Virus-specific Protein Synthesis Research Articles

Evidence for the temporal regulation of protein synthesis in synchronized bacteriophage phi chi 174 infections.

SUMMARY Virus-specific protein synthesis has been studied in unsynchronized and in starvation-synchronized infections of u.v.-irradiated su − host cells by ϕX174wt and a set of ϕX conditional lethal mutants. Several virus-specific protein species were detected in association with the host cell membrane at various times during the virus reproduction cycle; virus-specific proteins were also detected in the host cell cytoplasm. The intracellular distribution, between the cell membrane fraction and the cell cytoplasm fraction, of virus proteins was measured under a variety of experimental conditions. The possible significance of the presence of virus proteins on the host cell membrane is discussed.

The influence of supraoptimal temperature on poliovirus type 1 Mahoney strain reproduction.

Experiments with poliovirus type 1, Mahoney strain, reproduction of which at 40° C in MiO cells is inhibited by 90–95 per cent showed that at higher temperatures synthesis of viral proteins and RNA was inhibited and degradation of RNA (mainly of single-stranded) occurred. No disorders in the cleavage of viral polypeptides or in individual stages of virion morphogenesis at 40° C were observed. The synthesis of virus-specific proteins was found to be more sensitive to high temperature than RNA synthesis; inhibition of protein synthesis occurred immediately after the beginning of incubation of the infected cells at 40° C. At high temperature there was no change in the rate of translation or degradation of viral polyribosomes. It is suggested that the primary effect of high temperature on poliovirus reproduction consists in disturbance of early stages of translation.

Virus Replication in Enucleate Cells: Vesicular Stomatitis Virus and Influenza Virus

The requirement of the presence of a nucleus for the replication of vesicular stomatitis virus and influenza virus has been examined by following the growth and development of these viruses in enucleate BS-C-1 cells. Vesicular stomatitis virus replicates normally in enucleate cells with the rate of production of infectious virus, the amount of virus-specific protein synthesis, and the type of proteins produced being essentially the same in nucleate and enucleate cells. Influenza virus does not replicate in enucleate cells, no virus gene products can be detected, and there is no inhibition of cellular protein synthesis.

Picornaviral gene order: comparison of a rhinovirus with a cardiovirus.

We describe conditions for unmasking virus-specific protein synthesis in cells infected with rhinoviruses 1A and 2. The gene order and cleavage pattern of rhinovirus 1A show many similarities to those of encephalomyocarditis virus and of poliovirus.

Elektronenoptische Untersuchungen zur Wirkung des Streptomycetenantibioticums Borrelidin auf das Pseudorabiesvirus

The influence of borrelidin on the replication of the Pseudorabies virus grown in chicken embryonal fibroblasts was studied by electron microscopy. Electron micrographs taken of various stages of virus replication revealed an effect on the early steps of replication (eclipse). Incomplete viral particles defect in capsids were found in the cytoplasm at times where the regular viral replication cycle was finished in controls. Consequently, it is suggested that antiviral activity of borrelidin probably depends on a block in virus specific protein synthesis.

Elektronenoptische Untersuchungen zur Wirkung des Streptomycetenantibioticums Borrelidin auf das Pseudorabiesvirus

The influence of borrelidin on the replication of the Pseudorabies virus grown in chicken embryonal fibroblasts was studied by electron microscopy. Electron micrographs taken of various stages of virus replication revealed an effect on the early steps of replication (eclipse). Incomplete viral particles defect in capsids were found in the cytoplasm at times where the regular viral replication cycle was finished in controls. Consequently, it is suggested that antiviral activity of borrelidin probably depends on a block in virus specific protein synthesis.

Kinetics of synthesis and cleavage of encephalomyocarditis virus-specific proteins

A mathematical model is presented which describes the kinetics of synthesis and cleavage of encephalomyocarditis (EMC) virus-specific proteins as observed in pulse-chase and progressive labeling experiments. Although there is evidence that each ribosome completes translation of the entire protein coding region of the viral RNA, at 4 hr postinfection no polypeptide or nascent chain larger than the 100,000 dalton capsid precursor chain was observed. Hence, little or no “polyprotein” (mass 220,000–260,000 daltons) was produced. Rather, each of the three known primary products (polypeptides A, F, and C) was released upon completion of its synthesis. The precursor of the capsid polypeptides, polypeptide A, was always completed before it was cleaved. This resulted in the initial sequential labeling of the capsid polypeptides following the addition of radioactive amino acids to an infected cell suspension, and provided a means for ordering the capsid polypeptides within A. Chain F was stable, confirming previous reports. Polypeptide C cleaves to form D, which in turn cleaves to form E. However, only 15% of the C chains were released in the intact form. Thirty percent of the time, the nascent C chain had undergone one cleavage thus releasing D as the primary product, and 55% of the time both cleavages had occurred during translation, thus releasing E as a stable primary product. The half-life of the A chain was about 6.7 min, that of the C chain was 10 min, and that of the D chain was 12 min.

Protein synthesis in pox-infected cells treated with interferon

Incorporation of labeled amino acids into total protein is rapidly inhibited in Vaccinia-infected L-cells. This inhibition is enhanced after pretreatment with mouse interferon. In chick embryo fibroblasts, even at a multiplicity of infection of 50 PFU/cell, the incorporation of radioactive amino acids is only gradually inhibited and homologous interferon has no influence on the rate of protein synthesis inhibition. In interferon-treated CEF (1000–2000 units/ml), virus-specific proteins can no longer be detected by immunological techniques. Also, at least one of the viral DNases is not induced in these cells after interferon treatment. In contrast, in interferon-treated L-cells (1000–3000 units/ml) synthesis of Vaccinia virus-specific proteins is still detectable at very early stages of infection (1–2 h.p.i.). At later stages (2–4 h.p.i.), immunodiffusion failed to show the presence of virus-specific proteins in interferon-treated L-cells. This inhibition of viral protein synthesis is likely to be connected to the enhanced cytopathic effect of Vaccinia virus-infected and interferon-treated L-cells.

Synthesis and cleavage of virus-specifici proteins in Krebs II carcinoma cells infected with encephalomyocarditis virus.

Synthesis and cleavage of proteins in actinomycin D treated encephalomyocarditis virus-infected Krebs II cells were studied by acrylamide gel electrophoresis. In extracts of the infected cells, 8 major and at least 3 minor peptides of molecular weights ranging from 13×103 to 100×103 daltons, were discerned. Two of them were identified as structural proteins of the virion, one structural protein being absent from the cell extracts. High molecular weight proteins were shown to be precursors of other, stable peptides. These high molecular weight precursors, but not stable proteins, could be degraded by enzymes present in the extracts of infected or non-infected cells. The products of degradation differed, however, from those obtained during the cleavagein vivo. The processing of the precursor proteins in the cell was inhibited by diisopropyl fluorophosphate while the degradationin vitro was relatively insensitive to the drug.

Synchronization of Rous sarcoma virus production in chick embryo cells

The growth of chick embryo fibroblasts transformed by and producing the Schmidt-Ruppin strain of Rous sarcoma virus was synchronized by exposure to serum-free medium. It was found that virus-specific RNA and protein synthesis and virus release are synchronized in these cells and occur in the early S and G 1 phases of the cell cycle, respectively. These findings support the hypothesis that RSV synthesis is dependent upon specific host cell processes. The duration of the phases of the cell cycle in transformed cells could not be distinguished from the pattern in uninfected cells. Evidence is presented which indicates that the synchrony of transformed cells is achieved by the low pH of the serum-free medium rather than by the absence of serum.

Polysomal localization of R17 bacteriophage-specific protein synthesis.

Synthesis of viral ribonucleic acid (RNA) polymerase, maturation protein, and coat protein in Escherichia coli infected with bacteriophage R17 occurs mainly on polysomes containing four or more ribosomes. The 30S ribosomal subunits through trimer-size polysomes, which are associated with all of the R17-specific proteins and are predominant in the infected cell, synthesize only coat protein. These structures may accumulate as products derived from larger polysomes as a result of failure in the release of nascent polypeptides after termination of chain growth. Appreciable amounts of viral coat protein remain attached to ribosomes and polysomes during R17 bacteriophage replication, supporting the hypothesis of the repressor role of this protein. The time course of synthesis of virus-specific proteins obtained from the polysomes of infected cells demonstrated regulated R17 messenger RNA translation consistent with the idea that coat protein is preferentially synthesized whereas the synthesis of noncoat proteins is suppressed.

Proteins of vesicular stomatitis virus: III. Intracellular synthesis and extracellular appearance of virus-specific proteins

An examination of the kinetics of synthesis and release of virus-specific proteins from VSV-infected cells showed that the rate of virus protein synthesis was maximal about 4 hr post infection, a constant proportion of the newly synthesized protein being released from the cell at all times. Five virus-specific proteins VP1, VP2, VP3, VP4, and NS1 were identified in infected cells. The intracellular protein VP2 was shown to be distinct by its mobility on polyacrylamide gels from both the virion glycoprotein VP2 and the soluble antigen glycoprotein VP2a and may be a precursor to these proteins. Exposing cells to actinomycin D for 20 hr prior to infection showed that the relative rate of synthesis of NS1 was greatest in the first 2 hr of infection and decreased at later times. In contrast, the virion envelope proteins VP2 and VP4 formed an increasing proportion of virus protein synthesis with time. The result suggests that the protein NS1 may have an early intracellular function in replication. Pulse-chase experiments showed that the proteins VP1 and VP4 could be released from the cell soon after synthesis while newly synthesized VP2 and VP3 appeared in extracellular virus only after some delay.

Sendai virus RNA as messenger RNA determining the synthesis of early virus specific proteins.

Sendai virus irradiated by UV light, the dose of which is sufficient for inactivation of infectivity, is capable of inducing synthesis of early protein only. The synthesis of early virus-specific proteins was observed in the absence of virusspecific RNA-synthesis. Consequently, RNA of virion (plus-strand) is the m-RNA for the synthesis of early virus-specific proteins of Sendai virus. Since viral structural proteins do not synthesize under these conditions, it is assumed, that the synthesis of virus capsid proteins is coded by a minus-strand. The possible significance of such processes for an understanding of the nature of viral oncogenesis and stable antiviral immunity is discussed.

Process of infection with bacteriophage phiX174. XXXVI. Measurement of virus-specific proteins during a normal cycle of infection.

Double-labeling techniques in which (14)C-labeled, phiX174-infected cells and (3)H-labeled, uninfected cells were used permitted the identification of the virus-specific proteins after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis without prior inhibition of host-cell protein synthesis by ultraviolet irradiation. It was also possible to detect previously undescribed components of high molecular weight which may represent induced host proteins. The gel regions specifically corresponding to cistron II protein and the chloramphenicol-resistant VI protein were identified, and a third new, small peak of unknown origin was detected. Studies of the rate of synthesis of virus-specific proteins at various times after infection indicated that the product of cistron I (lysis) is made only late in infection, but the other proteins seemed to be synthesized at the same relative rates throughout infection (although in different amounts). Studies of the proteins obtained from uniformly labeled phiX virus particles indicated that all of the spikes are identical and allowed a formulation of the structure of the phage capsid.

Virus-specific proteins in Escherichia coli infected with phage Q β

The synthesis of virus-specific proteins following infection of Escherichia coli with the RNA phage Q β was studied using spheroplasts exposed to low concentrations of actinomycin D or rifampicin in the presence of an amino acid mixture containing radioactive leucine or histidine. Fractionation of the phage-induced proteins by acrylamide-gel electrophoresis revealed the presence of four peaks (I, IIa, IIb and III). Peak III contains neither histidine nor tryptophan and is identified as Q β coat. Besides coat protein, Q β particles have two more components, with the electrophoretic mobility of Peaks IIa and IIb, the nature of which is unknown, although presumably one or both of them correspond to attachment proteins.

Sindbis virus infection of chick and hamster cells: Synthesis of virus-specific proteins

In monolayers of chick embryo fibroblasts or BHK cells infected with Sindbis virus in the presence of actinomycin, host protein synthesis is inhibited by at least 80–90% between 2 and 3 hours after infection. Thereafter, Sindbis specified polypeptide chains form 60–70% of total protein synthesis. In particular, synthesis of the membrane protein of the virus constitutes 25% of total protein synthesis. Twelve to 16 polypeptides induced by virus infection have been identified in infected cells by acrylamide gel electrophoresis. The pattern of virus-specified proteins observed depends upon the length of the labeling period, the host cell, and mutations in the virus genome. The molecular weight of the membrane protein of the Sindbis virion has been estimated to be 53,000 and the core protein is estimated to be 30,000 in molecular weight.

DNA-dependent in vitro synthesis of bacteriophage enzymes.

The mechanism by which the bacteriophage T4 regulates the sequential synthesis of virus-specific proteins has not been elucidated as yet. The phasing of protein synthesis in T4-infected Escherichia coli may be controlled by either transcriptional or translational constraints. In order to study these regulatory mechanisms we have developed an in vitro system which, in response to bacteriophage DNA, synthesizes both early and late enzymes. Since both transcription and translation are required for enzyme synthesis in this system, it affords an unusual opportunity for the study of bacteriophage development. The in vitro system, which is prepared from uninfected E. coli, has been fractionated into ribosomes and supernatant proteins. The ribosomes are the source of initiation factors, and the supernatant proteins contain the amino acid activating enzymes, elongation factors, and RNA polymerase. The system is relatively free of host transfer RNA (tRNA) and DNA; enzyme synthesis depends on added tRNA, and RNA...

Wachstumsgeschwindigkeit der Peptidkette im Tabakmosaikvirus

The rate of incorporation of labelled amino acids into the complete tobacco mosaic virus (TMV), into soluble virus protein and into soluble cell proteins has been determined in discs of infected and healthy tobacco leaves. The rate of overall protein synthesis is increased by 50% in the infected leaves. At least 60% of the increase derives from the synthesis of virus-specific proteins and the synthesis of cellular proteins is not inhibited. The virus protein synthesis is strongly temperature dependent and shows a maximum at 28 °C. The exchange of free labelled amino acids between the external medium and the inner cellular pool reaches equilibrium within ten minutes. The influence of the exchange rate on the measurement of the kinetics of peptide chain synthesis is discussed in detail. Discs from infected leaves were incubated for short periods at low temperatures in media containing 3H-tyrosine or 3H-proline. Peptides isolated after 5 minutes incubation at 15 °C were found to be uniformly labelled with no apparent gradient of radioactivity from the N- to the C-terminus. The results indicate that the growth rate of the peptide chain at 15 °C is probably higher than 2 - 3 amino acids/sec and at 28 °C higher than 20 amino acids/sec. These values are higher than those for animal cells and similar to those for protein synthesis in Escherichia coli. Comparison of the growth rate of TMV protein with rate of total protein synthesis and the number of ribosomes in the tobacco leaves indicate that only a small portion of the ribosomes takes part in cell protein synthesis.

Synthesis of virus-specific proteins in Escherichia coli infected with the RNA bacteriophage MS2.

The synthesis of virus-specific proteins following infection of Escherichia coli with the RNA phage MS2 was studied using spheroplasts exposed to low concentrations of actinomycin D in the presence of an amino acid mixture containing radioactive leucine or histidine. Under the conditions employed there was about 75% inhibition of virus production and 99% inhibition of host protein synthesis by actinomycin. Fractionation of the phage-induced proteins by acrylamide gel electrophoresis revealed the presence of three major peaks, I, II, and III. The largest one (peak III) was characterized as coat protein. The rate of synthesis of proteins I and II declines before that of the coat protein has reached its maximum.

Reovirus-Specific Polyribosomes in Infected L-Cells

Polyribosomes were isolated from reovirus-infected L-cells. Virus-induced RNA isolated from these polyribosomes was single-stranded, heterogeneous in size, and capable of extensive hybridization with viral double-stranded RNA. Reovirus RNA, therefore, specifies in infected cells the formation of a single-stranded messenger RNA for virus-specific protein synthesis.