Specific secretion of polypeptides from cells infected with vaccinia virus.

The pattern of polypeptides specifically secreted by cells after infection with vaccinia virus has been analyzed. A complex pattern of apparently virus-specified polypeptides exhibiting temporal control of the type seen with intracellular polypeptides after virus infection was observed. Some of the specifically secreted polypeptides were shown to be modified by glycosylation and sulfation. The possible significance of these results is discussed.     

Characterization of polypeptides immunoprecipitable from Pichinde virus-infected BHK-21 cells

Using hamster anti-Pichinde virus serum, we immunoprecipitated polypeptides from BHK-21 cells infected with Pichinde virus. Seven immunoprecipitable polypeptides exhibited a time- and multiplicity of infection-dependent appearance when the cultures were pulse-labeled with L-[35S]methionine for 1 h. The predominant polypeptide was a nucleoprotein (NP) of 64,000 daltons. Components of 48,000, 38,000, and 28,000 daltons, when analyzed by two-dimensional tryptic peptide mapping, were found to be derived from NP. After a 3-h chase period, polypeptides of 17,000, 16,500, and 14,000 daltons were evident, and peptide mapping revealed that these three polypeptides were also related to NP. During a series of pulse-chase experiments, a 79,000-dalton glycoprotein (GPC) was cleaved to glycoproteins of 52,000 and 36,000 daltons. Radiolabel in a polypeptide of approximately 200,000 daltons (L) did not chase into smaller cleavage products. L, GPC, and NP were found to be unique by two-dimensional tryptic peptide mapping. Comparison of polypeptides immunoprecipitated from infected cells with structural components of purified virus revealed that L protein was evident in both. This is the first report of a high-molecular-weight polypeptide in Pichinde virus particles and infected cells.     

Plaque purification of cricket paralysis virus using an agar overlay on Drosophila cells

Cricket paralysis virus, an insect picorna-like virus, grown and assayed on Drosophila malanogaster cells, gave titers in excess of 10⁹ PFU/ml. The virus was plaque purified using an agar overlay and the intracellular polypeptides induced by “crude” and plaque-purified virions were demonstrated to be very similar.     

Herpesvirus sylvilagus I. Polypeptides of virions and nucleocapsids.

Herpesvirus sylvilagus was propagated in juvenile cotton tail rabbit kidney cells and purified from the cytoplasmic fraction of the infected cells. The purification procedure included zonal centrifugation through a 5 to 30% dextran t-10 gradient, followed by equilibrium centrifugation in a 5 to 50% potassium tartrate gradient. H. sylvilagus formed one band after centrifugation through the tartrate gradient at a density of 1.22 g/cm3. Contamination of the purified virus preparation by cellular proteins was less than 0.2% as determined by the removal of radioactivity from an artificially mixed sample containing [35S]methionine-labeled control cells and nonlabeled infected cells. H. sylvilagus nucleocapsids were isolated from infected cell nuclei and purified by sedimentation through a 36% sucrose cushion, followed by equilibrium centrifugation in 5 to 50% tartrate gradient. Forty-four polypeptides ranging in molecular weight from 18,000 to 230,00 were resolved when [35S]methionine-labeled enveloped H. sylvilagus was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Seventeen polypeptides found within the enveloped virus were also identified with the nucleocapsid. Six additional nucleocapsid polypeptides han no counterparts within the enveloped virus. The major polypeptide within both the virus and the nucleocapsid had a molecular weight of 150,000.     

Characterization of Measles Virus-Specific Proteins Synthesized In Vivo and In Vitro from Acutely and Persistently Infected Cells

Measles virus protein synthesis has been analyzed in acutely and persistently infected cells. To assess the role of measles in subacute sclerosing panencephalitis (SSPE), measles viral proteins synthesized in vivo or in vitro were tested for reactivity with serum from a guinea pig(s) immunized with measles virus and sera from patients with SSPE. Guinea pig antimeasles virus serum immunoprecipitates the viral polypeptides of 78,000 molecular weight (glycosylated [G]), 70,000 molecular weight (phosphorylated [P]), 60,000 molecular weight (nucleocapsid [N]), and 35,000 molecular weight (matrix [M]) from cells acutely infected with measles virus as well as from chronically infected cells, but in the latter case, immunoprecipitated M protein has a reduced electrophoretic migration. Sera of SSPE patients immunoprecipitated all but the G protein in acutely infected cells and only the P and N proteins from chronically infected cells. In immunoprecipitates of viral polypeptides synthesized in a reticulocyte cell-free translation system, in response to mRNA from acutely or persistently infected cells, the 78,000-molecular-weight form of the G protein was not detected among the cell-free products of either mRNA. Guinea pig antimeasles virus serum immunoprecipitated P, N, and M polypeptides from the products of either form of mRNA, whereas SSPE serum immunoprecipitated the P and N polypeptides but not the M polypeptide. The differences in immunoreactivity of the antimeasles virus antiserum and the SSPE serum are discussed in terms of possible modifications of measles virus proteins in SSPE.     

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.     

Mechanism of Interferon Action: Inhibition of Viral Messenger Ribonucleic Acid Translation in L-Cell Extracts

Encephalomyocarditis (EMC) virus ribonucleic acid (RNA) stimulated the incorporation of (14)C-amino acids into polypeptides in cell-free systems using preincubated S10 extracts from L cells. Incorporation was linear for over 2 hr. Analysis of the tryptic peptides derived from the polypeptide products formed in response to EMC RNA showed them to be virus specific. The major product, a polypeptide of 140,000 in molecular weight, migrated on sodium dodecyl sulfate-polyacrylamide gels with one of the virus-specific polypeptides present in EMC-infected cells. A minor component of molecular weight about 230,000 may correspond to the product of complete translation of the EMC virus genome. Little or no effect of interferon or vaccinia virus infection was observed in the preincubated, cell-free system. The EMC RNA-stimulated incorporation of (14)C-amino acids into polypeptides was not inhibited in extracts derived from L cells early in virus infection, from interferon-treated cells, or from cells subjected to both treatments. Interferon treatment did appear to have a slight inhibitory effect on chain elongation in this system. However, treatment of cells with highly purified interferon before virus infection caused a decrease of about 80% in the capacity of non-preincubated cell extracts to translate added EMC RNA. This effect did not extend to the translation of polyuridylic acid and could be reversed by preincubation of the extracts at 37 C for 20 min. The inhibition of translation was manifest at interferon concentrations as low as 5IU/ml, and in this respect closely paralleled the inhibition of virus growth. Inactivation of the antiviral activity of the interferon by heating or digestion with trypsin also abolished the effect on cell-free protein synthesis. The EMC-specific polypeptides formed in reduced amounts in extracts of interferon-treated vaccinia-infected cells were smaller than those formed in extracts of untreated, vaccinia-infected cells. Thus, inhibition of initiation or elongation of polypeptides, or both, can be demonstrated in cell-free systems employing non-preincubated extracts from interferon-treated, virus-infected cells. These results indicate that antiviral activity of interferon is directed against the translation of viral messenger RNA.     

Cells from newborn rat brain established in primary tissue culture will support rubella virus replication

We have initiated an in vitro study comparing the susceptibility of newborn and adult animals to rubella virus (RV) associated encephalitis. Glial cells from injured adult rat brain (RG cells) have been established in continuous culture and these cells were reported to restrict RV replication. When RG cells were infected, no infectious progeny virus particles were detected in tissue culture media and only five intracellular viral polypeptides could be detected using immune precipitation techniques (p75, p60, VP44, VP41, and VP19). Two polypeptides normally associated with a productive infection. VP24 and p30, could not be detected. In this report we have applied these techniques to an investigation of RV replication in newborn brain cells and have shown that relatively normal yields of all seven polypeptides found in RV-infected cells could be detected. These data indicate that some glia from newborn brain in primary culture are permissive for RV replication unlike the restricting RG cells and that this difference in restriction is critical in determining the outcome of their infection by RV.     

Serological studies with low-molecular-weight polypeptides from the Moloney strain of murine leukemia virus.

Major virion low-molecular-weight polypeptides were isolated from the Moloney strain of murine leukemia virus (type C) by agarose chromatography in 6M guanidine hydrochloride and were shown to have molecular weights of 15,000 (p15), 12,000 (p12), and 10,000 (p10) by their elution volumes and by their relative mobilities in sodium dodecyl sulfate-polyacrylamide gels. Each polypeptide could be iodinated and employed in double antibody radioimmunoassay procedures. All three polypeptides demonstrated a high degree of type-specificity in serologic immunoprecipitation analysis and in corresponding competition immunoassays. The p15 was immunologically distinct from other viron polypeptides including p12 and p10; the p12 and p10 were highly related to each other but not to other virion polypeptides and were even more type-specific than the p15 in serologic tests. Competition immunoassays with p15 and p10 indicate that the Moloney strain of MuLV is only a distant relative of the Friend-Rauscher group. The combined use of the Kirsten and Moloney low-molecular-weight polypeptide immunoassays suggest that xenotropic viruses constitute yet another group(s) of murine leukemia virus with distinct type-specific antigens, further expanding an already heterogeneous group of mouse type C viruses.     

Studies on the polypeptides of varicella-zoster (V-Z) virus. 1. Detection of varicella-zoster virus polypeptides in infected cells.

Virus-induced polypeptides in cells infected with varicella-zoster virus (VZV) were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. When human embryonic lung (HEL) cells infected with the Oka strain of VZV were labelled with 35S-methionine or 14C-glucosamine from 40 hr to 46 hr after infection, at least 18 VZV-induced polypeptides and 10 glycoproteins could be identified in the infected cells. The molecular weights of the polypeptides and glycoproteins ranged from about 145,000 to 23,000, and from about 105,000 to 48,000, respectively. Lysates of VZV-infected cells were treated with specific antisera prepared in green monkeys or guinea-pigs, and analysed by SDS-PAGE and fluorography. In all, 33 polypeptides (with molecular weight of about 145,000 to 22,000) and 13 glycoproteins (molecular weight, about 105,000 to 38,000) were found in the immunoprecipitates. None of these polypeptides and glycoproteins were detected when infected cells cultured in the presence of phosphonoacetic acid (PAA) were treated in the same way.     

The glycoprotein products of varicella-zoster virus gene 14 and their defective accumulation in a vaccine strain (Oka).

Many characteristics of the putative protein encoded by varicella-zoster virus (VZV) open reading fram (ORF) 14 indicate that it is a glycoprotein, which has been designated gpV. To identify the protein products of the gene, the coding sequences were placed under the control of the vaccinia virus p7.5 promoter and recombinant vaccinia viruses were constructed. Heterogeneous polypeptides with molecular weights of 95,000 to 105,000 (95K to 105K polypeptides) were expressed in cells infected by a vaccinia virus recombinant (vKIP5) containing ORF 14 from VZV Scott but were not expressed by control vaccinia viruses. These polypeptides were recognized by antibodies present in human sera that contained high levels of anti-VZV antibodies. Conversely, antisera raised in rabbits inoculated with vKIP5 reacted specifically with heterogeneous 95K to 105K polypeptides present in VZV Scott-infected but not uninfected cells; these polypeptides show a patchy plasma membrane fluorescence pattern in VZV Scott-infected cells. These same antisera neutralized VZV strain Scott infectivity in the absence of complement. Endoglycosidase F treatment of isolated gpV polypeptides and tunicamycin treatment of cells infected with the vKIP5 recombinant indicated that the polypeptides were glycosylated. Three sets of data imply that the VZV strain Oka, which has been used to produce a live attenuated virus vaccine, accumulates low levels of gpV polypeptides relative to wild-type strains: (i) blocking of antibodies in human sera with excess VZV Oka-infected cell antigen yielded residual antibodies which were reactive with the 95K to 105K gpV polypeptides expressed in cells infected by VZV strain Scott and by the vKIP5 vaccinia virus recombinant, but not with Oka-infected cell polypeptides; (ii) antisera raised to vKIP5 detected very low levels of reactive polypeptides made in VZV Oka-infected cells and neutralized VZV Oka virus much less efficiently than VZV Scott; and (iii) comparisons of the reactivity of sera from live attenuated virus vaccine vaccinees with sera derived from patients recovering from wild-type infections indicated greatly reduced levels of gpV-specific antibodies in some vaccinees.     

Immunological Cross-Reactions Between Two Low-Molecular-Weight Polypeptides from A Murine Type C Virus

Two low-molecular-weight type-specific virion polypeptides from the Kirsten strain of Murine leukemia virus, polypeptides p10 and p12, are immunologically related by radioimmunoassay competition techniques.     

Viral polypeptides detected by a complement-dependent neutralizing murine monoclonal antibody to human cytomegalovirus.

Murine monoclonal antibodies were produced which coimmunoprecipitated, under reducing conditions, 130,000- and 55,000-dalton (Da) polypeptides from cells infected with human cytomegalovirus (CMV) strain AD169. A 92,000-Da species, possibly a biosynthetic intermediate, was also detectable. One of the monoclonal antibodies, 15D8, neutralized CMV AD169 only in the presence of guinea pig complement. A second monoclonal antibody, 14E10, coimmunoprecipitated the 130,000- and 55,000-Da polypeptides but did not neutralize viral infectivity. By sequential immunoprecipitation, both monoclonal antibodies have been shown to recognize the same polypeptides. Monoclonal antibody 15D8 detected the 130,000- and 55,000-Da polypeptides in five of six clinical strains and three laboratory strains tested. The 14E10 monoclonal antibody detected the 130,000-Da protein in four of six CMV clinical isolates and in strain AD169 but did not immunoprecipitate any polypeptides from extracts of cells infected with either Towne or Davis laboratory strains. In kinetic studies, the synthesis of the 130,000-Da polypeptide preceded the appearance of the 55,000-Da polypeptide. In infected cells radiolabeled with a pulse of L-[35S]methionine, the isotope was initially detected in the 130,000-Da polypeptide but could be chased into the 55,000-Da polypeptide. These polypeptides exist in the intracellular and extracellular virus as disulfide-linked multimers. Extracellular virus contained a high-molecular-weight (greater than 200,000 Da) multimer composed entirely of 55,000-Da polypeptides. In extracts from infected cells an additional high-molecular-weight multimer was detected consisting of disulfide-linked 130,000-Da polypeptides.     

Proteins of Epstein-Barr Virus. II. Electrophoretic analysis of the polypeptides of the nucleocapsid and the glucosamine- and polysaccharide-containing components of enveloped virus.

Two series of experiments were undertaken to identify the topological location of the structural polypeptides of Epstein-Barr virus. In the first series of experiments, nucleocapsids prepared by detergent treatment of enveloped virus with Nonidet P-40 and sodium deoxycholate were found to be composed of seven polypeptides, VP2, 6, 7.5, 24, 27, 31, ANd 33, which ranged in molecular weight from over 200 X 10(3) to 28 X 10(3). Nine other polypeptides, VP 4, 7, 8, 10, 15, 16, 23, 28, and 29, could be identified in preparations of Epstein-Barr virus nucleocapsids, but the relative amount of this second group of polypeptides was less in preparations of nucleocapsids than in preparations of enveloped virus. The incomplete removal of these polypeptides from enveloped virus by detergent treatment suggests that some of these polypeptides may be components of the envelope or tegument that lie in close proximity to the outer surface of the nucleocapsid In the second series of experiments periodic acid-Schiff-staining and glucosamine-containing components were identified with similar electrophoretic mobility to several of the polypeptides of enveloped virus (VP 5, 8, 9, 11, 12, 13, 14, 15, 16, 17, 28, and 29) that were completely or incompletely removed from purified virus preparations by detergent treatment. The similarity between the polypeptide composition of the nucleocapsids of Epstein-Barr virus and herpes simplex virus was in contrast to the dissimilarity between the nonnucleocapsid polypeptides of Epstein-Barr virus and herpes simplex virus.     

Polypeptide Synthesis in Simian Virus 5-Infected Cells

Polypeptide synthesis in three different cell types infected with simian virus 5 has been examined using high-resolution polyacrylamide slab gel electrophoresis, and all of the known viral polypeptides have been identified above the host cell background. The polypeptides were synthesized in infected cells in unequal proportions, which are approximately the same as they are found in virions, suggesting that their relative rates of synthesis are controlled. The nucleocapsid polypeptide (NP) was the first to be detected in infected cells, and by 12 to 14 h the other virion structural polypeptides were identified, except for the polypeptides comprising the smaller glycoprotein (F). However, a glycosylated precursor (F(0)) with a molecular weight of 66,000 was found in each cell type, and pulse-chase experiments suggested that this precursor was cleaved to yield polypeptides F(1) and F(2). No other proteolytic processing was found. In addition to the structural polypeptides, the synthesis of five other polypeptides, designated I through V, has been observed in simian virus 5-infected cells. One of these (V), with a molecular weight of 24,000, was found in all cells examined and may be a nonstructural viral polypeptide. In contrast, there are polypeptides present in uninfected cells that correspond in size to polypeptides I through IV, and similar polypeptides have also been detected in increased amounts in cells infected with Sendai virus. These findings, and the fact that the synthesis of all four of these polypeptides is not increased in every cell type, suggest that they represent host polypeptides whose synthesis may be enhanced upon infection. When a high salt concentration was used to decrease host cell protein synthesis in infected cells, polypeptides IV and (to a lesser extent) I were synthesized in relatively greater amounts than other cellular polypeptides, as were the viral polypeptides. The possibility that these polypeptides may play some role in virus replication is discussed.     

Biosynthesis of Rauscher leukemia viral proteins

Antisera to disrupted Rauscher leukemia virus (RLV) or to the purified Rauscher viral 30,000 dalton polypeptide were used to specifically precipitate newly synthesized intracellular viral polypeptides from extracts of infected NIH Swiss mouse cells (JLS-V16). Analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of extracts from cells pulse-labeled for 10–20 min with ³⁵S-methionine showed that immune precipitates contained none of the nonglycosylated internal structural polypeptides of mature viruses. The major viral-specific polypeptides labeled in 10 min included polypeptides of 180,000, 140,000, 110,000, 80,000, and 60,000 daltons with minor polypeptides of 65,000, 50,000, and 40,000 daltons. Labeling the intracellular virus-specific polypeptides with ¹⁴C-glucosamine indicated that the 180,000, 110,000, 80,000, and 60,000 dalton polypeptides were glycosylated, and all but the 110,000 dalton polypeptides are contained in the mature virions. Based on pulse-chase experiments, it appears that at least 3 of the large polypeptides (140,000, 65,000, and 50,000 daltons) are precursors to the three major internal structural polypeptides of the mature virions.     

Characteristics of chromatin preparations from herpes simplex virus infected cells

Chromatin isolated from herpes simplex virus type 1-infected baby hamster kidney cells contains a number of tightly associated virus-induced polypeptides. A subset of these proteins bind to immobilized DNA in vitro (Vmw 175, 155, 130, 63, 43, 38/39). Virus-induced polypeptides extractable with acid from infected cell chromatin include Vmw 155, the major capsid protein of herpes simplex virus type 1 virions, and Vmw 63 and 38/39 which are heterogeneous with respect to charge and are phosphorylated. These chromatin preparations, in the presence of deoxynucleoside triphosphates and MgCl2 were capable of synthesizing viral and cell DNA in a reaction which was stimulated by the addition of ATP, riboNTPs and potassium acetate. In vitro synthesized viral DNA co-sedimented with prelabelled parental DNA but the single-stranded product was smaller than parental DNA. Density labelling indicated that extensive synthesis was taking place and all BamHI fragments of viral DNA were represented by the DNA synthesized in vitro.