Location of the sequences coding for capsid proteins VP1 and VP2 on polyoma virus DNA.

The 19S and 16S polyoma virus late mRNAs have been separated on sucrose-formamide density gradients and translated in vitro. The 16S RNA codes only for polyoma capsid protein VP1, while the 19S RNA codes in addition for capsid protein VP2. Since the 19S and 16S species have been previously mapped on the viral genome, these results allow us to deduce the location of the sequences coding for VP1 and VP2. Comparison of the chain lengths of the capsid proteins with the size of the viral mRNAs coding for them suggests that VP1 and VP2 are entirely virus-coded. Purified polyoma 19S RNA directs the synthesis of very little VP1 in vitro, although it contains all the sequences required to code for the protein. The initiation site for VP1 synthesis which is located at an internal position on the messenger is probably inactive either because it is inaccessible or because it lacks an adjacent "capped" 5' terminus. Similar inactive internal initiation sites have been reported for other eucarotic viral mRNAs (for example, Semliki forest virus, Brome mosaic virus, and tobacco mosaic virus), suggesting that while eucaryotic mRNAs may have more than one initiation site for protein synthesis, only those sites nearer the 5' terminus of the mRNA are active.     

Characterization of the mRNA''s for the polyoma virus capsid proteins VP1, VP2, and VP3.

Polyadenylated cytoplasmic RNA from polyoma virus-infected cells can be translated in the wheat germ system to yield all there polyoma virus capsid proteins, VP1, VP2, and VP3. The translation products of RNA selected from total cytoplasmic RNA of infected cells by hybridization to polyoma virus DNA showed a high degree of enrichment for VP1, VP2, and VP3. The identity of the in vitro products with authentic virion proteins was established in two ways. First, tryptic peptide maps of the in vitro products were found to be essentially identical to those of their in vivo counterparts. Second, the mobilities of the in vitro products on two-dimensional gels were the same as those of viral proteins labeled in vivo. VP1, VP2, and vp3 were all labeled with [35S] formylmethionine when they were synthesized in the presence of [35S] formylmethionyl-tRNAfmet. We determined the sizes of the polyadenylated mRNA's for VP1, VP2, and VP3 by fractionation on gels. The sizes of the major mRNA species for the capsid proteins are as follows: VP2, 8.5 X 10(5) daltons; VP3, 7.4 X 10(5) daltons; and VP1, 4.6 X 10(5) daltons. We conclude that all three viral capsid proteins are synthesized independently in vitro, that all three viral capsid proteins are virally coded, and that each of the capsid proteins has a discrete mRNA.     

Polyoma virus complementary RNA directs the in vitro synthesis of capsid proteins VP1 and VP2.

Polyoma virus complementary RNA, synthesized in vitro by using highly purified Escherichia coli RNA polymerase and nondefective form I polyoma DNA, was translated in a wheat germ cell-free system. Polypeptides were synthesized that comigrated on sodium dodecyl sulfate-polyacrylamide gels with the polyoma capsid proteins VP1 and VP2, although most of the cell-free products were of smaller molecular weights. The VP1-size protein specifically immunoprecipitated with anti-polyoma virus serum, and upon digestion by trypsin yielded [35S]methionine-labeled tryptic peptides that co-chromatographed with the [3H]methionine-labeled tryptic peptides of virion-derived VP1 on both cation-exchange and anion-exchange resins. The VP2-size in vitro product contained all the virion VP2 methionine-labeled tryptic peptides, as shown by cation- and anion-exchange chromatography and two-dimensional fingerprinting on cellulose. We conclude that full-length polyoma VP1 and VP2 are synthesized in response to complementary RNA and consequently that the viral capsid proteins VP1, VP2, and VP3 are entirely virus coded.     

Chromatographic separation of the polyoma virus proteins and renaturation of the isolated VP1 major capsid protein.

Treatment of purified polyoma virions with 6 M guanidine-hydrochloride and 0.01 M beta-mercaptoethanol resulted in the immediate loss of both hemagglutinating and plaque-forming ability. Gel filtration through Sepharose CL-6B beads allowed separation of the dimer, VP1, VP2, VP3, and histone proteins VP4-7 in highly purified form. Renaturation of the purified VP1 protein resulted in the formation of subunits that were morphologically, biophysically, and immunologically similar to native virion capsomeres.     

Cell-free synthesis of measles virus proteins.

Polyadenylated mRNA extracted from cytoplasm of measles virus-infected Vero cells was translated in a cell-free system. Three of the polypeptides obtained corresponded to nucleocapsid protein, phosphoprotein, and membrane protein of measles virions. A fourth polypeptide, present in measles virus-infected cells, could be generated by addition of Vero cytoplasmic extract and was identified as a cleavage product of the nucleocapsid protein.     

Cell-free synthesis of herpes simplex virus proteins.

Polyribosomes isolated from herpes simplex virus type I (HSV-1)-infected cells have been used to program a eucaryotic cell-free translation system. At least 10 HSV-specific polypeptides, with apparent molecular weights of 25,000 to 160,000, are synthesized by wild-type HSV-infected polyribosomes. Polyribosomes prepared from thymidine kinase-negative mutants of HSV direct the synthesis of three putative nonsense termination polypeptides. HSV-specific polypeptides synthesized in vitro are precipitated with antiserum to HSV-infected cell proteins.     

Partial amino-terminal sequences of the polyoma nonhistone proteins VP1, VP2, and VP3 synthesized in vitro.

The three polyoma virus capsid proteins VP1, VP2, and VP3 were synthesized in vitro in the presence of several radiolabeled amino acids and, after purification on sodium dodecyl sulfate-polyacrylamide gels, were subjected to sequential Edman degradation. The partial amino-terminal amino acid sequences obtained were compared with the sequence of amino acids predicted from the polyoma virus DNA sequencing (Arrand et al., J. Virol. 33:606--618, 1980). Together, these results showed that the 5' ends of the VP1, VP2, and VP3 coding sequences are located 1,217, 289, and 634 nucleotides, respectively, from the junction of HpaII restriction fragments 3 and 5.     

Type-specific and cross-reactive antigenicity of capsid proteins VP1 and VP2 of echovirus type 7

After disruption of echovirus type 7 virions with urea and heat, VP1 and VP2 were separated by isoelectric focusing in urea-containing sucrose gradients. Antisera to these two polypeptides were produced in guinea pigs. In complement fixation, antiserum to VP1 reacted with native and heated virions (N and H antigens, respectively) of homologous virus, and also cross-reacted with heated virions of some other enteroviruses used. Antiserum to VP2 was reactive only with heated virions of homologous and heterologous viruses. Interestingly, the anti-VP2 serum reacted neither with native nor even with heated procapsids (naturally-occurring empty capsids). Antiserum to VP1, but not VP2, showed neutralizing and hemagglutination-inhibiting activities. These results suggest that 1) both VP1 and VP2 possess cross-reactive antigenic determinants which are exposed on the surface of heated virions, and 2) type-specific determinants of VP1 are located on the surface of native virions.     

Alternate mRNA splicing is required for synthesis of adeno-associated virus VP1 capsid protein.

Fine-structure mapping of the capsid-specific mRNAs from adeno-associated virus (AAV) revealed an alternate splicing pattern in these RNAs. S1 nuclease and primer extension analyses showed that splicing of these mRNAs occurs at acceptor sites at nucleotide 2228 (major splice) or 2201 (minor splice). Both splice acceptors were ligated to the same 55-nucleotide leader in mature mRNAs. Both species were present in equal amounts in mRNA derived from AAV plasmid-transfected cells. However, when adenovirus infection accompanied the DNA transfection, the major splice predominated over the minor splice. Using cDNA clones of both the major and minor spliced mRNAs, we demonstrated that the largest AAV capsid protein, VP1, was derived from the minor spliced mRNA. The other capsid proteins, VP2 and VP3, came predominantly from the major spliced mRNA. These results, which describe the previously undetected minor splice, provide a mechanism for the production of all three AAV virion proteins.     

Cell-free assembly of the herpes simplex virus capsid.

Herpes simplex virus type 1 (HSV-1) capsids were found to assemble spontaneously in a cell-free system consisting of extracts prepared from insect cells that had been infected with recombinant baculoviruses coding for HSV-1 capsid proteins. The capsids formed in this system resembled native HSV-1 capsids in morphology as judged by electron microscopy, in sedimentation rate on sucrose density gradients, in protein composition, and in their ability to react with antibodies specific for the HSV-1 major capsid protein, VP5. Optimal capsid assembly required the presence of extracts containing capsid proteins VP5, VP19, VP23, VP22a, and the maturational protease (product of the UL26 gene). Assembly was more efficient at 27 degrees C than at 4 degrees C. The availability of a cell-free assay for HSV-1 capsid formation will be of help in identifying the morphogenetic steps that occur during capsid assembly in vivo and in evaluating candidate antiherpes therapeutics directed at capsid assembly.     

Myristoylation of budgerigar fledgling disease virus capsid protein VP2.

We present evidence that the structural protein VP2 of budgerigar fledgling disease virus, an avian polyomavirus, is specifically modified by covalent attachment of myristic acid. The fatty acid linkage is insensitive to hydroxylamine treatment and thus represents the amide type of fatty acylation of proteins.     

Expression of the polyomavirus minor capsid proteins VP2 and VP3 in Escherichia coli: in vitro interactions with recombinant VP1 capsomeres.

The polyomavirus VP2 and VP3 capsid proteins were expressed in Escherichia coli. The majority of the expressed proteins were in an insoluble fraction, and they were extracted and initially purified in 8 M urea before renaturation. Soluble VP2 and VP3 were mixed with purified recombinant VP1 capsomeres, and their interactions were assayed by immunoprecipitation and ion-exchange chromatography. Coimmunoprecipitation could be demonstrated with antibodies to either VP1 or VP2/VP3. Mixing recombinant VP1 with VP2 and VP3 modified the recognition of VP1 by domain-specific antipeptide antibodies and altered the chromatographic behavior of the individual proteins. Similar results were observed when a truncated VP1 protein, delta NCOVP1, with 62 amino acids deleted from the carboxy terminus was mixed with VP2/VP3. After the mixing, equilibrium dissociation constants for their binding to either VP1 or delta NCOVP1 were determined to be 0.37 +/- 0.23 microM for VP2 and 0.18 +/- 0.21 microM for VP3. These studies demonstrate that the recombinant VP2 and VP3 proteins interact with VP1 to affect the biochemical properties of VP1 capsomeres and to change the epitope accessibility of VP1 pentamers. These changes may reflect conformational alterations in VP1 capsomeres which are necessary for viral genome encapsidation.     

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.     

Simian virus 40 and polyoma virus induce synthesis of heat shock proteins in permissive cells.

During the lytic infection of monkey and mouse cells with simian virus 40 and polyoma virus, respectively, the preferentially increased synthesis of two host proteins of 92,000 and 72,000 Mr was observed by 15 to 20 h after infection besides the general stimulation of most cellular proteins. The incubation of uninfected monkey and mouse cell cultures for 30 to 60 min at 43.5 degrees C induced the enhanced synthesis of at least three proteins of 92,000, 72,000 and 70,000 Mr, the last one being the major heat shock protein of mammalian cells. Two-dimensional gel electrophoresis and partial proteolytic digestion confirmed that the same 92,000- and 72,000-Mr proteins are stimulated by virus infection and thermal treatment. In simian virus 40-infected CV-1 cells, we also observed the weak stimulation of a 70,000-Mr protein comigrating in gel electrophoresis with the major heat shock protein. The 92,000-, 72,000- and 70,000-Mr proteins of monkey cells are structurally very similar to the corresponding proteins of mouse cells. In immunoprecipitations, no specific association of these proteins to simian virus 40 T antigens was noticed.     

Phosphorylation of the budgerigar fledgling disease virus major capsid protein VP1.

The structural proteins of the budgerigar fledgling disease virus, the first known nonmammalian polyomavirus, were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major capsid protein VP1 was found to be composed of at least five distinct species having isoelectric points ranging from pH 6.45 to 5.85. By analogy with the murine polyomavirus, these species apparently result from different modifications of an initial translation product. Primary chicken embryo cells were infected in the presence of 32Pi to determine whether the virus structural proteins were modified by phosphorylation. SDS-PAGE of the purified virus structural proteins demonstrated that VP1 (along with both minor capsid proteins) was phosphorylated. Two-dimensional analysis of the radiolabeled virus showed phosphorylation of only the two most acidic isoelectric species of VP1, indicating that this posttranslational modification contributes to VP1 species heterogeneity. Phosphoamino acid analysis of 32P-labeled VP1 revealed that phosphoserine is the only phosphoamino acid present in the VP1 protein.     

Correlation between genetic loci and structural differences in the capsid proteins of polyoma virus plaque morphology mutants.

Two plaque morphology variants of polyoma virus (A-2 and 208) showed marked differences in agarose gel electrophoresis of the whole particles, isoelectric focusing of the major capsid protein VP1 (45,000 daltons) and three tryptic peptides (A, B and C) of VP1. No major difference in apparent molecular weight on NaDodSO₄ gels, amino acid composition or carbohydrate detectable by Schiff staining was revealed between the capsid proteins of the two viruses.Correlations have been made between phenotype, portions of the primary amino acid sequence of VP1 and the physical map of polyoma virus DNA by analysis of this protein from large plaque A-2 virus, minute plaque 208 virus and large plaque 208 virus selected after marker rescue with a fragment of polyoma virus DNA generated by the Hpa II restriction enzyme. The interrelationship of these properties was established by taking advantage of the observations of Miller, Cooke and Fried (1976)that heterozygous markers present on heteroduplex DNA are found in 100% of selected progeny and in only 50% of unselected progeny.All five marker rescued isolates selected for large plaque morphology showed only two A-2-specific characters, the absence of peptide C in tryptic maps of VP1 and the aggregation of VP1 on isoelectric focusing. The other four characters which distinguish A-2 and 208 were present or absent in 40–60% of the five isolates, which is close to the expected 50% for unselected markers. Three of the four A-2-specific characters (the presence of peptide A, absence of peptide B and isoelectric point of VP1) have been found to occur coordinately in the marker rescued isolates. The fourth character (electrophoretic mobility of virus particles in agarose gels) segregated independently.The techniques used in this study should find wide application in correlating primary amino acid sequence, nucleotide sequence and phenotype in other systems.     

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.     

The capsid determinant of fibrotropism for the MVMp strain of minute virus of mice functions via VP2 and not VP1.

The minute virus of mice, prototype strain MVMp, productively infects cultured murine fibroblasts but not T cells. The immunosuppressive strain, MVMi, shows the converse tropism. These reciprocal tropisms are mediated by the viral capsids, in which their determinants have been mapped to a few specific amino acids in the primary sequence shared by VP1 and VP2. Which of these proteins is relevant in presenting these determinants during infection is not known. We have approached this question using a recombinant parvovirus system in which a LuIII-derived transducing genome, containing the luciferase reporter in place of viral coding sequences, can be packaged by capsid proteins from separate helper sources. We generated transducing virions by using helper constructs expressing either VP1 or VP2, containing the MVMp or MVMi tropic determinant region, in various combinations. The virions were used to infect human NB324K cells and murine A9 fibroblasts. Transduction of the human cells (permissive for both MVMp and MVMi) required both VP1 and VP2 and was successful with all combinations of these proteins. In contrast, significant transducing activity for A9 cells was detected only with recombinant virions containing VP2 of MVMp, while the use of either source of VP1 had little effect. We conclude that VP2 from MVMp is necessary to enable infection of murine A9 fibroblasts.     

Cell-free synthesis of encephalomyocarditis virus

We developed a system for complete replication of encephalomyocarditis virus (EMCV) in a test tube by using an in vitro translation extract from Krebs-2 cells. Efficient virus synthesis occurred in a narrow range of Mg(2+) and EMCV RNA concentrations. Excess input RNA impaired RNA replication and virus production but not translation. This suggests the existence of a negative-feedback mechanism for regulation of RNA replication by the viral plus-strand RNA or proteins.