6, depending on the conditions of degradation. Presumably, VP1 and VP2, comprising about 80% of the protein, form the capsid of the virus. VP4, 5, and 6 may form a nucleoprotein in the virion, and VP3 may serve as an intermediate structural component.     

Temperature-sensitive mutants of foot-and-mouth disease virus with altered structural polypeptides. I. Identification by electrofocusing

The structural polypeptides of foot-and-mouth disease virus were analyzed by electrofocusing in a polyacrylamide gel containing 9 M urea. Three versions of the technique were used to accomodate the widely differing isoelectric points of the four polypeptides. VP2 was identified by comparing mature virus with procapsids. The selective actions of proteases on virions of two serotypes and on their 12S particles were examined. From this emerged a simple test for distinguishing the similarly sized polypeptides: VP1, VP2, and VP3. The effects of carbamylation and succinylation on the charge of the polypeptides were investigated. From the properties of polypeptides modified either chemically or by mutation, it was concluded that all amino acid substitutions that might be expected to cause a charge change would be detected except for neutral-to-histidine substitutions in the most basic polypeptide, VP1. In a sample of 73 temperature-sensitive mutants, 11 classes of variant polypeptides were distinguished on the basis of charge. Their molecular weights were unchanged. Alterations were found in all structural polypeptides except VP4. Mutations affecting VP2 caused similar shifts in the precursor, VP0.     

Virion polypeptide composition of the human papovavirus BK: comparison with simian virus 40 and polyoma virus.

The polypeptide composition of labeled BK virus was compared with that of simian virus 40 (SV40) and polyoma virus by co-electrophoresis of disrupted virions in polyacrylamide gels containing approximately 73% of the capsid protein and had a molecular weight of 39,000. It was smaller than VP1 of SV40 and polyoma virus. The other polypeptides of BK virus were similar in molecular weight to those of SV40. A comparison of the proteins of BK virus and SV40 iodinated with chloramine T before and after disruption in alkaline buffer at pH 10.5 revealed differences between the two viruses in the number and distribution of tyrosines available for iodination. The tryptic peptides of VP1, VP3, VP4, and VP5 combined of SV40 were compared with those of the same polypeptides of BK virus. Among the 19 peptides of VP1 resolved, only two were common to both viruses. The analyses of VP4 and VP5, the histone-like proteins, however, showed more similarity between the viruses, with 6 of 15 resolved peptides in common. The tryptic digests of VP3 were completely different.     

Characterization of cricket paralysis virus-induced polypeptides in Drosophila cells

Cricket paralysis virus purified from Galleria mellonella larvae was shown to be similar to virus purified from Drosophila melanogaster cells. Cricket paralysis virus contained three major structural polypeptides of similar molecular weight (around 30,000), had a buoyant density of 1.344 g/ml, and had a capsid diameter of 27 nm. Twenty virus-induced polypeptides could be detected in CrPV-infected Drosophila cells. Two major polypeptides found in the infected cells corresponded to two structural viral polypeptides (VP1 and VP3), whereas the third major intracellular polypeptide was the apparent precursor of the third viral structural polypeptide (VP2). Three of the primary virus-induced polypeptides had molecular weights of 144,000, 124,000, and 115,000. These and other polypeptides were chased into lower-molecular-weight proteins when excess cold methionine was added after a short [³⁵S]methionine pulse. Although cricket paralysis virus has a number of characteristics in common with the mammalian enteroviruses, the extremely fast processing of high-molecular-weight polypeptides into viral proteins seems atypical. Also, no VP4 (8,000 to 10,000 molecular weight) has been found in the virus particles.     

Structural proteins of simian virus 40. I. Histone characteristics of low-molecular-weight polypeptides.

The DNA-associated polypeptides of simian virus 40 (SV40), VP4 (mol wt 14,000), VP5 (mol wt 12,000), and VP6 (mol wt 11,000), have several properties characteristic of cell histones. After extraction from purified SV40 with dilute acids, these three polypeptides co-electrophoresed on low pH polyacrylamide gels with monkey-kidney cell histones F3, F2b, and F2a1. No virus polypeptide co-electrophoresed with histone F1. Polypeptides VP4, 5, and 6 lacked tryptophan, and only VP4 contained cysteine, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis of virus labeled in vivo with (3H)lysine and either (14C)tryptophan or (35S)cystine. All of the capsid polypeptides VP1, 2, and 3 contained tryptophan whereas only VP1 and 2 contained cysteine. In addition, VP4, 5, and 6 are rich in arginine and lysine when compared with virus labeled with a mixture of amino acids. Analysis of virus grown in cells labeled prior to infection showed that VP4, 5 and 6 were labeled fivefold greater than the major capsid polypeptide, VP1, which indicates that they were partially derived from preexisting cell histones. Based on these data and on previously determined molecular weight estimates, we conclude that VP4, 5, and 6 are histones F3, F2b, and F2a1, respectively, although the possibility that SV40 contains a small amount of F2a2 could not be excluded.     

Proteins of Epstein-Barr virus. I. Analysis of the polypeptides of purified enveloped Epstein-Barr virus.

Epstein-Barr virus (EBV) was purified from the extracellular fluid of HR-1 and B95-8 cell lines. The preparations of purified virus consisted of enveloped particles and had EBV-specific antigneic reactivity. Comparison of the amount of labeled protein in preparations of virus purified from cultures incubated in [35S]methionine with the amount of labeled protein in preparations obtained following a mixture of unlabeled virus with [35S]methionine-labeled cellular proteins indicated that less than 2% of the labeled protein in the purified virus preparation could be attributed to contamination with labeled cellular proteins. No extraneous membranous material was seen in thin sections of the purified virus preparations. Analysis of the polypeptides of purified enveloped EBV indicated the following. (i) Eighteen polypeptides could be resolved in Coomassie brilliant blue-stained electropherograms of extracellular virus purified from HR-1 and B95-8 cultures. (ii) Thirty-three polypeptides could be resolved in fluorograms of labeled EBV purified from B95-8 cultures and subjected to electrophoresis in acrylamide gels cross-linked with diallyltartardiamide. The molecular weight of the EBV polypeptides was estimated by co-electrophoresis with the polypeptides of purified herpes simplex virus and purified polypeptides of known molecular weight to range from 28 x 10(3) to approximately 290 x 10(3) (iii) The polypeptides of EBV could be grouped by their relative molar abundancy into three classes: VP6, 7, and 27 present in high abundance; VP1, 12, 20, 23, and 29 present in moderate abundance; and a third class of less abundant polypeptides, VP4, 5, 8, 9, 10, 11, 15, 16, 21, and 22. The remainder of the polypeptides could not be precisely quantitated. (iv) The polypeptides of purified EBV, although similar in number and in range of molecular weight to the polypeptides of purified herpes simplex virus, differ sufficiently from those of herpes simplex virus so as to preclude comparison of individual polypeptide components.     

Structure and Function of the Polypeptides in Simian Virus 40 I. Existence of Subviral Deoxynucleoprotein Complexes

Velocity sedimentation analysis of simian virus 40 degraded in alkaline buffers, pH 10.5, yields two components: soluble protein containing the largest polypeptides, VP1 and VP2, of the virion, and a deoxynucleoprotein complex (DNP-I) containing the viral deoxyribonucleic acid (DNA) and the small polypeptides, VP4, 5, and 6, and all or part of VP3. Dissociation of DNP-I by equilibrium centrifugation in CsCl yields a complex (DNP-II) of the viral DNA and residual, tightly bound polypeptide; VP4, 5, and 6, but not VP3, are recovered after separation from DNP-II. Treatment of the virus with beta-mercaptoethanol and iodination experiments suggest that VP1 and VP2 might exist as compact structures cross-linked with disulfide bonds, perhaps forming the capsid. VP4, 5, and 6 form a relatively stable complex with the viral DNA and are supposed to be the internal proteins. The location of VP3 is not well defined; at least a portion of it is tightly bound to the viral DNA.     

Stable association of viral protein VP1 with simian virus 40 DNA.

Mild dissociation of simian virus 40 particles releases a 110S virion core nucleoprotein complex containing histones and the three viral proteins VP1, VP2, and VP3. The association of viral protein VP1 within this nucleoprotein complex is mediated at least partially through a strong interaction with the viral DNA. Treatment of the virion-derived 110S nucleoprotein complex with 0.25% Sarkosyl dissociated VP2, VP3, and histones, leaving a stable VP1-DNA complex. The VP1-DNA complex had a sedimentation value of 30S and a density of 1.460 g/cm3. The calculated molecular weight of the complex was 7.9 x 10(6), with an average of 100 VP1 molecules per DNA. Agarose gel electrophoresis of the VP1-DNA complex demonstrated that VP1 is associated not only with form I and form II simian virus 40 DNAs but also with form III simian virus 40 DNA generated by cleavage with EcoRI.     

Processing of structural polypeptides of infectious flacherie virus of the silkworm, Bombyx mori: VP1 and VP4 are derived from VP0.

Infectious flacherie virus of the silkworm, Bombyx mori, contains five major polypeptides termed VP0, VP1, VP2, VP3, and VP4 in the order of descending molecular weight. Immunoblot analysis with specific antisera against each of these structural polypeptides showed that antisera against VP1 and VP4 unequivocally reacted with VP0 as well as their homologous structural polypeptides. Limited proteolysis of VP0 and VP1 by Staphylococcus aureus V8 protease gave several common polypeptide fragments. Amino acid sequence analysis showed that VP0 and VP4 shared a common NH2-terminal amino acid sequence. These results indicate that VP1 and VP4 are generated from VP0 and that VP4 occupies the NH2-terminal portion of VP0.     

Proteolytic enhancement of rotavirus infectivity: molecular mechanisms

The polypeptide compositions of single-shelled and double-shelled simian rotavirus particles were modified by exposure to proteolytic enzymes. Specifically, a major outer capsid polypeptide (VP3) having a molecular weight of 88,000 in double-shelled particles was cleaved by trypsin to yield two polypeptides, VP5* and VP8* (molecular weights, 60,000 and 28,000, respectively). The cleavage of VP3 by enzymes that enhanced infectivity (trypsin, elastase, and pancreatin) yielded different products compared to those detected when VP3 was cleaved by chymotrypsin, which did not enhance infectivity. The appearance of VP5* was correlated with an enhancement of infectivity. Cleavages of the major internal capsid polypeptide VP2 were also observed. The VP2 cleavage products had molecular weights similar to those of known structural and nonstructural rotavirus polypeptides. We confirmed the precursor-product relationships by comparing the peptide maps of the polypeptides generated by digestions with V-8 protease and chymotrypsin. The remaining rotavirus structural polypeptides, including the outer capsid glycoproteins (VP7 and 7a), were not altered by exposure to pancreatic enzymes. Cleavage of VP3 was not required for virus assembly, and specific cleavage of the polypeptides occurred only on assembled particles. We also discuss the role of cleavage activation in other virus-specific biological functions (e.g., hemagglutination and virulence).     

In vitro morphogenesis of foot-and-mouth disease virus.

Foot-and-mouth disease virion RNA is translated efficiently and completely in a rabbit reticulocyte lysate cell-free system. Treatment of cell-free lysates with monospecific serum prepared against the individual viral structural proteins or with monoclonal antibodies prepared against the inactivated virus or against a viral structural protein precipitated all of the structural proteins, suggesting that structural protein complexes were formed in vitro. Sucrose gradient analysis of the cell-free lysate indicated that complexes sedimenting at 5, 14, 60 to 70, and ca. 110S were assembled in vitro. Structural proteins VP0, VP1, and VP3 were the major polypeptides found in these complexes. The material sedimenting at 110S, i.e., containing VP0, VP1, and VP3, was precipitated by a 140S-specific monoclonal antibody but not by a 12S subunit-specific monoclonal antibody, suggesting that this capsid structure contained at least one epitope present on the intact virus.     

Steady-state infection by echovirus 6 associated with nonlytic viral RNA and an unprocessed capsid polypeptide.

We established a human cell line which was persistently infected (PI) by the normally cytolytic echovirus 6. All of the cultured PI cells contained genome-size viral RNA which was synthesized continuously and incorporated into virus particles. This steady-state infection has been maintained for more than 6 years. In contrast to RNA of wild-type echovirus 6, the viral RNA from PI cells was not lytic when transfected into uninfected, susceptible cells. The capsid polypeptides of the virus particles produced during lytic infections were compared with those of virus particles from PI cells. Wild-type virions contained five polypeptides with molecular masses of 31.5, 27, 25.8, 21.2, and 9.5 kilodaltons. Comparison of polypeptide profiles of virions and empty immature capsids along with peptide analyses by immunoblotting and partial proteolysis of isolated viral proteins identified the cleavage products of the 31.5-kilodalton polypeptide (VP0) as the two smaller polypeptides (VP2 and VP4). The virus particles produced by PI cells as well as cellular extracts of PI cells contained only the three largest proteins (VP0, VP1, and VP3), indicating that VP0 was not processed during persistent infection. The lack of VP2 and VP4 in the defective virus particles coincided with their inability to attach to uninfected, susceptible cells. The maintenance of the steady-state infection of echovirus 6 was not dependent upon the release of virus particles from PI cells.     

Birnavirus precursor polyprotein is processed in Escherichia coli by its own virus-encoded polypeptide.

The cDNA fragment of the large RNA segment of infectious bursal disease virus 002-73, when expressed in Escherichia coli, produces precursor polyprotein (N-VP2-VP4-VP3-C), most of which is then processed to generate constituent polypeptides. Using cDNA fragments containing site-specific mutations and two monoclonal antibodies that are specific to VP2 and VP3 of mature virus particles, we demonstrated that the VP4 protein is involved in processing of the precursor polyprotein to generate VP2 and VP3 and excluded the possibility of internal initiation for the generation of VP3.     

Structure of the Fab-labeled "breathing" state of native poliovirus

At 37°C, the structure of poliovirus is dynamic, and internal polypeptides VP4 and N terminus of VP1 (residues 1 to 53) externalize reversibly. An Fab fragment of a monospecific antibody, which binds to residues 39 to 55 of VP1, was utilized to locate the N termini of VP1 in native (160S) particles in this "breathing" state. Fab and virus were mixed and imaged via cryogenic electron microscopy. The resulting reconstruction showed the capsid expands similarly to the irreversibly altered cell entry intermediate (135S) particle, but the N terminus of VP1 is located near the 2-fold axes, instead of the "propeller tip" as in 135S particles.     

Polypeptides of the surface projections and the ribonucleoprotein of avian infectious bronchitis virus.

Purified avian infectious bronchitis virus was digested with bromelain (0.7 mg/ml), and the surface projections were removed. Polyacrylamide gel electrophoresis of the polypeptides from these bromelain-treated particles showed that VP1, VP2, and VP5 were missing from the seven polypeptides. VP1 to VP7, that were present in untreated virus preparations. Milder bromelain treatment (0.07 mg/ml) left visible surface projections and polypeptides comprising VP1 and VP2 intact, but removed VP5. Thus, there are apparently two types of surface projections on the virus particle. The ribonucleoprotein complex was released from virus particles disrupted with 1% Nonidet P-40. The proportion of VP6 in such preparations was greatly reduced, implying that VP6 is the structural polypeptide of the ribonucleoprotein. Polypeptides VP1, VP2, VP4, and VP5 are glycosylated, but none of the polypeptides contains lipid.     

Immunological Reactivity of Antisera Prepared Against the Sodium Dodecyl Sulfate-Treated Structural Polypeptides of Adenovirus-Associated Virus

The preparation of antisera to the three purified sodium dodecyl sulfate (SDS)-treated polypeptide components (VP1, VP2, VP3) of adenovirus-associated virus (AAV) type 3H is described. In immunofluorescence tests (FA), these antisera stained heat-stable antigens with distinct morphologies in cells co-infected with either adenovirus or herpes simplex virus. Kinetic studies of antigen formation showed that VP1 antiserum first stained the cytoplasm (14 hr) and later (by 18 hr) stained both cytoplasmic and intranuclear areas. VP2 antiserum stained only discrete intranuclear areas, and VP3 antiserum stained nearly the entire nucleus. All three VP antigens appeared at about the 14th hr postinfection, about 2 hr prior to the appearance of whole virion antigen. The VP antisera cross-reacted in FA with AAV types 1 and 2 (all at one-eighth of the homologous titer), but did not react with other parvoviruses, i.e., rat virus, hemadsorbing enteric virus of calves, minute virus of mice, or H-1 virus. These non-neutralizing antisera reacted specifically with SDS-treated AAV virion antigens in complement fixation and immunodiffusion tests, and antiserum prepared against SDS-treated helper adenovirus structural polypeptides reacted with adenovirus polypeptide antigens. All antisera to SDS-treated polypeptides were specific for new antigens revealed on the dissociated peptides and did not react with whole virions, whereas whole-virion antisera did not cross-react with the polypeptide antigens. These findings suggest that antigens unique to the polypeptides of AAV are revealed by SDS treatment and that these antigens can be detected in cells prior to the folding of the polypeptides into the molecular configuration they possess as virion subunits. These results also indicate that at least one AAV polypeptide component is synthesized in the cell cytoplasm.     

Structural Proteins of Adenovirus-Associated Virus Type 3

Three major structural proteins were found in adenovirus-associated virus (AAV) type 3H virions which were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights of the polypeptides were determined to be approximately 66,000 (VP1), 80,000 (VP2), and 92,000 (VP3). The component having a molecular weight of 66,000 comprised about 80% of the total virion protein in the major AAV-3H particle, and the other two components comprised about 10% each. Proteins of the same molecular weight were found in the minor dense AAV-3H virion, but the 80,000- and 92,000-molecular-weight components were present at about one-half the concentration. The AAV-3H virion contains about 72 molecules of VP1 and 8 and 7 molecules of VP2 and VP3, respectively.     

Proteins in intracellular simian virus 40 nucleoportein complexes: comparison with simian virus 40 core proteins.

Intracellular nucleoprotein complexes containing SV40 supercoiled DNA were purified from cell lysates by chromatography on hydroxyapatite columns followed by velocity sedimentation through sucrose gradients. The major protein components from purified complexes were identified as histone-like proteins. When analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels, complex proteins comigrated with viral core polypeptides VP4, VP5, VP6, and VP7. (3H) tryptophan was not detected in polypeptides from intracellular complexes or in the histone components from purified SV40 virus. However, a large amount of (3H) tryptophan was found in the viral polypeptide VP3 relative to that incorporated into the capsid polypeptides VP1 and VP2. Intracellular complexes contain 30 to 40% more protein than viral cores prepared by alkali dissociation of intact virus, but when complexes were exposed to the same alkaline conditions, protein also was removed from complexes and they subsequently co-sedimented with and had the same buoyant density as viral cores. The composition and physical similarities of nucleoprotein complex and viral cores indicate that complexes may have a role in the assembly of virions.     

Structural polypeptides of rabbit, bovine, and human papillomaviruses.

The number and apparent molecular weight of the structural polypeptides of Shope rabbit papilloma virus (RPV), bovine papilloma virus (BPV), and human papilloma virus (HPV) were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Up to 10 polypeptides were detected in highly purified BPV and HPV full particles; a close homology was found between the polypeptide composition of both viruses. Purified RPV virions gave a similar polypeptide pattern. The main components of the three papillomaviruses are the major polypeptide (VP1) with a mol wt of approximately 54,000 and the three smaller polypeptides (VP8, 9, 10) with mol wt of about 16,500, 15,500 and 12,500, respectively. VP8, VP9, and VP10 are never detected in empty capsids. When BPV virions were disrupted with alkaline buffer, the six lower-molecular-weight polypeptides (VP5 to 10) remained associated with viral DNA. This suggests that they are internal components of the virions and that the four higher-molecular-weight polypeptides (VP1 to 4) may represent external components. The polypeptide compositions of BPV and polyoma virus, another papovavirus, have been compared. The number of BPV and polyoma virus components (10 and 6, respectively) and the molecular weight of their major polypeptide (54,000 and 44,500, respectively) are different; however, the three main DNA-associated polypeptides of BPV (VP8, 9, 10) and the three histone-like components of polyoma virus (VP4, 5, 6) were shown to have identical apparent molecular weights. The possibility that some of the minor components of papillomaviruses may be proteolytic degradation products or cell protein contaiminants is discussed.