Virus-specific Protein Synthesis Research Articles

Synthesis of virus-specific proteins in simian sarcoma virus-transformed primate cells

Marmoset monkey cells productively and nonproductively transformed by simian sarcoma virus (SSV) were evaluated for expression of simian sarcoma associated virus (SSAV)-specific structural polypeptides and SSV transformation-specific polypeptides. Virus-producing cells contained, in addition to virus structural proteins, a 62,000-dalton polypeptide (pr62 gag) precipitable by antisera specific for SSAV p28 or p10, and a polypeptide of approximately 80,000 daltons (pr80 env) which could be glucosamine-labeled and which was precipitated by antisera specific for SSAV envelope proteins. Results of pulse-chase experiments provided evidence for a precursor-product relationship between pr62 gag and p28 and between pr80 env and the envelope proteins gp70, p15E, and p12E. Pr62 and p12 were the only detectable phosphorylated virus-specific polypeptides. Nucleic acid hybridization techniques demonstrated SSAV-related DNA and RNA in clonal SSV-transformed nonproducer marmoset cell lines (HF/SSV-NP), but synthesis of SSAV-related protein was demonstrated in only one of five of these lines. This cell line expressed pr62 which was not processed to yield p28 or other gag gene products. Gel electrophoresis of immunoprecipitates demonstrated that SSV does not express a transformation-specific polyprotein comprised in part of either p28, p10, or p12 and a nonstructural component. Antibodies which precipitated a protein specific for SSV-transformed cells were not detected in sera from marmoset monkeys bearing SSV-induced tumors, or in sera from a goat or marmosets inoculated with autologous SSV-transformed cells or HF/SSV-NP cells.

The measles virus-specific protein synthesis of persistently and lytically infected cells studied in vivo and in vitro.

The virus-specific protein synthesis of the hamster brain cells persistently infected with a wild-type measles virus (M-MB/MVB) was investigated in vivo and in vitro and compared to the protein synthesis of the Vero-cells lytically infected with the wild-type measles virus and the SSPE-measles virus strain LEC. The virus-specific protein synthesis in the M-HB/MVB-cells in vivo was weak. Only two virus-specific polypeptides, corresponding to polypeptide G and nucleocapsid polypeptide NP, were detected. On the other hand, the in vitro translation products, coded by the RNA extracted from the actinomycin D-treated M-HB/MVB-cells, were similar compared to those products coded by the RNAs from the lytically infected Vero-cells. The polypeptides with MWs of 75 000, 61 000, 40 000, 37 000 and 18 000 were coded with all three RNA-extracts. So the similar mRNAs were induced in the actinomycin D-treated M-HB/MVB-cells, but their translation in vivo was limited. In addition to those polypeptides mentioned above the polypeptide with a MW of 42 000 was detected among the in vitro translation products of the M-HB/MVB-cells.

Frog virus 3 requires RNA polymerase II for its replication.

The involvement of host cell RNA polymerase II in the replication of frog virus 3 (FV 3) was examined in alpha-amanitin-sensitive or -resistant Chinese hamster ovary (CHO) cells in the presence and absence of alpha-amanitin. In the presence of alpha-amanitin, FV 3 replicated normally in resistant CHO cells but failed to do so in sensitive CHO cells. Synthesis of virus-specific RNAs and proteins was inhibited in sensitive cells infected in the presence of alpha-amanitin, but in alpha-amanitin-resistant cells, as expected, virus-specific protein synthesis and, by implication, virus-specific RNA synthesis were not affected by the presence of the drug. Inhibition of FV 3 replication was maximum when alpha-amanitin was added to sensitive CHO cells before virus adsorption, but the drug had no effect on virus replication if added after the adsorption. These data indicate that host RNA polymerase II was required for early transcription of the FV 3 genome and confirm a nuclear requirement for FV 3 RNA synthesis (R. Goorha et al., Virology 82:34-52, 1978).

Protein Synthesis in Cowpea Mosaic Virus-infected Cowpea Protoplasts: Detection of Virus-related Proteins

SUMMARY A study was made of the proteins synthesized in cowpea protoplasts infected with cowpea mosaic virus (CPMV). Protoplasts were labelled with 35S-methionine at different times during the infection cycle and the proteins synthesized were analysed by SDS-polyacrylamide slab gel electrophoresis of different subcellular fractions. Since no shut-off of host protein synthesis occurred, virus-specific protein synthesis had to be distinguished from a high background of protoplast proteins. Eleven polypeptides with mol. wt. of 170, 130, 112, 110, 87, 84, 68, 37, 30, 24 and 23 (all × 103) were detected whose synthesis was either induced or stimulated by CPMV infection. No differences were observed between the electrophoretic patterns of samples from uninfected and CPMV-infected protoplasts during the first 8 h of infection (latent phase). The 170 × 103 and 30 × 103 species were the first virus-related polypeptides detectable by labelling between 9 and 15 h after inoculation. From about 16 h after inoculation, all other virus-related proteins appeared to be synthesized at increasing rates. The polypeptides with mol. wt. of 170, 130, 112, 110, 68, 37 and 23 (all × 103) could be designated as virus-induced; the others may well be host-coded proteins, the synthesis of which is stimulated by CPMV infection. Although CPMV replication is known to occur in close association with virus-induced cytopathic membrane structures the 112 × 103 and 68 × 103 mol. wt. polypeptides were the only virus-related proteins specifically found in the particulate fractions of the protoplasts. From the 11 virus-related proteins, the 37 × 103, 24 × 103 and 23 × 103 mol. wt. proteins were identified as virus coat proteins. The 170 × 103 and 30 × 103 mol. wt. polypeptides also appeared to be virus-specific and to be coded for by CPMV bottom component RNA.

Different patterns of replication in influenza virus-infected KB cells.

When KB cells were infected either with the fowl plague (FPV) Rostock strain (Hav1N1) or the WSN (H0N1) strain of influenza A virus the yield of cell-associated haemagglutinin and neuraminidase polypeptides was essentially comparable, but virus particles were not produced in the FPV-KB system. WSN virus-infected KB cells synthesized normal amounts of mature virus particles and had all the characteristics of a permissive replication cycle. Biosynthesis and transport of RNP antigen from nucleus to cytoplasm of infected cells were traced by immunofluorescent staining at 4 and 8 hours after the beginning of infection. While the fluorescent-stained material was totally confined to the nuclei in FPV-infected KB cells, RNP antigen migrated out of the nucleus during the replicative cycle of WSN virus in the same host cell. Patterns of virus-specific protein synthesis were studied by pulse-labelling with 35S-methionine. The most significant feature concerned the amplification of synthesis of virus-induced matrix (M) protein which did not occur in FPV-infected cells but occurred normally during WSN infection. The different patterns of replication in the same host cell when infected by different influenza A viruses is discussed.

Sites of synthesis of viral proteins in avian sarcoma virus-infected chicken cells.

We determined the sites of synthesis of avian sarcoma virus-specific proteins in infected chicken cells by immunoprecipitation of the products synthesized in vitro by free and membrane-bound polyribosomes; 85% of Pr76, the precursor of the viral internal structural proteins (group-specific antigens), was synthesized on free polyribosomes, and 15% was synthesized on membrane-bound polyribosomes. Pr92, the lycosylated precursor of the viral glycoproteins (gp85 and gp35), was synthesized exclusively on membrane-bound polyribomes, which is consistent with its role as a membrane protein. When we investigated the site of synthesis of pp60src, the product of the avian sarcoma virus src gene, we found that 90% was synthesized on free polyribosomes, whereas 10% was detected on membrane-bound polyribosomes. The implications of these results with respect to the subcellular location of pp60src are discussed.

Neurovirulence mutant of vesicular stomatitis virus with an altered target cell tropism in vivo.

Intracerebral infection of weanling Swiss mice with a temperature-sensitive (ts) mutant of vesicular stomatitis virus (VSV), ts pi364, resulted in a unique neuropathological syndrome not previously described with other VSV mutants. Mice infected with wild-type VSV died from an acute encephalitis characterized by neuronal necrosis and efficient virus replication in both brain and spinal cord. In contrast, with VSV ts pi364, the most prominent histopathological feature was destruction of the ependyma of the lateral ventricles. Virus antigen was also limited to the leptomeninges and the lateral ventricles. Infected mice survived and developed hydrocephalus. Replication of ts pi364 in the brain was 10- to 100- fold less than that of wild-type VSV, and appearance of virus in the spinal cord was delayed. VSV ts pi364 was isolated from mouse cells persistently infected with VSV. Another VSV ts pi mutant, isolated from the same persistent infection, behaved in vivo like wild-type VSV, even though both mutants were very similar in plaque size, reversion frequency, cut-off temperature, and synthesis of virus-specific proteins at semipermissive temperature. These results strongly suggest that VSV ts pi364 has a second, non-ts mutation which results in a restricted target cell range in vivo; wild-type VSV can infect both neurons and ependymal cells, whereas ts pi364 does not replicate in neurons.

Inductive requirements for the generation of virus-specific T lymphocytes. II. Poxvirus and H-2 antigens associate without cellular or virus-directed protein synthesis, and remain immunogenic in cell membrane fragments.

The nature of the interaction between vaccinia virus (VAC) and fibroblastic cells that renders the latter capable of being recognized by virus-specific, H-2 identical murine T lymphocytes has been studied. L cells exposed for 10 min to VAC rendered noninfectious by exposure to ultraviolet light became susceptible targets for cytotoxic T lymphocytes (CTL) without the synthesis of new viral proteins. Susceptibility was retained even if cellular protein synthesis was irreversibly inhibited with pactamycin before virus exposure. Immobilization of cell-surface membranes by glutaraldehyde fixation before (but not after) exposure to virus severely impaired the formation of the "virus + self" complex that in vitro stimulated secondary CTL responses by H-2 identical virus-primed memory cells even though virus attachment to fixed cells were unaffected. This stimulatory complex, once formed, was maintained in membrane fragments prepared from cells previously exposed to VAC. These findings indicate that VAC-specific CTL or their immediate precursors can recognize only those viral envelope antigens that become membrane integrated and that this event requires neither host cell-specific nor virus-specific protein synthesis.

Action of Interferon on Aged Chick Embryo Cells

In primary chick embryo cells aged <i>in vitro, </i>vaccinia virus replication is drastically inhibited by pretreatment with low doses of interferon. The antiviral state was established with doses about ten times smaller than those required with normal primary chick cells. Viral protein synthesis is also highly sensitive to chick interferon in these cells. Cell-free lysates from aged chick cells translated several viral and globin mRNAs efficiently. While drastic inhibition of vaccinia virus-specific protein synthesis by interferon treatment could be detected in the intact aged cells, the translation in the cell-free lysate was not significantly affected by pretreatment of the cells with interferon or interferon followed by infection with vaccinia WR.

Transcription and replication of influenza virus RNA

Methods are described for measuring the amounts of virus-specific polyadenylated (A+) cRNA, nonpolyadenylated (A−) cRNA, and virus genome RNA (vRNA) in nucleus and cytoplasm of cells infected with influenza virus. The amounts of A(+) cRNA to individual virus genes accumulated during infection at different rates, not related to gene molecular weight, indicating transcriptional control. The pattern of accumulation of each gene transcript was the same in the nucleus as in the cytoplasm. Addition of cycloheximide during infection abolished the transcriptional control and resulted in linear accumulation of each gene transcript. In cells treated with 0.1 μg/ml actinomycin D, which blocked late but not early virus-specific protein synthesis, there was a significant accumulation of all gene transcripts in the nucleus and a relative decrease in transcripts of genes coding for late proteins (M and HA) in the cytoplasm. Analysis of A(−) cRNA and vRNA accumulations in infected cells showed that the A(−) cRNA synthesis precedes vRNA synthesis and net accumulation of A(−) cRNA ceased by 3 hr postinfection (pi). Significant amounts of vRNA in excess of input vRNA were detected by about 2 hr pi and continued to accumulate in both nucleus and cytoplasm at least up to 6 hr pi. Protein synthesis was required for A(−) cRNA production, since in the presence of cycloheximide very little A(−) cRNA, and no vRNA, could be detected. Actinomycin D (0.1 μg/ml) reduced the amount of A(−) cRNA, and completely inhibited the increase in vRNA which occurred from 2 hr pi in normal infection.

Interaction of influenza A virus with human peripheral blood lymphocytes.

Peripheral blood lymphocytes cultured in the presence of phytohemagglutinin, concanavalin A, or pokeweed mitogen were exposed in vitro to influenza A virus. The synthesis of several virus-specific proteins, including the nucleoprotein, membrane protein, and nonstructural 1 protein were detected, although no infectious virus was produced by the lymphocyte cultures. Evidence was obtained that only a subpopulation of mitogen-transformed cells would support virus protein synthesis. A comparison of the interactions of influenza A virus with lymphocytes from normal individuals and from rheumatoid arthritis patients showed that the same range of virus-specific proteins were made, in similar quantities, regardless of the source of lymphocytes.

Methylation of vaccinia virus-specific mRNA in the interferon-treated chick cell

Results obtained with cell-free protein-synthesizing systems imply a mediating role of the methylated 5′-terminal “cap” structure in the regulation of poxvirus-specific protein synthesis. In order to study a possible involvement of methylation in the interferon-induced inhibition of virus-specific protein synthesis in Vaccinia-infected cells, the extent of methylation of Vaccinia mRNA synthesized in interferon-pretreated and control chick embryo fibroblasts was compared. Induction of the antiviral state by pretreatment of chick cells with homologous interferon does not markedly reduce the degree of overall methylation of virus-specific mRNAs. Besides the two predominant “cap I” structures m 7GpppAmpN and m 7GpppGmpN, “incomplete” structures m 7GpppA and m 7GpppG can be detected in Vaccinia mRNA isolated from interferon-treated as well as from control cells. The ratio of complete to incomplete “cap I” structures is decreased in the viral RNA from interferon-treated cells. This qualitative change in “cap” methylation seems not to be limited to virus-specific mRNA. Possible implications of these findings on translational control in the interferon-treated cell are discussed.

Suppression of the Synthesis of Cellular Macromolecules by Herpes Simplex Virus

Synthesis of cellular protein was substantially inhibited within 1 h of infection with herpes simplex virus, type 2, strain G (HSV-2). The inhibition also occurred, although no virus-specific protein synthesis was detected, after infection with u.v. irradiated virus and in cytoplasts that had been enucleated before infection. The inhibitory activity could not be distinguished from infectivity by dilution, sedimentation or reaction with gamma-globulin. HSV-2 also suppresssed the synthesis of Sendai virus proteins, but not those specified by HSV-1. Host protein synthesis was no more sensitive than virus protein synthesis to an increased concentration of NaCl in the medium, nor could the suppression of host synthesis be prevented by adding excess MgCl2 to the medium or by omitting CaCl2 or NaCl. It was accompanied by the breakdown of polyribosomes, which also occurred in the presence of cycloheximide but not at 4 degrees C. The breakdown yielded ribosomes that were sensitive to a high salt concentration, unlike those produced by treatment of polyribosomes with RNase. The synthesis of cellular DNA and RNA was also inhibited following infection with u.v.-inactivated virus. It is concluded that the suppression of host protein synthesis (and probably also of host DNA and RNA synthesis) is caused by a constituent of the infecting virus particles. The mechanism is obscure but probably does not depend on the leakage out of the cell of Mg2+ or into the cell or Ca2+ or Na+ ions, nor on the specific inhibition of initiation of host polypeptide chains, nor on RNase-like attack on host polyribosomes.

Globin mRNAs are primers for the transcription of influenza viral RNA in vitro

Because influenza viral RNA transcription in vitro is greatly enhanced by the addition of a primer dinucleotide, ApG or GpG, we have proposed that viral RNA transcription in vivo requires initiation by primer RNAs synthesized by the host cell, specifically by RNA polymerase II, thereby explaining the alpha-amanitin sensitivity of viral RNA transcription in vivo. Here, we identify such primer RNAs, initially in reticulocyte extracts, where they are shown to be globin mRNAs. Purified globin mRNAs very effectively stimulated viral RNA transcription in vitro, and the resulting transcripts directed the synthesis of all the nonglycosylated virus-specific proteins in micrococcal nuclease-treated L cell extracts. The viral RNA transcripts synthesized in vitro primed by ApG also directed the synthesis of the nonglycosylated virus-specific proteins, but the globin mRNA-primed transcripts were translated about 3 times more efficiently. The translation of the globin mRNA-primed, but not the ApG-primed, viral RNA transcripts was inhibited by 7-methylguanosine 5'-phosphate in the presence of S-adenosylhomocysteine, suggesting that the globin mRNA-primed transcripts contained a 5'-terminal methylated cap structure. We propose that this cap was transferred from the globin mRNA primer to the newly synthesized viral RNA transcripts, because no detectable de novo synthesis of a methylated cap occurred during globin mRNA-primed viral RNA transcription. Preliminary experiments indicate that other purified eukaryotic mRNAs also stimulate influenza viral RNA transcription in vitro.

Heat inactivation of vaccinia virus particle-associated functions: properties of heated particles in vivo and in vitro.

The heat inactivation characteristics of several vaccinia virus particle-associated functions known to be involved in the transcription of the genome were examined. All functions were more resistant to heat than infectivity. Noninfectious particles were generated which exhibited significant levels of activity of all enzymes examined, and their properties were investigaed both in vitro and in vivo. RNA was synthesized in vitro by such particles, although transport of the RNA into the surrounding medium was defective. This RNA was larger than that made in normal particles but it was polyadenylated and functioned in vitro as a message coding for normal early proteins. The sequences transcribed were similar to those transcribed in normal particles, and we suggest that the production of abnormally large RNA is due to a defect in transcriptional termination. We could not detect any virus-specific protein or RNA synthesis in cells exposed to these inactivated particles and conclude that the loss of infectivity caused by heating is due to a general decline in the activities of a number of particle functions.

Replication of mycoplasmavirus MVL51 V. In vivo synthesis of virus-specific proteins

MVL51 is a nonlytic bullet-shaped mycoplasmavirus containing circular single-stranded DNA of 2 × 10 6 daltons. In vivo synthesis of virus-specific proteins has been studied in Acholeplasma laidlawii wild-type and REP − cells. REP − cells propagate double-stranded DNA but not single-stranded DNA mycoplasma viruses. Cells were uv irradiated to suppress synthesis of cell proteins, infected, and incubated in medium containing radioisotopically labeled leucine. Proteins were extracted and analyzed by gel electrophoresis. Six virus-specific proteins have been identified, with molecular weights of 70,000, 50,000, 30,000, 19,000, 14,000, and 10,000. The four larger proteins correspond to the four viral structural proteins. The 14,000 dalton protein may have a role in the replication of double stranded DNA viral replicative intermediates. The amount of 10,000 dalton protein is maximal during progeny viral single-stranded DNA synthesis.

The effect of tobacco mosaic virus infection on host and virus-specific protein synthesis in protoplasts

The nature and rate of virus-specific protein synthesis were determined in tobacco mosaic virus-infected protoplasts as a function of time after inoculation. Samples of infected and mock-infected protoplasts were exposed to radioactive amino acid for relatively short sequential time periods and the consequent labeled proteins were assessed following SDS-polyacrylamide gel electrophoresis and fluorography. The synthesis of three virus-specific proteins of molecular weights 160,000, 135,000, and 17,500 was confirmed. Synthesis of all three proteins was first detected during the 5- to 7-hr postinoculation period at which time the synthetic rate of the 135,000-dalton protein was greatest. This was soon overtaken by the 17,500-dalton capsid protein, the synthetic rate of which kept increasing until it accounted for a major portion of total protoplast protein synthesis. At 1 day postinoculation, it accounted for 50% and, at not quite 2 days, 70% of the total protein synthesis. Evidence is presented to suggest that virus-specific protein synthesis occurs in addition to, rather than at the expense of, normal cellular protein synthesis.

Host range restriction of vesicular stomatitis virus on duck embryo cells

Vesicular stomatitis virus (VSV) plaque formation and replication are restricted in duck embryo cells compared to chick embryo cells. Mutants of VSV which replicate normally on both chick and duck cells were isolated. The duck-adapted mutant, when passed through chick cells, retains its ability to grow normally on duck cells. This indicates that the ability of this virus to grow on duck cells is due to mutation rather than to host-controlled modification. VSV is restricted in duck cells but not in quail or pheasant embryo cells which follows the evolutionary relationship of these species. Newcastle disease virus and Rous sarcoma virus are restricted in duck cells also. Virus-specific protein synthesis of the wild-type VSV is greatly reduced in duck cells compared to chick cells. A structural protein of the duck cell-adapted mutant is altered.

Viral polyproteins in chick embryo fibroblasts infected with avian sarcoma leukosis viruses

Chicken embryo fibroblasts infected by avian RNA tumor viruses have been analyzed by immune precipitation for the synthesis of virus-specific proteins. Three virus-specific precursor polyproteins with molecular weights of 180,000, 95,000, and 76,000 were detected following pulse-labeling of the cells. The 76,000-dalton polyprotein is the precursor to the nonglycosylated gag proteins of the virion as shown earlier by Vogt and Eisenman (1973, Proc. Nat. Acad. Sci. USA 70, 1734–1738). By using monospecific antisera it was possible to identify the 180,000-dalton polyprotein as having sequences in common with the virion polymerase and the gag proteins and the 95,000-dalton polyprotein as being related to the virion glycoproteins gp85 and gp37. Although the 180,000-dalton protein contains sequences for the gag proteins, pulse-chase experiments indicated that it did not represent a precursor to the major 76,000 dalton gag precursor polyprotein and is most likely synthesized by an occasional read-through of the messenger RNA for the gag precursor polyprotein.

On the mechanism of inhibition of influenza virus replication by amantadine hydrochloride.

The results of analyses of fowl plague virus-specific RNA and protein synthesis in infected chick embryo fibroblasts incubated in amantadine hydrochloride are reported. They indicate that provided amantadine is present from the time of virus addition no expression of the virus genome occurs and that the synthesis of even the first detectable transcripts catalysed by the polymerase of the infecting virus particles is prevented. In agreement with previous reports it is concluded that amantadine prevents an unknown event which occurs immediately following virus infection.