Dengue virus induced polypeptide synthesis

Summary Dengue 2 virus polypeptide synthesis was investigated in BHK₂₁ cells. Nine or ten virus induced polypeptides were identified, three of which are glycoproteins as demonstrated by tunicamycin treatment of infected cells. We performed pulse chase experiments, experiments with amino-acid analogs, protease inhibitors or pactamycin treatment of infected cells, to determine whether or not large polypeptide processing occurs. In some of these experiments a large polypeptide (P130) was immunoprecipitated by an anti-dengue 2 serum. We observed a transfer of label between small molecular weight polypeptides which might be the result of restricted proteolytic cleavage.With 5 Figures     

The synthesis of polypeptides in influenza C virus-infected cells

The synthesis of virus-specific polypeptides was analyzed in MDCK cells infected with the JJ/50 strain of influenza C virus. In addition to three major structural proteins gp88, NP, and M, the synthesis of five polypeptides with molecular weights of 29,500 (C1), 27,500 (C2), 24,000 (C3), 19,000 (C4), and 14,000 (C5) was found in infected cells. None of these polypeptides were detected either in virions or in immunoprecipitates obtained after treatment of infected cell lysates with antiviral serum, suggesting that they are not viral structural proteins. Polypeptides C1-C5 were found to be synthesized in MDCK cells infected with different influenza C virus strains as well as in different host cell types infected with C/JJ/50. Further, it was observed that cellular protein synthesis was greatly reduced under hypertonic conditions, whereas the synthesis of C1-C5 was relatively unaffected. These results suggest that polypeptides C1-C5 are virus coded rather than host cell coded. Peptide mapping studies showed that each of polypeptides C3, C4, and C5 had a peptide composition similar to the M protein. The amount of C2 synthesized in infected cells was insufficient for mapping. This polypeptide was, however, found to rapidly disappear in pulse-chase experiments, suggesting that C2 is probably not unique but biosynthetically related to one of the other proteins. In contrast to these polypeptides, polypeptide C1 showed a map which is largely different from any major structural polypeptide. It therefore appears likely that C1 is a nonstructural protein of influenza C virus similar to the NS1 protein of influenza A and B viruses.     

The synthesis of Sendai virus polypeptides in infected cells. II. Intracellular distribution of polypeptides.

The intracellular distribution and the kinetics of association of Sendai virus polypeptides with cytoplasmic fractions and plasma membranes have been studied. The viral surface glycoproteins HN and F₀ have been found in pulse-chase experiments to migrate from rough to smooth membranes and to the plasma membrane. The M protein was found in varying amounts in most cell fractions but was predominantly associated with smooth membranes; there was no evidence of its migration from rough to smooth membranes. The L, P, and NP polypeptides were found with the rough membrane and free ribosome fractions. Polypeptides B and C, which were previously found in extracts of whole infected cells, were found to be unstable during cell fractionation. In the presence of protease inhibitors, polypeptide C, which is thought to be a virus-specific nonstructural protein, was found with the rough membrane fraction. Thus its instability in cell fractions appears to be due to proteolytic digestion. Polypeptide B was not found in cell fractions even in the presence of protease inhibitors. Evidence reported in the following paper has indicated that B is a phosphorylated form of polypeptide M and that its instability is presumably due to loss of phosphate. Polypeptides I–IV, which the available evidence suggests are cellular polypeptides whose synthesis may be enhanced in infected cells, were found in significant amount in soluble form after cell fractionation, although IV was also associated with most membrane fractions.     

Characterization of the polypeptides synthesized in cells infected with a temperature-sensitive mutant derived from an HVJ (Sendai virus) carrier culture

Summary The intracellular synthesis of virus-specific polypeptides in cells infected with the wild-type virus of HVJ (HVJ-W) (haemagglutinating virus of Japan—the Sendai strain of parainfluenza 1 virus) and with a temperature-sensitive(ts) mutant (HVJ-pB) derived from an HVJ carrier culture has been analysed by polyacrylamide gel electrophoresis. At the permissive temperature (32° C), all of the known virus structural polypeptides were identified in cells infected with each strain of virus and in addition to the non-structural polypeptides B and C, another polypeptide at the region with a molecular weight of 26,000 to 27,000 (26 to 27K) could be detected in infected cells. At the non-permissive temperature (38° C), the synthesis of the polypeptide M was markedly restrained in cells infected with HVJ-pB, while other major virus polypeptides were present in approximately comparable amounts to cells infected with the wild-type virus. A non-structural polypeptide with a molecular weight of 105K was dominant ints mutant infected cells at higher temperatures and disappeared after temperature-shift from 38° to 32° C. The production of the non-structural polypeptides B and 27K was also temperature-sensitive. The molecular weights of the polypeptides B, M and 27K in HVJ-pB infected cells were larger than those of the corresponding polypeptides in HVJ-W infected cells. The synthesis of the M protein in HVJ-pB infected cells started just after lowering the incubation temperature and the newly made M protein was successfully incorporated into virus particles.With 4 Figures     

Control of vesicular stomatitis virus protein synthesis.

The five structural polypeptides of vesicular stomatitis virus are synthesized in infected cells at nonequimolar rates which correspond to their relative amounts in the virus particle. These experiments are concerned with the mechanism(s) responsible for the relative rates of viral polypeptide synthesis.Treatment of infected cells with amino acid analogs in order to prevent formation of functional viral proteins resulted in inhibition of viral genome replication and virion assembly. This treatment, however, did not alter the relative rates of viral polypeptide synthesis. This suggested that the mechanisms determining these rates do not require the function of newly-synthesized viral proteins and do not involve direct coupling between protein synthesis and virion assembly.Relative translation frequencies of viral messenger RNA molecules coding for each polypeptide were compared by treating cells with low levels of cycloheximide and anisomycin, inhibitors of protein synthesis that interfere preferentially with polypeptide chain elongation. Under these conditions the rate of synthesis of each viral polypeptide should be proportional only to the amount of its messenger RNA available for translation. The relative rates of viral polypeptide synthesis were not altered by this treatment, suggesting that viral messenger RNA molecules coding for each polypeptide are translated with similar average frequencies.Rates of peptide chain growth were compared by following the incorporation of ³H-amino acids into completed viral polypeptides following a pulse-label. The time necessary to achieve maximum incorporation of isotope into each completed viral polypeptide was taken as its translation time, the time necessary to synthesize and release a completed molecule. We were able to determine translation times for the M and G polypeptides by this method and found that they correlated directly with their molecular weights, suggesting that nascent chains of these two polypeptides are propagated at similar rates.     

Proteins induced by infection with caliciviruses.

Three polypeptides with mol. wt. 100 (P100), 80 (P80) and 65 (P65) X 10(3) were found in calicivirus infected cells. P100 and P80 were present in sub-molar amounts compared with P65 and no precursor product relationship between the three polypeptides could be demonstrated using pulse-chase experiments or selective inhibitors of protein synthesis and of proteases. In the presence of protease inhibitors a polypeptide with mol. wt. 120 X 10(3) (P120) was demonstrated which appeared to be the precursor of P100. Possible mechanisms of translation in the caliciviruses are discussed.     

Machupo virus polypeptides: Identification by immunoprecipitation

Summary The most abundant protein in purified Machupo virions (Corvallo strain) labelled with¹⁴C-Protein hydrolysate is a 64K polypeptide which is associated with virion RNAs. Another structural polypeptide, 37K, solubilized by nonionic detergent seems to be a major surface glycoprotein. In addition to this, a 78K polypeptide and a minor 50K polypeptide have been detected.In Machupo virus infected cells three virus-specific polypeptides similar in size to those described for structural polypeptides were immunoprecipitated with anti-Machupo virus serum. The most abundant virus-specific polypeptide was nonglycosylated (64K, NP), and the others were glycosylated polypeptides (78K and 37K). The synthesis of NP and 78K polypeptides was recognized at the beginning of a log phase of virus replication. Pulse-chase experiments as well as experiments with an arginine analogue, canavanine (to block proteolytic processing) suggest that 78K is a precursor for structural glycoproteins of Machupo virions.With 4 Figures     

Antigens specified by herpesviruses. 3. Viral-induced nuclear polypeptides

Polyacrylamide gel electrophoresis was used to follow the sequential appearance within the nucleus of polypeptides synthesized in cells infected with herpes simplex virus type 1. Coelectrophoresis of ³H-labeled uninfected cell polypeptides and ¹⁴C-labeled nuclear polypeptides from cells pulse-labeled for 2 hr at various times after infection, showed that the synthesis of the major host polypeptides had been shut off by 4 hr post-infection. Pulse-harvesting and pulse-chase experiments revealed the presence of viral induced polypeptides in nuclei from cells labeled between 0 and 2 hr post-infection. At this early time period it was possible to detect the presence of the major structural polypeptide. Coelectrophoresis of ³H-labeled purified viral nucleocapsids obtained by treatment of enveloped virus particles with NP-40 with ¹⁴C-labeled nuclear extracts revealed that the majority of the nuclear polypeptides were components of the nucleocapsid. The molecular weight of the nuclear viral-induced polypeptides ranged from 11,500 to 175,000.     

The polypeptides induced in Drosophila cells by Drosophila C virus (strain Ouarzazate)

The Ouarzazate strain of Drosophila virus (DCV0) was grown in Drosophila melanogaster tissue culture cells, and [35S]methionine-labeled virions were found to contain a group of major structural proteins with a molecular weight of approximately 30,000 as well as several minor proteins of higher molecular weight and a protein of approximately 10,000 daltons. Using a range of pulses, chases and gel systems, examination of the intracellular proteins induced by DCV0 showed the presence of 17 polypeptides not found in uninfected cells. The synthesis of virus-induced polypeptides was extremely asymmetric with a rapid appearance of the major virus structural proteins and a much slower appearance of the lowest molecular weight structural protein (VP4). Processing of virus-induced proteins including the appearance of VP4 was demonstrated using pulse-chase after pulsing with [35S]methionine. While the highest molecular weight induced protein found in infected cells was 146,000, pretreatment of cells with iodoacetamide resulted in the appearance of a protein with a molecular weight of approximately 200,000. The evidence presented in this paper supports the inclusion of DCV0 in the Picornaviridae group.     

Polypeptides of bovine rotavirus.

Polyacrylamide gel electrophoresis of bovine rotavirus or neonatal calf diarrhoea virus (NCDV) grown in cell culture resolved eight species of polypeptide. The inner shell particles contained five polypeptides and the outer shell three polypeptides. A major polypeptide of the outer shell was glycosylated. The infectivity of NCDV was enhanced by treatment with trypsin in vitro. All eight polypeptides were affected by trypsin treatment as judged by diminished intensity of polypeptide bands by radiography and several new bands appeared. The intracellular synthesis of NCDV polypeptides was studied by pulse and pulse-chase experiments. Infected cells contained all eight virus capsid proteins and, in addition, three presumably virus-specific polypeptides which were non-capsid polypeptides (NCVP). There was no evidence that any of these polypeptides was processed after synthesis. It is suggested, therefore, that all these polypeptides are primary gene products.     

Effect of impaired glycosylation on the synthesis of envelope proteins of rauscher murine leukemia virus

The effects of 2-deoxy-d-glucose (deoxyglucose) and cytochalasin B on the synthesis of Rauscher murine leukemia viral envelope proteins were investigated. JLS-V9 cells infected with and producing Rauscher murine leukemia virus (R-MuLV) were cultured in the presence of deoxyglucose (15 mM) or of varying concentrations of cytochalasin B (0.1–25 μg/ml) and the synthesis of virus-specific polypeptides was examined in pulse-chase experiments. Overall protein synthesis was inhibited to some extent by cytochalasin B; at a concentration of 25 μg/ml an inhibition of about 70% was observed. Incorporation of d-[1-³H]glucosamine, however, was almost completely inhibited (97%) at the same concentration of the drug. Newly formed virus-specific polypeptides were analyzed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis after immuno-precipitation with polyvalent and monospecific antisera against R-MuLV proteins. No specific effect on the synthesis of the gag gene product p30 was observed. The synthesis of the precursor of the envelope polypeptides, env-pr82, however, was prevented in the presence of cytochalasin B. Instead, the synthesis of a new glucosamine-deficient 70,000-molecular weight polypeptide (env-pr70¹) was observed. Env-pr70¹ could be immunoprecipitated with anti-gp69/71 serum and anti-p15(E),p12(E) serum and, therefore, probably represents the protein moiety of env-pr82. In pulse-chase experiments in the presence of cytochalasin B, env-pr70¹ was converted to a polypeptide with a molecular weight of about 75,000 which was slowly lost during the chase period. Production of virus-specific gp69/71, p15(E), and p12(E) was inhibited under these conditions. Similar results were obtained with deoxyglucose, except that the inhibition of glycosylation by the latter compound is irreversible.     

Polypeptides specified by the influenza virus genome: I. Evidence for eight distinct gene products specified by fowl plague virus

The structural polypeptides of fowl plague virus (influenza A) and those synthesized in fowl plague virus-infected chick embryo fibroblasts have been analyzed by high resolution polyacrylamide gel electrophoresis. We detected eight distinct virus gene products: three polymerase-associated polypeptides (P₁, P₂, P₃), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), membrane polypeptide (M), and a nonstructural polypeptide (NS). The molecular weights of these polypeptides correlate closely with the molecular weights of the eight genome RNA species found in fowl plague virus.The three high molecular weight polypeptides, P₁, P₂, and P₃, were detected both in virions and infected cells, and their separate identity established by a two-dimensional tryptic peptide mapping procedure. An active RNA polymerase enzyme complex isolated from virions and virus-infected cells contained all three P proteins together with the NP protein. The nonstructural polypeptide (NS), together with the P proteins and the NP, appeared early in the infectious cycle, while the M protein and HA protein appeared later in infection. The NS and M polypeptides, which have similar molecular weights, were separated on SDS-polyacrylamide gels and shown to be distinct by tryptic peptide mapping.     

Coding assignments of Drosophila X virus genome segments: in vitro translation of native and denatured virion dsRNA

Both dsRNA genome segments of Drosophila X virus (DXV) were denatured and translated in vitro using nuclease-treated rabbit reticulocyte lysates. The synthesis of all four primary gene products was detected by polyacrylamide gel electrophoresis and autoradiography. Genome segment A (mol wt 2.3 X 10(6)) encoded polypeptides with molecular weights of 67,000 (67K), 34K, and 27K, whereas segment B (mol wt 22 X 10(6)) encoded the 110K polypeptide. The proteolytic processing of 67K which generates a 49K polypeptide in infected cells was also observed in vitro. Pulse-chase experiments indicated that synthesis of the three polypeptides encoded by genome segment A initiated independently and simultaneously, suggesting that segment A is polycistronic. Native (undenatured) DXV dsRNA could also be translated with high fidelity (vitro). The messenger activity of native dsRNA was abolished by S1 nuclease treatment but completely restored on subsequent denaturation. In vitro "pulse-chase" experiments using native dsRNA as messenger, indicated that the order of translation of polypeptides on genome segment A was 5'-67K-27K-34K-3'.     

Measles virus polypeptide synthesis in infected cells

The synthesis of measles virus polypeptides has been studied using an inhibitor of virus-induced cell fusion, carbobenzoxy-d-phenylalanyl-l-phenylalanyl-nitro-l-arginine (SV4814). Cells infected at a multiplicity of 10 fuse extensively by 17 hr and die shortly thereafter, making it difficult to detect viral polypeptides. Cells protected from fusion by SV4814 survive and continue to produce virus, and the synthesis of viral polypeptides can be followed for 4 days. The previously described measles virion polypeptides G, 2, NP, 5, and M have been identified in infected cells, and, in addition, a polypeptide (L) with a molecular weight of ～200,000 has been found in infected cells and in small amounts in virions. (New designations suggested for polypeptides G, 2, and 5 are H, P, and F₁, respectively.) A glycosylated polypeptide (F₀, MW ～62,000) has also been found in infected cells, but not in virions. This polypeptide is thought to be the precursor of two polypeptides which appear under pulse-chase conditions: F₁ (MW ～40,000), which is not glycosylated, and F₂, a small glycosylated polypeptide detected with [³H]glucosamine labeling. In addition to facilitating studies of measles virus polypeptide synthesis, the use of SV4814 has shown that cell fusion is the major factor in early cell death caused by measles virus, but that cell death ultimately ensues in the absence of cell fusion, indicating another mechanism of measles virus-induced cell damage.     

Translational strategies in potexviruses: products encoded by clover yellow mosaic virus, foxtail mosaic virus, and viola mottle virus RNAs in vitro

We have examined the template properties of the genomic RNAs from three potexviruses: clover yellow mosaic virus (CYMV), foxtail mosaic virus (FMV), and viola mottle virus (VMV). All three RNAs encode a large (160,000 to 182,000 molecular weight) nonstructural protein when translated in the rabbit reticulocyte cell-free system. Only CYMV RNA is able to direct the synthesis of significant quantities of a product whose electrophoretic mobility, antigenic determinants, and partial peptide map resemble those of authentic coat protein. All three viral RNAs also encode a number of discrete polypeptide products whose molecular weights are intermediate between those of the large nonstructural proteins and those of their respective coat proteins. We have examined the kinetics of synthesis in vitro of these intermediate products. For each of the three viral templates, as well as for papaya mosaic virus RNA, pulse-chase experiments suggest that most, if not all, the intermediate polypeptides are incomplete ("paused") or prematurely terminated precursors to the corresponding large nonstructural protein. A partial proteolytic map of the in vitro products encoded by CYMV RNA supports this interpretation. Our data, and those of others, are consistent with a model in which all potexviruses would encode a large nonstructural protein.     

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 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.     

Biosynthesis of Rauscher leukemia viral proteins: presence of p30 and envelope p15 sequences in precursor polypeptides.

Viral structural polypeptides p30 and a 17,000-dalton polypeptide, termed envelope p15, are formed in Rauscher leukemia virus (RLV)-infected N.I.H. Swiss mouse embryo fibroblasts by cleavage of high molecular weight precursor polypeptides. The evidence for this conclusion is based on the analysis of polypeptides precipitated from RLV-infected cells by antiserum directed against RLV structural proteins. High resolution sodium dodecyl sulfate (SDS)-polyacrylamide-gel electrophoresis (PAGE) of such immune precipitates from infected cells pulse-labeled with [³⁵S]methionine or pulse-labeled and then chased in unlabeled medium provides evidence that three size classes of unstable polypeptides are precursors to virion p30. They are: two polypeptides with an approximate molecular weight of 200,000 (termed Pr1a and b), an 80,000-dalton polypeptide (Pr3) and a 65,000-dalton polypeptide (Pr4). Ion-exchange chromatography of tryptic digests showed that methionine-containing tryptic peptides of p30 are present in these precursor polypeptides. Methionine-labeled tryptic peptide sequences of envelope p15 were present in a 90,000-dalton peptide fraction containing two components (Pr2a and b). The latter polypeptides comigrated with viral specific fucose-free glycoproteins not present in virions or uninfected cells.