Polypeptides of a viral DNA-protein complex form polyoma virus-infected cells.

A nascent viral DNA-protein complex was isolated from cells productively infected with polyoma virus (Py), by a conventional extraction procedure. This complex was purified by velocity sedimentation through neutral sucrose gradients, followed by ion-exchange chromatography. After in vitro labeling with ¹²⁵I, the protein moiety of the purified complex was analyzed by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. Four polypeptides were thus detected: viral structural polypeptides VP1, VP2, and VP3, and a nonstructural polypeptide of a molecular weight of approximately 70,000. The latter represented as much as 48% of the ¹²⁵I radioactivity associated with all four polypeptides.     

Characterisation of murine pneumonia virus proteins.

Twelve [³⁵S]methionine-labelled polypeptides ranging in molecular weight from 12,000 to 191,000, which were not present in mock-infected BS-C-1 cells, were detected in cells infected with murine pneumonia virus (PVM). All but two of these polypeptides were present in cell-released virus concentrated by polyethylene glycol precipitation and partially purified by equilibrium centrifugation. Two major glycopolypeptides, VPII (79,000 mw) and VPIII (68,000 mw) were present in partially purified virus but only VPII was prominent intracellularly. Nucleocapsids were isolated from lysates of infected cells by centrifugation in a gradient of 15–50% metrizamide and contained a major (VPIV) and a minor (VPVI) polypeptide with molecular weights of 42,300 and 35,700, respectively. Polypeptides with the electrophoretic mobilities of six of the PVM polypeptides (VPIV, VPV, VPVII, VPIX, VPX, and VPXI) were detected by in vitro translation of cytoplasmic RNA from PVM-infected cells in a cell-free system prepared from rabbit reticulocytes. PVM resembled RS virus, the other member of the pneumovirus genus, in its ability to multiply in enucleate cells. It is distinguished, however, by its four low molecular weight polypeptides (VPIX, VPX, VPXI, and VPXII), which appear to be primary gene products, and by the different electrophoretic mobility of the glycopolypeptides (VPII and VPIII).     

Proteins of equine herpesvirus type 3. I. Polypeptides of the purified virion.

Enveloped virions of equine herpesvirus type 3 (EHV-3) were purified from the extracellular fluids of infected horse embryo fibroblast cell cultures (KyED) by sequential banding in dextran-10 and potassium tartrate density gradients. The preparations of purified virus consisted of enveloped herpesvirus particles with little extraneous cellular material demonstrable by electron microscopy or by overt addition of labeled cellular proteins prior to purification. Structural polypeptides of the purified EHV-3 virions were analyzed by electrophoresis in SDS-polyacrylamide slab gels cross-linked with N,N′-diallytartardiamide. Thirty-four polypeptides, ranging in molecular weight from 14 × 10³ to 220 × 10³ were resolved in Coomassie brilliant blue-stained electropherograms of the purified virions. Ten of these proteins were larger than 100 × 10³ and two were larger than 200 × 10³. A 148 × 10³ capsid protein was a major structural polypeptide of the EHV-3 virion. Coelectrophoresis of the proteins of EHV-3 virions with those of the genetically unrelated equine herpes-virus type 1 (EHV-1) revealed a similarity in size range and number of the virion structural proteins; however, the molecular weights and proportional composition of the majority of EHV-3 polypeptides differed significantly from those of the virion proteins of EHV-1.     

Genetic characterization of plasmid formation by N- mutants of bacteriophage lambda.

Cells of plants infected with any of three strains of tobacco mosaic virus (the cowpea, common, and a wheat strain) contain, in addition to full-length virus rods, a heterodisperse population of rod fragments. RNA extracted from purified virus showed that some of these rods were of discrete sizes; the RNAs ranged in molecular weight from about 0.28 to 1.7 × 10⁶, containing 3′-ends identical to the original 3′-end of viral RNA, as judged by their capacities to bind a specific amino acid. Only the cowpea strain produced a rod containing a small (0.28 × 10⁶M_r) RNA, which is known to be the mRNA for coat protein. Each strain produced an RNA of 0.68 × 10⁶M_r (intermediate-length RNA) which coded in vitro (using a wheat germ system) for an ～30,000 M_r polypeptide. Other RNAs (0.9 to 1.7 × 10⁶M_r) were less similar in molecular weights among the three strains, but the predominant in vitro product of each was also the ～30,000 M_r polypeptide. peptide maps comparing the translation products of the short and intermediate RNAs of the cowpea strain showed they were distinct polypeptides. We conclude that at least some of the less-than-full-length viral rods found in preparations of the common and wheat strains of TMV, as well as those previously reported for the cowpea strain, represent the encapsidation of subgenomic portions of the viral RNA which are engendered during viral replication.     

A comparison of the polypeptides of human and bovine respiratory syncytial viruses and murine pneumonia virus

Human and bovine respiratory syncytial (RS) viruses and pneumonia virus of mice (PVM) are morphologically similar viruses and are the only known members of the metamyxovirus (or pneumovirus) group. Comparison of the polypeptides of these viruses by polyacrylamide-gel electrophoresis (PAGE) has shown that the serologically related human and. bovine RS viruses have similar polypeptide profiles. However, PVM does not resemble the other two viruses closely. The molecular weights of RS virus polypeptides were established by discontinuous SDS-PAGE in gradient slab gels and were comparable to previous determinations by continuous SDS-PAGE (Wunner and Pringle, 1976), apart from the detection of an additional 10,000-MW polypeptide. Comparison of 11 strains of human RS virus isolated from different localities, or from the same locality at different dates, showed that the relative mobility of VP32 (a nonglycosylated polypeptide of unknown function) was a stable characteristic of each strain. The mobilities of the other viral polypeptides showed little variation. The 11 strains fell into three groups in which the molecular weights of VP32 were estimated to be 31,000, 32,000, and 35,000, but there was no clear correlation with date or place of origin.     

Equine herpesvirus in vivo: cyclic production of a DNA density variant with repetitive sequences.

The DNA of a strain of equine herpesvirus type 1 passed more than 500 times in Syrian hamsters (EHV-1ha) has been analyzed by CsCl equilibrium density gradient ultracentrifugation, analytical sedimentation, and DNA-DNA reassociation kinetics. The viral DNA consisted of light and heavy species having densities in CsCl of 1.716 and 1.724 g/cm³, which correspond to guanine plus cytosine contents of 56 and 64%, respectively. These values were confirmed by T_m measurements. Similar molecular weight values were obtained by analytical sedimentation for the light (87.9 × 10⁶) and heavy (81.8 × 10⁶) DNA species. The heavier species was produced in a cyclic manner. Hamsters infected with virus containing a high proportion of the heavy species gave reduced virus yields and survived longer. The genetic relatedness of the two viral DNA species of EHV-1ha was compared by examining the ability of each to reanneal with ³²P-labeled viral DNA of the tissue culture strain (L-M cell) of EHV-1 (EHV-1tc). The lighter (1.716 g/cm³) species of EHV-1ha was composed of unique sequences completely homologous to the entire EHV-1tc genome, while the heavier species (1.724 g/cm³) consisted of sequences homologous to approximately 50% of the EHV-1tc genome. Of these homologous sequences, 40–60% (20–30% of the entire EHV-1tc genome) were reiterated. Further, analyses of the EHV-1tc genome (fragmented and unfragmented) by thermal chromatography on hydroxylapatite and in neutral preparative CsCl equilibrium density gradients revealed considerable intramolecular heterogeneity in nucleotide distribution. Finally, analysis of the structural polypeptides of virions of EHV-1ha which contained the heavy and light DNA species revealed that the following two major viral proteins were missing from virions containing the heavier DNA species: VP8, an envelope protein with a molecular weight of 173,000, and VP23, a nucleocapsid protein with a molecular weight of 38,000.     

The polypeptides of human respiratory syncytial virus: products of cell-free protein synthesis and post-translational modifications.

The properties and inter-relationships of virus-induced polypeptides synthesized in BS-C-1 cells infected with human respiratory syncytial (RS) virus have been investigated in vivo and in vitro. Analysis of the kinetics of synthesis of RS virus-induced polypeptides in vivo provided no evidence for temporal controls, a situation comparable to that observed with other negative-stranded RNA viruses with unsegmented genomes. In vitro translation of cytoplasmic RNA extracted from infected cells resulted in the synthesis of six virus-induced polypeptides, four of which were of similar molecular weights to species produced in infected cells. Synthesis of all of these polypeptides was directed by polyA)-containing RNA. The identity of the major nucleocapsid polypeptide (VP 41) synthesized in vitro was confirmed by peptide mapping. A prominent polypeptide in infected cells, VP 38, was not synthesized in vitro. The relationship of this polypeptide to other RS virus polypeptides was investigated in detail, and it was observed that VP 38 and VP 41 exhibited a precursor-product relationship in pulse-chase experiments and appeared to be related by peptide mapping. However, addition of the protease inhibitors tolylsulfonyl-lysyl chloromethylketone (TLCK) and tolylsulfonyl-phenylalanyl chloromethylketone to infected cells during labeling enhanced the recovery of VP 41 and correspondingly decreased VP 38. A similar effect was obtained if addition of TLCK was delayed until the time of cell lysis. Taken together, these observations suggest that during the course of infection, the nucleocapsid polypeptide VP 41 undergoes a transition from a protease-sensitive form (detected by the occurrence of VP 38) to a protease-resistant form. Two other hitherto unreported post-translational modifications of RS virus-induced polypeptides, sulfation and phosphorylation, are described.     

The polypeptide composition of avian infectious bronchitis virus particles

Summary Egg grown avian infectious bronchitis virus (IBV) centrifuged on sucrose density gradients was found to consist of a major virus peak of density 1.17 to 1.18 g/cm³ and occasionally two minor virus peaks of density 1.21 to 1.22 g/cm³ and 1.13 g/cm³. Three different IBV strains were examined and no morphological differences were detected between virus particles of different densities or from different strains. The polypeptides of the different density virus particles from the three IBV strains were analysed on polyacrylamide gels. In all cases 7 polypeptides were observed, although there were differences in the proportions of these polypeptides in particles of different densities and those from the different strains. The polypeptides have been called VP1 (molecular weight 130,000), VP2 (105,000), VP3 (97,000), VP4 (81,000), VP5 (74,000), VP6 (51,000) and VP7 (33,000). Additional polypeptides were produced if slightly harsher treatments were used.With 4 Figures     

Charge heterogeneity in polypeptides of negative strand RNA viruses

We surveyed charge heterogeneity in the polypeptides of three enveloped RNA viruses, Sendai virus, influenza virus (WSN strain), and vesicular stomatitis virus (VSV). Isoelectric focusing in polyacrylamide gel was followed by electrophoresis in a second dimension after denaturation of the polypeptides with sodium dodecyl sulfate. Nucleocapsid polypeptides P and NP of Sendai virus exhibited charge heterogeneity which may correspond to various extents of post-translational phosphorylation (R. A. Lamb and P. W. Choppin, 1977). The synthesis of Sendai virus polypeptides in infected cells. 111. Phosphorylation of polypeptides. Virology81, 382–397. Nucleocapsid polypeptide N of VSV was more homogeneous, whereas the NP polypeptide of influenza virus appeared to be too basic (isoelectric point greater than 8.0) to be resolved in the isoelectric focusing system employed. Glycosylated envelope polypeptides of all three viruses separated into multiple (3 to 8) acidic species with isoelectric points in the range of 4 to 5 for VSV glycopolypeptide G and 5 to 6 for the glycopolypeptides of Sendai virus and influenza virus. Although some of the heterogeneity in VSV glycopolypeptide G may stem from variations in content of N-acetyl neuraminic acid (NANA), a different basis for the heterogeneity is suggested by failure of extensive neuraminidase treatment to abolish it. Moreover, NANA is removed from the glycopolypeptides of Sendai virus and influenza virus by virus-specified neuraminidases. A possible relationship between content of amino sugars (other than NANA) and charge heterogeneity was suggested by the finding that the amount of radioactive glucosamine incorporated biosynthetically into G of VSV and HN of Sendai virus was greater in the more electropositive species. The functional consequences of this variety of post-translational modifications remain to be determined.     

Measles virus-specified polypeptides in infected cells

Summary The synthesis of wild-type measles virus-specified polypeptides in Vero cells in pulse-chase experiments, in cells with synchronized protein synthesis by high salt concentration, and in the presence of proteolytic enzyme inhibitors was analyzed by polyacrylamide slab-gel electrophoresis. Six major (L, G, 2, NP, 5 and M) structural polypeptides were identified in infected cells. The results of pulse-chase experiments suggested that most of the structural polypeptides were synthesized at their final length. Polypeptide M was found to be sensitive to trypsin. In TLCK-treated cells its molecular weight was about 1000–2000 daltons higher than in untreated cells. A minor virus-specific polypeptide with a molecular weight of about 23,000 was found as a very faint and diffuse band. In addition, three nonstructural polypeptides with molecular weights of 65,000, 38,000 and 18,000 were also detected. The experiments with proteolytic enzyme inhibitors and with synchronized protein synthesis suggested that the polypeptide with a molecular weight of 65,000 might be a precursor of the structural polypeptide 5.With 4 Figures     

Two disulfide-linked polypeptide chains constitute the active F protein of paramyxoviruses.

The paramyxovirus glycoprotein which is required for virus-induced cell fusion, hemolysis, and the initiation of infection (F protein) has been found with three different viruses to consist of two disulfide-linked glycopolypeptide chains (F₁ and F₂) which are derived from the precursor glycopolypeptide (F₀) by proteolytic cleavage. The larger glycopolypeptide chain (F₁) previously identified in SV5, Sendai, and Newcastle disease virions has an estimated molecular weight of 48,000–54,000. The smaller polypeptide chain (F₂) has been found to have a molecular weight of ～10,000–16,000, depending on the virus. The identification of the F₂ polypeptide was accomplished by isolating the disulfide-linked complex (F_1,2) under nonreducing conditions, followed by reduction of the disulfide bonds and separation of the two polypeptide chains. Although both polypeptides are glycosylated, the F₂ polypeptide contains more carbohydrate per unit protein than F₁. No free N-terminus could be detected on the F₀ or F₂ polypeptides of Sendai virus, whereas N-terminal phenylalanine was found on F₁. This suggests that the order of the F₀ polypeptide is X-NH-F₂—Phe-F₁—COOH. The finding that with three different paramyxoviruses the biologically active virions possess F₁ and F₂ polypeptides suggests that this is a general feature of paramyxoviruses, and that the activation of infectivity, cell fusion, and hemolysis is due to a conformational change in the F protein resulting from proteolytic cleavage to form an active complex of two disulfide-linked polypeptide chains.     

Baculovirus structural polypeptides

The structural polypeptides of eight insect baculoviruses were studied using vertical slab polyacrylamide gel electrophoresis. All viruses revealed a complex but unique composition of 15 to 25 bands with molecular weights ranging from 15,000 to 160,000. Since certain baculoviruses have more than one nucleocapsid per viral envelope (multiples), comparisons were made of the multiples and singles (enveloped single nucleocapsids) for each virus. Quantitative and qualitative differences were documented to exist in polypeptide composition. Where possible, the envelopes of certain baculoviruses were selectively removed in order to identify the major capsid proteins. Autographa californica MNPV (NPV: nuclear polyhedrosis virus) capsids contained two major polypeptides, VP18.5 and VP37. Rachiplusia ou MNPV capsids contained several polypeptides of which VP16, VP17, VP18, VP30, and VP36 were considered major constituents. Anticarsa gemmatalis MNPV contained one major capsid protein, VP29, and several minor polypeptides. Major capsid proteins of Heliothis zea SNPV were VP16, VP28, and VP63; as were VP16, VP17, and VP31 of Trichoplusia ni granulosis virus (GV).     

Evidence for endogenous reovirus-like particles in a tissue culture cell line from Drosophila melanogaster

Virus-like particles were isolated from the Blood IIIBlood III cell line of Drosophila melanogaster by sucrose and cesium chloride density centrifugation. The particles have a buoyant density of 1.37 g/cm³ in cesium chloride and a diameter of 568 Å The viral genome contains double-stranded RNA by the criteria of resistance to ribonuclease A and nuclease S₁. Polyacrylamide gel electrophoresis resolves the RNA into 10-segments and an additional minor component rangina in molecular weight from 0.55 × 10⁶ to 2.75 × 10⁶.     

Comparison of capsid polypeptides of group B coxsackieviruses and polypeptide synthesis in infected cells

Summary Capsid polypeptides of all six types (B1–6) of group B coxsackieviruses were compared by high-resolution gel electrophoresis, and synthesis of protein and RNA in B4- or B5-infected HeLa cells was analyzed. Four polypeptides, VP1–4, were detected in each type. Another polypeptide, VP0, slightly larger than VP1, was also detected in trace amounts in some types. VP1–3 showed different but characteristic molecular weights (VP1, 34,500 to 37,000; VP2, 31,000 to 36,000; VP3, 26,000 to 32,500), and presented well-defined and reproducible differences in electrophoretic mobility. The molecular weight of VP4 ranged from 5,000 to 5,500. VP1 was largest in B2 and B4, smallest in B1, and of intermediate size in the other types. VP2 was largest in B4 and smallest in B2; VP3 was largest in B5 and B6 and smallest in B4. In B4- or B5-infected HeLa cells, host protein synthesis began to decline after 2 hours postinfection and was less than 20 percent of the control by 6 hours postinfection. Actinomycin D-resistant viral RNA synthesis started at about 2 hours postinfection, peaked by 5 hours, and then declined rapidly. Virus-specific protein synthesis began while host protein synthesis was declining, increased during the ensuing period, and declined in late infection. A number of virus-specific proteins with molecular weights from 23,500 to >92,500 were detected in the host cytoplasm. At least three of these proteins were also present in the nucleus. The kinetics of processing of virus-specific proteins were examined by pulse-chase experiments in B5-infected cells. The relative intensities of [³⁵S]-methionine-labeled polypeptides suggest that a number of smaller, stable chains (MW 23,500 to 38,000) are generated by cleavage of a precursor polypeptide (MW 92,500 to 100,000).With 7 Figures     

The polypeptide structure of transmissible gastroenteritis virus.

The polypeptides of purified preparations of the coronavirus responsible for transmissible gastroenteritis of pigs have beem examined by polyacrylamide gel electrophoresis. Four major polypeptides, VPI (mol. wt. 200000), VP2 (50 000), VP3 (30000) and VP4 (28500) and two minor polypeptides, VPIa (105000) and VPIb (80500) have been reproducibly demonstrated in the virion, of which VPI, VP3 and VP4 contain carbohydrate. Treatment of the virion with the proteolytic enzyme bromelain removes the surface projections and VPI, thus identifying this glycopolypeptide as the major structural component of the projection.     

Membrane-bound proteins of Japanese encephalitis virus-infected chick embryo cells

The seven Japanese encephalitis virus specific polypeptides found in infected chick embryo cells were all bound to membranes. None were completely released from the membranes by treatment with neutral salt, alkaline salt, or dilute detergent, but two of them were partially released by both the neutral and alkaline salts. The polypeptides were released or attacked by trypsin at unequal rates and in the sequence: NV-5≥ NV-4 > V-3. NV-5 was released as a relatively undegraded soluble polypeptide, NV-4 was extensively degraded, and V-3 was degraded but part of its trypsinderived fragment (TF-2) remained membrane bound. We suggest that the three largest viral polypeptides are bound in such a manner that the larger the polypeptide, the more exposed and superficial it is. Treatment of virions with trypsin produced low molecular weight material and three discrete polypeptide fragments, probably all derived from the large virion envelope protein V-3; two (TF-1 and TF-3) had electrophoretic mobilities similar to the two naturally occurring nonvirion virus-specified polypeptides, NV-1 and NV-3.     

Herpes simplex virus protein synthesis in the presence of 2-deoxy-D-glucose.

The proteins and glycoproteins induced by herpes simplex virus type 1 (HSV-1) were labeled with [¹⁴C]amino acids or [¹⁴C]glucosamine in the presence or absence of 2-deoxy-d-glucose (deoxyglucose) and analyzed by slab gel electrophoresis. In the presence of 0.1% deoxyglucose (6.1 mM), the major envelope glycoprotein (VP123, MW 123,000) labeled with [¹⁴C]glucosamine was shifted to a component of an apparent lower molecular weight (VP123′). In the presence of increasing concentrations of deoxyglucose, there was a progressive decrease in the amount of [¹⁴C]amino acids incorporated into polypeptides which normally band in the VP123 region. Concomitant with this decrease was an increase in [¹⁴C]amino acids incorporated into a polypeptide(s) of greater electrophoretic mobility and of an apparently lower molecular weight. The polypeptide(s) was designated DG92 (MW 92,000) and was found to be predominantly associated with the nuclear fraction of HSV-1-infected cells cultured in the presence of deoxyglucose. The effects of deoxyglucose on HSV-1 polypeptide synthesis could be prevented by the addition of mannose.     

Proteins specified by African swine fever virus

Summary At least 28 polypeptides have been identified in intracellular virus, with molecular weights ranging from 11,500 to 243,000 daltons. By treatment with Nonidet P-40 and 2-mercaptoethanol it is possible to obtain subviral particles that have lost some proteins and have a density in CsCl of 1.31 g/cm³ which is higher than that of the complete virus (1.23 g/cm³). After addition of NaCl the virus loses its major protein VP73 which indicates that it is localized in the viral envelope. Cores obtained after this treatment are made up of at least 14 proteins. Incorporation of³H-fucose and³H-glucosamine in intracellular virus occurs in three minor components. The protein VP42 is possibly the cell actin and appears to be strongly associated with the virus. It is not possible to eliminate it under conditions where the viral envelopes desappear morphologically. At least the proteins VP172, VP162, VP146 and VP73 act as antigens in the natural infection.With 5 Figures     

An analysis of type-C retrovirus polypeptides and their associations in the virion.

The polypeptide compositions of [³H]leucine-labeled Prague-Rous sarcoma virus-subgroup C (Pr-RSV-C) and Friend murine leukemia virus (FLV) were investigated using guanidine hydrochloride gel filtration and a high-resolution SDS-polyacrylamide-gel system. These techniques resolved seven major structural components in Pr-RSV-C (gp85, gp35, p27, p19, p12, p15, and p10), as reported previously for other avian leukosis-sarcoma viruses. However, in the case of FLV two previously unresolved proteins (p15E and p12E) were clearly demonstrated in addition to gp71, p30, p15C, p12, and p10. FLV p15E, p12E, and gp71 were removed when intact virions were digested with bromelain, whereas the remaining components were not affected. These and other studies support the notion that gp71, p15E, and p12E are situated on the surface of the virion. The linkages (covalent and noncovalent) between viral polypeptides were also examined. The results of these studies indicate that more than 90% of avian gp85 and gp35 are disulfide linked as a viral glycoprotein complex (VGP). The data also suggests that p19 exists as a network of disulfide-linked molecules, some of which may be further linked to VGP. In contrast to the avian system, only about 10–15% of FLV gp71 is disulfide linked to p15E in the VGP complex; the remaining gp71 is apparently loosely attached to the virus, perhaps by a noncovalent interaction with p12E. The implications of these associations to virus structure and assembly are discussed.