Protein synthesis in cells infected by Autographa californica nuclear polyhedrosis virus (Ac-NPV): the effect of cytosine arabinoside

Twenty-two virion polypeptides (VP) were detected reproducibly when the occluded form (OV) of [35S]methionine-labeled Ac-NPV, purified from polyhedral inclusion bodies (PIB), was analyzed by polyacrylamide gel electrophoresis and autoradiography. Ten of the VPs and the polyhedral protein (PP) were phosphorylated and 6 VPs were glycoproteins. Purified, nonoccluded virus (NOV) revealed 25 polypeptides. During a single cycle of virus replication, the synthesis of 33 infected cell polypeptides (1CP) was detected in different relative proportions at different times. They were arbitrarily designated as early (0-12hr), middle (12-18 hr), and late 18-24 hr) polypeptides. Most of the middle and late proteins seemed to be viral structural proteins. Rapid post-translational cleavage of IPCs was not observed; however, pulse-chase experiments revealed post-translational modifications of at least four polypeptides. Inhibition of DNA synthesis at the time of infection did not prevent the synthesis of ICPs, VPs, or the formation of PIBs. Progeny PIB from both control and cytosine arabinoside (Ara C)-treated cells that were infected with radiochemically pure [3H]thymidine-labeled NOV contained parental virus DNA. Electron micrographs of thin sections showed that, whereas PIBs from control cultures contained complete virions, those from Ara (C-treated cells contained both full and empty virus.     

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.     

Vaccinia virus antigens on the plasma membrane of infected cells. I. Viral antigens transferred from infecting virus particles and synthesized after infection

SDS-polyacrylamide gel electrophoretic analysis of plasma membranes prepared from L cells infected with radioiodinated vaccinia virus particles showed that at 2.0 hr postinfection, 125I-labeled virion polypeptides with molecular weights of 58K-60K, 32K-34K, 17K, and 12K-14K were associated with infected cell plasma membranes. By 4 hr postinfection, only the 32- to 34-kDa polypeptide, derived from infecting virus particles, could be detected on infected cell surfaces. A variety of techniques were applied to analyzing purified plasma cell membranes to define the viral antigens expressed on cell surfaces after infection, including (a) surface radioiodination of infected cells; (b) immune or Western blotting; (c) specific immunoprecipitation of viral proteins present in nonionic detergent extracts of membranes purified from [35S]methionine-labeled, virus-infected cells. It was determined that vaccinia virus-specified polypeptides with molecular weights of 78K-82K, 65K, 50K, 42K-45K, 35K-37K, 32K-34K, 30K, 20K, and 17K-18K were expressed by 3 hr postadsorption, on the plasma membranes of infected cells and were accessible to binding by exogenous antiviral antibodies. Viral antigens with molecular weights similar to those expressed on cell surfaces were secreted or shed from infected cells and could be detected in the medium harvested from virus-infected mouse L-cell cultures.     

Tryptic and chymotryptic methionine peptide analysis of the in Vitro translation products specified by the transforming region of adenovirus type 2

Early region 1 (El) cytoplasmic RNAs of adenovirus type 2 were translated in vitro. Structural relationships between these proteins were then established by tryptic and chymotryptic digestion and two-dimensional peptide mapping. This analysis also allowed a direct comparison of these proteins with polypeptides isolated from early infected cell extracts and previously assigned to E1 by indirect means (M., Green, W. S. M. Wold, K. H. Brackmann, and M. A. Cartas, 1979, Virology 97, 275–286). E1a (0–4.5%) RNA encodes five proteins in vitro: 35K, 41K, 47K and 53K at early times, and a 28K protein at later times in infection. These five proteins are all highly related by peptide mapping analysis, and are also related to a group of four proteins isolated from cell extracts prepared early in infection. E1b (4.5–11.0%) early RNA encodes 52K and one or more 15K proteins in vitro. The 52K protein shares tryptic peptides with a 53K immunoprecipitated T antigen. A 15K protein shares some peptides with the 52K protein and with several proteins isolated from infected cells. At intermediate to late times, E1b RNA encodes a 12K protein that corresponds to the virion structural polypeptide IX. These data also permit the correlation of E1 proteins synthesized in vitro with the polypeptides predicted by E1 sequence analysis.     

Equine cytomegalovirus: structural proteins of virions and nucleocapsids

Enveloped virions and nucleocapsids of equine cytomegalovirus (ECMV; equine herpesvirus type 2) have been purified from the supernatants and the nuclear extracts of infected rabbit kidney (RK) cells, respectively, and their structural protein compositions have been analyzed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that ECMV nucleocapsids were composed of nine proteins (average molecular weights = 148K, 52K, 49.5K, 46K, 43.5K, 38.5K, 27K, 20K, and 18K), which together constituted 89% of the total nucleocapsid protein on the basis of incorporated 3H-labeled amino acids. The 148K protein comprised 47.3% of the total protein and thus appeared to be similar in molecular weight and proportional composition to the major capsid proteins of other herpesviruses. Purified virions were composed of 37 proteins whose average molecular weights ranged from 14K to greater than 200K. Three intense glycoprotein bands (83K, 78K, and 73.5K) as well as four less intensely labeled glycoproteins were detected in [3H]glucosamine-labeled virion preparations. At least 14 structural proteins were readily detected in extracts of infected cells which had been [35S]methionine labeled late in infection, and 11 of these were immunoprecipitated by rabbit antiserum against purified virions. The protein composition of ECMV differs substantially from those of equine herpesvirus type 1 and type 3 as well as from those of other herpesviruses.     

Presence in infected cells of nonvirion proteins encoded by adenovirus messenger RNAs of the major late transcription regions L0 and L1

The adenovirus major late promoter functions at early and intermediate times to produce a limited set of mRNAs that appear in the cytoplasm of productively infected HeLa cells. These mRNAs may be translated in cell-free systems to produce two unrelated polypeptides of approximately 13,500 Mr (L0-13.5K and L0-13.6K) and a pair of related polypeptides of approximately 55,000 Mr (the L1-52K/55K proteins). Radiochemical protein sequence analysis of in vitro synthesized proteins has identified the N-terminal sequences of the L0-13.5K and L0-13.6K proteins (J. B. Lewis and C. W. Anderson (1983), Virology 127, 112-123). Additional sequence analyses confirmed the identification of the open reading frame for the L0-13.5K protein, and identified the ATG encoded by nucleotides 11,040 to 11,042 from the left end of the adenovirus genome as the initial codon of the L1-52K/55K protein. Antisera raised against synthetic peptides homologous to these three amino termini were used to demonstrate the presence of the L0-13.5K protein, the L0-13.6K protein, and the L1-52K/55K proteins in extracts of HeLa cells infected by adenovirus 2. The L0-13.5K protein was detected at early, intermediate, and late times after infection. The L0-13.6K and L1-52K/55K proteins were detected only at late times. Immunofluorescence microscopy indicated that the L0-13.6K protein is distributed around the periphery of the nucleus and along fibers running the length of the cell. Nonpermeabilized infected cells were stained by anti-L0-13.6K peptide serum at a single spot on the cell surface. Neither the L0-13.6K nor the L1-52K/55K proteins were detected in purified virus.     

Studies on human parainfluenza virus 3: characterization of the structural proteins and in vitro synthesized proteins coded by mRNAs isolated from infected cells

The structural proteins of human parainfluenza virus 3, a member of the paramyxovirus family, were characterized by SDS-polyacrylamide gel electrophoresis of radiolabeled virus. The purified virion contains at least eight structural proteins, with estimated molecular weights of 251K, 90K, 71K, 68K, 65K, 51K, 35K, and 21K, respectively. Three of the polypeptides (71K, 65K, and 51K) were identified as glycoproteins based on their incorporation of [3H]glucosamine. Disruption of the virus by Triton X-100 in the presence of increasing salt concentrations indicated that the polypeptides of molecular weights 251K, 90K, 68K, and 21K were components of the nucleocapsid. In parainfluenza virus 3 infected BS-C-1 cells, seven virus structural polypeptides were identified. Six structural proteins (90K, 71K, 68K, 51K, 35K, and 21K) were detected in the cell lysate at 7 hr after infection, while at 10 hr an additional polypeptide (251K) was also observed. At least two nonstructural polypeptides of molecular weights 30K and 25K were also detected in infected cells. mRNAs isolated from virus-infected cells were translated in a cell-free protein-synthesizing system. The in vitro translation products were identical to the authentic virion polypeptides as determined by partial digestion with staphylococcal V8 protease.     

Intracellular distribution and phosphorylation of virus-induced polypeptides in frog virus 3-infected cells

Analysis of the intracellular localization of frog virus 3 (FV 3)-infected cell polypeptides (ICPs) showed that although the largest amount of newly synthesized proteins were recovered, at any time postinfection, in the cytoplasmic fraction, most of the early viral-induced ICPs were present in the nuclei and the relative molar ratio of ICPs 90, 42, 31 was higher in the nuclei than in the cytoplasm. After 5–8 hr late polypeptides species were found concentrated in the nucleus (ICPs 70, 63, 12). The absence of viral DNA replication did not prevent the appearance of ICPs in the nucleus. Under abortive conditions produced by the replacement of arginine by canavanine, only a restricted set of ICPs was induced (early polypeptides) and the nuclear concentrations of ICPs 31 and 42 was modified. Newly phosphorylated proteins in infected cells were predominantly found in the nucleus, most species being viral induced. Moreover, following the induction of protein kinase, the specific activity of this enzyme was about 50 times higher in the nuclei than in the cytoplasmic fraction. These results extend the evidence for specific steps of FV 3 replication in the host nucleus, emphasizing the importance of the nucleus as the predominant site of viral polypeptide phosphorylation.     

Respiratory syncytial virus glycoproteins

The proteins of respiratory syncytial (RS) virus were analyzed by SDS-polyacrylamide gel electrophoresis. Eight virion structural proteins with molecular weights of 180,000, 89,000, 48,000, 42,000, 34,000, 28,000, 25,000, and 21,000 were identified. These proteins were given tentative designations of L (180,000), G (89,000), F1 (48,000), NP (42,000), P (34,000), M (28,000), Vp25 (25,000), and F2 (21,000). The 89,000-, 48,000-, and 21,000-dalton polypeptides were glycosylated and could be purified on lentil-lectin sepharose columns. All three glycoproteins could be immunoprecipitated from extracts of infected cells but not from uninfected cells, suggesting that they are viral specified. The host cell affected the apparent molecular weights of the largest and smallest glycosylated polypeptides possibly by differences in glycosylation. The 48,000- and 21,000-dalton glycopolypeptides were disulfide linked subunits of a 68,000-dalton glycoprotein that was seen on unreduced gels. The 68,000-dalton glycoprotein was thus similar to the fusion (F) protein of paramyxoviruses. Treatment of infected cultures with tunicamycin, a drug that blocks glycosylation, inhibited syncytial formation and resulted in over a 1000-fold reduction of extracellular infectious virus. Virions purified from tunicamycin-treated cells had reduced amounts of all three glycosylated proteins. No new forms of these proteins were conclusively identified, suggesting that unglycosylated forms of RS glycoproteins were not incorporated into virion membranes.     

Heat-inactivated frog virus 3 selectively inhibits equine herpesvirus type 1 translation in a temporal class-dependent manner

Superinfection of equine herpesvirus type 1 (EHV-1)-infected rabbit kidney cells with heat-inactivated frog virus 3 (delta FV3) differentially blocked EHV-1 protein synthesis. The extent of inhibition varied with the specific EHV-1 message, but in general late protein synthesis was inhibited more than early and immediate early translation. Since FV3 has been shown to block heterologous RNA and protein synthesis, it was necessary to determine whether the observed reduction in herpesvirus protein synthesis was primarily due to a block in translation or to an earlier inhibition of EHV-1 mRNA synthesis. To distinguish between these alternatives, replicate cultures of EHV-1 infected cells were either superinfected with delta FV3 or treated with 10 micrograms/ml actinomycin D at 6 hr after infection, and EHV-1 protein synthesis monitored 3 hr later. We found that addition of actinomycin D to EHV-1 infected cultures had only a slight effect on EHV-1 translation, whereas superinfection with delta FV3 markedly reduced EHV-1 protein synthesis. This result suggested that the observed decline in EHV-1 protein synthesis was not due to the inhibition of herpesvirus mRNA synthesis. In addition, we showed that RNA extracted from delta FV3-superinfected cells directed the synthesis of full-size EHV-1 proteins in vitro indicating that shut-off was not caused by the degradation of EHV-1 mRNAs. Taken together these results show that delta FV3 selectively inhibited EHV-1 protein synthesis and are consistent with earlier observations which suggest that translational shut-off occurs at initiation.     

Synthesis of tacaribe viral proteins.

The synthesis of Tacaribe virus-specific proteins in infected BHK-21 cells has been analyzed by polyacrylamide slab gel electrophoresis and fluorography. The two major structural polypeptides of the virion were observed above the host cell background by pulse labeling with amino acid or sugar precursors. In addition to the virion glycoprotein (G), another major virus-specific glycoprotein (MW = 70,000) was detected in infected cells. The nucleoprotein (N) was first detected in infected cells around 24–34 hr postinfection (pi). It appeared to be synthesized as a primary gene product, since no larger precursor was detected by short pulse labeling. The rate of synthesis of N increased until 48 hr pi followed by a slight decrease by 72 hr pi. The structural and nonstructural glycoproteins were detected around 48 hr pi and their rate of synthesis increased up to 60 hr pi. The nucleocapsid protein, the minor nonglycosylated virion protein (P), as well as the structural and nonstructural glycoprotein could be specifically precipitated from infected cells using serum from rabbits hyperimmunized with Tacaribe virions. These results as well as pulse-chase experiments experiments suggest that the nonstructural glycoprotein may be a precursor of the virion glycoprotein.     

Immunoprecipitation of virus-specific immediate-early and early polypeptides from cells lytically infected with human cytomegalovirus strain AD 169

By immunoprecipitation of human cytomegalovirus-infected cell-specific polypeptides (ICPs) with a variety of human cytomegalovirus-positive sera and analysis by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, we can identify at least 20 ICSP bands from lytic infections by 6 hr postinfection (pi). Three of these polypeptide bands (78K, 73K, and 68K) may represent more than one species of polypeptide. Four polypeptide bands (78K, 77K, 73K, and 31K) can be identified as immediate-early based on their synthesis in the presence of actinomycin-D after removal from a protein synthesis block mediated by cycloheximide (CH). An immediate-early 78K polypeptide and an early 49K polypeptide are synthesized only transiently during the first 4 hr pi. Most immediate-early polypeptide synthesis is enhanced after removal of a 5 hr CH block. Taken together, these results identify many previously undetected immediate-early and early ICSPs and suggest that several regulatory events are occurring during the early phase of the lytic cycle.     

Host cell and virus strain differences in synthesis of immediate early polypeptides in HSV-1 infected cells

The synthesis of the immediate early (IE) polypeptides was analysed in primary rabbit kidney (RK) cells and a stable line of rabbit lung (ZP) cells infected with the syncytial (syn) strain HSZP and the non-syn strain KOS of herpes simplex virus type 1 (HSV-1). Results showed the following: After cycloheximide reversal the infection of RK and ZP cells with HSZP strain led to synthesis of five IE polypeptides (175K, 136K, 87K, 68K, and 63K), while infection of both cell cultures with the KOS strain led to synthesis of significantly reduced amounts of the IE polypeptides. The ability to switch on the expression of non-alpha viral genes was impaired in RK cells infected with the HSZP strain. The IE polypeptides were still detectable without any sign of the non-IE polypeptide synthesis 4 hr after cycloheximide reversal. The observed failure of the IE HSZP polypeptides to undergo posttranslational modification in ZP cells may be the consequence of this phenomenon. In contrast to the KOS IE mRNAs, the HSZP IE mRNAs exhibited a pronounced functional stability in both cell cultures. The IE polypeptides were still synthesized in HSZP-infected cells which had been incubated for 19 hr after cycloheximide reversal in the presence of actinomycin D (Act D). The HSZP strain failed to suppress the host polypeptide synthesis in RK but not in ZP cells. However, the HSZP strain, in contrast to the KOS strain, proved to be defective with respect to the early shutoff of host polypeptide synthesis in both cell cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vaccinia virus proteins on the plasma membranes of infected cells. II. Expression of viral antigens and killing of infected cells by vaccinia virus-specific cytotoxic T cells

Evidence is presented that virion-derived antigens as well as viral antigens expressed on cell surfaces after infection, may participate in the formation of "target-antigen complexes" (TACs) which render vaccinia virus-infected cells susceptible to recognition and killing by syngeneic, vaccinia virus-specific cytotoxic T cells (VV-CTLs). By employing L cells infected with trypsin-treated and untreated virions, evidence was obtained that proteins with molecular weights of 32K and 37K may be among the virion-derived antigens which participate in TAC formation. Following virus infection, a sequential expression of virus-specified antigens on the plasma membrane of infected cells could be detected. At 1 hr p.i., polypeptides with molecular weights of 48K-50K and 36K-37K were present on infected cell surfaces; by 2 hr p.i., polypeptides with molecular weights of 48K-50K, 42K-44K, 36K-37K, 29K-30K, and 16K-17K were detected on plasma membranes. As measured by in vitro, 51Cr-release assays, vaccinia virus-infected L cells were completely susceptible to lysis by VV-CTLs (greater than or equal to 50% measured specific lysis) when (a) "early" but not late viral functions were expressed as measured with virus-infected cells which had been treated with hydroxyurea (5 X 10(-3) M) to block DNA replication or (b) when active protein synthesis was allowed to proceed for 90 min postadsorption and the infected cells were then treated with cycloheximide (100 micrograms/ml) to block further protein synthesis. Under these experimental conditions, polypeptides with molecular weights of 58K, 48K-50K, 42K, 36K-37K, 34K, 32K-33K, 27K-29K, and 16K-17K were expressed on the plasma membranes of vaccinia virus-infected cells but not uninfected cells. Whether each of the virion-derived and (or) virus-encoded polypeptides can associate with Class I, major histocompatibility antigens on the surfaces of virus-infected cells to form a primary or cross-reacting TAC recognized by VV-CTLs remains to be investigated.     

The 10K virion phosphoprotein encoded by gene US9 from herpes simplex virus type 1

Gene US9 of herpes simplex virus type 1 has been predicted, from DNA sequence analysis, to encode a protein of mol wt 10,026, designated 10K (D.J. McGeoch, A. Dolan, S. Donald, and F.J. Rixon (1985). J. Mol. Biol. 181, 1-13). We have investigated this protein by using a synthetic peptide corresponding to the 11 amino acids adjacent to the amino-terminal methionine and rasing antisera in rabbits. One antiserum was able to precipitate at least 12 electrophoretically distinct polypeptide species from extracts of BHK cells infected with HSV-1. The estimated molecular weights of these polypeptides ranged from 12K to 20K and immunoblotting showed them to be related proteins. The primary translation product has an apparent mol wt of 13K. The various forms of 10K differ in their relative abundance in the infected cell and also in their degree of phosphorylation. Lower molecular weight forms of the 10K protein can be precipitated from NP-40 extracts of HSV-1 virions, suggesting that these forms of 10K are contained in the virion tegument or envelope. An association between this protein and nucleocapsids has also been observed in the nuclei of infected cells by immunoelectron microscopy. These observations imply that the product of US9 is a tegument protein which becomes associated with nucleocapsids at, or soon after, their formation in the nuclei of infected cells.     

Covalent attachment of psoralen to a single site on vesicular stomatitis virus genome RNA blocks expression of viral genes

Mumps virus was adapted to growth in Vero cells, which yielded virus of high infectivity titers. The structural polypeptides of purified virions grown in Vero cells were similar to those described previously for egg-grown mumps virus: L (200K), HN (79K), NP (72K), F₁ (61K), P (45K), M (40K), and F₂ (16K). We have analyzed the synthesis of viral polypeptides in Vero cells by pulse labeling with radioactive amino acid precursors. The nucleoprotein (NP) was the first to be detected intracellularly above the cellular protein background at 6 h.p.i. By 12 h.p.i., all viral polypeptides were observed except for the glycoproteins F₁ and F₂, which are derived from a precursor designated F₀ (74K). Two low-molecular-weight polypeptides not present in purified virions were also detected in infected cells. They are designated pI (28K) and pII (19K). Peptide mapping revealed that these two polypeptides share regions of their amino acid sequence and that they are also related to the structural protein P. Polypeptides pI and pII were found in several cell types (Vero, CEF, MDBK cells) infected with mumps virus. Infection of Vero cells with other paramyxoviruses (SV5 and Sendai virus) did not induce the synthesis of proteins comparable to pI and pII, whereas in mumps virus-infected cells no counterpart to the nonstructural C protein of Sendai virus was detected. Pulse-chase experiments suggest that pI and pII may not be derived from P by proteolytic cleavage.     

Detection of cellular proteins associated with human adenovirus type 5 early region 1A polypeptides

Antisera prepared against synthetic peptides corresponding to the amino and carboxy termini of human adenovirus type 5 (Ad5) early region 1A (E1A) proteins were used to identify polypeptides that are associated with these viral species in lytically infected KB cells. Proteins were sought which coprecipitated with E1A polypeptides using both sera and which were not recognized in extracts from mock-infected cells by either serum. Four such species were identified with apparent molecular weights of 68K, 65K, and a doublet at about 105K. A fifth species migrating with a molecular weight in excess of 250K was also identified consistently with E1A-C1 but not E1A-N1 serum. Addition of an excess of the appropriate synthetic peptide to the immunoprecipitation mixtures prevented the precipitation of all of these species. Mixing experiments demonstrated that all species were cellular proteins expressed in normal uninfected KB cells and in addition showed that an association with E1A proteins could take place in vitro. Studies carried out with the mutants pm975 and hr1 indicated that while the 105K doublet and the greater than 250K species were found with the products of both the 1.1- and 0.9-kb E1A mRNAs, 65K and 68K appeared to be primarily associated with those of the 1.1-kb mRNA. Finally, the 105K doublet and greater than 250K were shown to be phosphoproteins. These data indicated that Ad5 E1A proteins may function in a complex with cellular polypeptides which includes species of 105K, 68K, 65K, and possibly a large protein of greater than 250K.     

Differences between translation products of tick-borne encephalitis virus RNA in cell-free systems from Krebs-2 cells and rabbit reticulocytes: involvement of membranes in the processing of nascent precursors of flavivirus structural proteins

Tick-borne encephalitis virus (TBEV) RNA was translated in extracts from Krebs-2 cells and in rabbit reticulocyte lysates. In the former system, two polypeptides, p53 and p13, corresponding to envelope (E) and core (C) proteins of the virion, respectively, were synthesized preferentially. In contrast, the major product in reticulocyte lysates was represented by a heterogeneous set of high-molecular-weight polypeptides which did not appear to include p53 or p13. The reticulocyte lysates, however, acquired the ability to produce structural proteins (p53 at least) after addition of purified membranes isolated from the rough endoplasmic reticulum of Krebs-2 cells. On the other hand, the ability of Krebs-2 extracts to generate identifiable viral structural proteins was lost after degradation of membranes by the nonionic detergent Triton X-100. These findings strongly suggest that membrane-dependent processing of protein precursors is involved in the formation of TBEV structural proteins. Evidence has been obtained that only nascent precursor polypeptides can be processed efficiently into structural proteins in the membrane-dependent reaction.