Virus-specific RNA Synthesis Research Articles

Temperature-sensitive mutants of influenza WSN virus defective in virus-specific RNA synthesis.

Influenza WSN virus temperature-sensitive (ts) mutants were examined for defects in viral complementary RNA (cRNA) synthesis. The synthesis of viral cRNA was determined by hybridizing RNA from infected cells to radiolabeled virion RNA of known specific activity. Mutants in complementation groups I and III synthesized little, or no, cRNA at the nonpermissive temperature (39.5 C). When cells infected by these mutants were incubated for 5 h at the permissive temperature (33 C) and were then shifted to 39.5 C, net synthesis of cRNA ceased. This strongly suggests that mutants in these two complementation groups possess a ts defect in the transciptase complex. Mutants in group II and group V synthesize reduced amounts of cRNA at 39.5 C. In contrast to the group I and group III mutants, cRNA synthesis in cells infected by a group II or a group V mutant continues after a shift-up. This indicated that these mutants do not possess a ts transcriptase complex and that these mutants are most probably defective in some step in the amplification of cRNA synthesis. As will be discussed, the most likely defect in these mutants is in the synthesis of virion-type RNA. These results suggest that there are two influenza viral gene functions required for transcription and most likely two additional gene functions required for RNA replication.

Studies on the synthesis of viral RNA-polymerase-template complexes in BHK 21 cells infected with Semliki Forest virus

Two main types of virus-specific RNA sedimenting at about 42 and 26 S, respectively, on sucrose density gradients are synthesized in animal cells infected with Semliki Forest virus (SFV). Two and one-quarter hours after infection of BHK 21 cells with SFV only about 10% of the total amount of virus-specific RNA that accumulates during the 8-hr growth cycle of the virus have been synthesized. If at this time an inhibitor of protein synthesis, cycloheximide, is added to infected cells, both of these RNA species are synthesized during the viral growth cycle with the same time course and in amounts similar to those in untreated cultures. This result suggests that the RNA polymerase-template complexes present at 2.25 hr post infection (p.i.) are stable and synthesize all virus-specific 42 and 26 S RNA accumulating during the viral growth cycle. These polymerase-template complexes are synthesized between 45 min and 2.25 hr p.i. During multiplication of SFV in BHK 21 cells the amount of radioactivity incorporated from radioactively labeled amino acids into protein during a series of labeling intervals decreases, and the ratio of synthesis of virus-specific proteins to cellular proteins increases. Both of these effects are not expressed to a significant extent at 2.25 hr post infection (p.i.). Sodium dodecyl sulfate-polyacrylamide-gel electrophoretic (SDS-PAGE) analysis of the polypeptides synthesized in infected cultures shortly after removal of cycloheximide, which had been present from 2.25 – 6 hr p.i. shows that cycloheximide does not block the development of these changes in cellular protein synthesis. Some implications of these findings concerning the synthesis of virus-specific RNA and the interference of virus multiplication with cellular protein synthesis are discussed.

Host-dependent restriction of mengovirus replication. II. Effect of host restriction on late viral RNA synthesis and viral maturation

Restricted mengovirus replication in Mandin-Darby bovine kidney (MDBK) cells is characterized by a 400-fold reduction in infectious virus yield and a 40-fold increase in the production of noninfectious virus. Using conditions which insure that all MDBK cells are infected, virus-specific RNA and protein synthesis were measured in the restrictive host and in a permissive host for mengovirus, HeLa cells. Labeling kinetics and sucrose gradient analysis of mengovirus-specific RNA from MDBK cells show a reduction of 10-fold in virion RNA, 5-fold in double-stranded RNA, and 12.5-fold in single-stranded RNA. The viral RNA biosynthetic processes which occur late in the replicative cycle and result in the production of 90% of the single-stranded viral RNA that is packaged into capsid proteins in the permissive host are absent in restrictive MDBK cells. Viral protein synthesis as measured by labeled viral-specific polysome is decreased, and there is an accumulation of 80S subviral particles in the restricted host. It is suggested that restriction may act at a number of stages of viral replication and maturation.

The synthesis and turnover of virus-specific polyadenylated RNA in polyoma-infected cells

Mouse embryo cells infected with the 3049 strain of polyoma virus contain several fold more virus-specific, polyadenylated RNA beginning between 4 and 8 hours after the onset of viral DNA synthesis than do cells infected with wild-type virus (lpS). Following infection with either virus strain, there is an identical small but significant enhancement of the level of total polyadenylated RNA measured by binding of 125I-labeled RNA to poly(dT)cellulose. The polyadenylation of “early” virus-specific RNA is inhibited 85–90% by cordycepin resulting in an “early” RNA preparation which competes fully with polyadenylated “early” virus-specific RNA in the ternary complex assay. Utilizing the nonpolyadenylated “early” RNA, competition hybridization demonstrated that approximately 78% of the enlarged pool of “late” 3049 polyadenylated RNA and 72% of the “late” lpS pool consisted of sequences unique to the “late” period. No significant difference in the rate of decay of 3049 and lpS-specific, “late” polyadenylated RNA following actinomycin D block was found. Infection by either strain of polyoma virus did not alter the rate of decay of total polyadenylated RNA.

Camptothecin: an inhibitor of influenza virus replication.

Summary Camptothecin, an inhibitor of cellular nucleic acid synthesis, prevents fowl plague virus replication in BHK 21/13 cells. In the presence of camptothecin the synthesis of haemagglutinin and neuraminidase antigens does not occur; the ribonucleoprotein antigen is synthesized and accumulates only in the cell nucleus; and the synthesis of virus-specific RNA (of which complementary RNA predominates) is reduced considerably.

Replication of measles virus: continued synthesis of nucleocapsid RNA and increased synthesis of mRNA in the presence of cycloheximide.

The effect of cycloheximide on virus specific RNA synthesis in Vero cells infected with either wild-strain (Edmonston) or subacute sclerosing panencephalitis strain measles virus was investigated. At 3 days postinfection, cells treated with cycloheximide (2.6 x 10(-4) M) and then exposed to [(3)H]uridine showed a marked increase in labeled virus-specific RNA. A major portion of this incremental labeled RNA was putative viral mRNA which sedimented at 16, 22, and 30S. Five distinct classes of polyribosomes, which were not evident in untreated cells, were found in cycloheximide-treated cells and each contained similar species of virus-specific RNA. Viral nucleocapsid RNA, 50 and 18S, was synthesized and encapsidated in the presence of cycloheximide. The latter observation is in apparent contrast to reports that cycloheximide inhibits replication of RNA of classical paramyxoviruses, and may indicate that mechanisms for replicating RNA of measles virus are different from those for replicating RNA of paramyxoviruses.

Iridescent virus replication: Patterns of nucleic acid synthesis in insect cells infected with iridescent virus types 2 and 6

The patterns of nucleic acid synthesis in insect cells infected with iridescent virus types 2 and 6 has been examined using nucleic acid hybridization techniques. Virus-specific RNA synthesis was detected 24 hr after infection. Virus-specific DNA synthesis was detected 96 hr after infection. Host-specific nucleic acid synthesis declined throughout infection, and host-specific nucleic acid synthesis was detected only in the first 48 hr of infection. The synthesis of iridescent virus progeny DNA molecules precedes the appearance of mature iridescent virus particles.

Semliki Forest virus replication in cultured Aedes albopictus cells: studies on the establishment of persistence.

SUMMARY Semliki Forest virus (SFV) established a persistent, non-cytopathic infection in cultured Aedes albopictus cells with no effect on cumulative cell number as compared with control cultures. All cells were initially infected by SFV as judged by infective centre and immunofluorescence assay and released approx. 50 to 70 p.f.u./cell in the initial 24 h after infection. At 12 h after infection there was a ‘shut-down’ of virus-specific RNA synthesis followed by a sharp decline in the percentage of cells infected, stabilizing at about 2% by 48 h. Significant amounts of virus were released from about 2% of cells in persistently infected cultures. At 8 to 10 days a small-plaque, attenuated variant of SFV appeared in infected cultures; it replaced ‘wild-type’ virus completely by day 16. The establishment of persistence could not be related to (i) the release of interferon-like activity from infected cells, (ii) the presence of a directly antiviral agent in infected cells or medium, or (iii) changes in the total levels or subcellular distribution of acid phosphatase. Subculturing persistently infected cells stimulated cell division and promoted a (reduced) burst of virus production; simultaneous superinfection with SFV gave no increase in virus yields.

In vitro synthesis of Rous sarcoma virus-specific RNA is catalyzed by a DNA-dependent RNA polymerase.

Synthesis of Rous sarcoma virus RNA was examined in vitro with a new assay for radioactive virus-specific RNA. Nuclei from infected and uninfected cells were incubated with ribonucleoside [alpha-(32)P]triphosphates, Mn(++), Mg(++) and (NH(4))(2)SO(4). Incorporation into total and viral RNA proceeded with similar kinetics for up to 25 min at 37 degrees . About 0.5% of the RNA synthesized by the infected system was scored as virus-specific, compared to 0.03% of the RNA from the uninfected system and 0.005% of the RNA synthesized by monkey kidney cell nuclei. Preincubation with DNase or actinomycin D completely suppressed total and virus-specific RNA synthesis. alpha-Amanitin, a specific inhibitor of eukaryotic RNA polymerase II, completely inhibited virus-specific RNA synthesis, while reducing total RNA synthesis by only 50%. We conclude that tumor virus-specific RNA is synthesized on a DNA template, most probably by the host's RNA polymerase II.

Failure of an influenza virus to initiate infection in enucleate BHK cells.

Studies employing indirect immunofluorescent staining, acrylamide gel electrophoresis of [(35)S]methionine-labeled cellular polypeptides, and RNA-RNA hybridization of [(3)H]uridine-labeled cellular RNA, failed to detect evidence of fowl plague virus infection of BHK cells enucleated with cytochalasin B, although virus-specific polypeptide and RNA synthesis was detected in nucleate BHK cells. The enucleate cells permitted the synthesis of Newcastle disease and vaccinia virus structural proteins. We conclude that influenza virus fails to initiate macromolecular synthesis in enucleate BHK cells, and the role of the nucleus in influenza virus replication is discussed.

Bovine enterovirus-1: characterization, replication and cytopathogenic effects.

SUMMARY The biological parameters of a bovine picornavirus, bovine enterovirus-1, are described. The virus is shown to have a rapid growth cycle and to be stable over an extended period at 56 °C and at pH 2 to 10. Within the first hour of infection of cells with BEV-1, cellular protein synthesis was inhibited to less than 40% of normal levels. Maximum release of progeny virus occurred 6 to 8 h post-infection, which coincided with the time of maximum cytopathogenic effects as seen by electron microscopy. The synthesis of virus-specific RNA commenced 1 to 2 h after infection and proceeded exponentially until 5 h after infection. Four virus capsid polypeptides were found together with procapsid proteins, one of which appeared to be a precursor of VP2 and VP4.

Cyclic production of vesicular stomatitis virus caused by defective interfering particles.

Purified standard and defective interfering (DI) particles of vesicular stomatitis virus (VSV) were reconstituted, and the mixture was continuously passaged for six 24-hr growth periods in Chinese hamster ovary cells in suspension cultures. The cultures were monitored for cellular viability, for intracellular virus-specific RNA, for total extracellular viral particles, and for production of plaque-forming standard VSV. During the six passages, there was a cyclic, overlapping pattern of synthesis of both DI and standard VSV particles; decline in the production of standard virus was always preceded by large amounts of DI particles. Intracellular virus-specific RNA synthesis reflected the pattern of production of extracellular particles. Viral antigens represented by nucleocapsids accumulated in cells when DI particles were being produced in large quantities. These nucleocapsids contained RNA of DI particles. Under other conditions, infected cells were able to survive longer than usual and were resistant to superinfection by standard VSV. Preliminary evidence indicates that this resistance was due to surface interference. Therefore, a wide variety of different interactions between virus and host occurs simply by varying the total concentration of viral particles and the relative concentrations of standard and OI particles in these preparations.

Effects of L-glutamine deprivation on growth of HVJ (Sendai virus) in BHK cells.

l-Glutamine requirement for viral maturation was found in BHK-HVJ cells, a cell line of baby hamster kidney cells persistently infected with HVJ (Sendai virus). Synthesis of envelope protein in BHK-HVJ cells was markedly suppressed by deprivation of l-glutamine, whereas development of nucleocapsid (S) antigen was less affected. More detailed examination of this phenomenon was carried out by using a cytolytic system. Growth of HVJ in BHK cells cultured in media deprived of various amino acids was investigated, and omission of l-glutamine from culture medium resulted in a marked inhibitory effect on the release of infectious virus and synthesis of envelope protein, although synthesis of virus-specific RNA and nucleocapsid antigen in the cells was readily detected. When l-glutamine was restored to the culture medium, infectious virus and envelope protein could be detected. l-Glutamic acid, l-aspartic acid, or l-alanine could be substituted for l-glutamine. Effects of l-glutamine deprivation on HVJ growth in several other cells were also investigated. The growth of HVJ in the cells other than BHK and FL cells was not suppressed by lack of l-glutamine. Growth of Sindbis virus in BHK cells was also markedly retarded in the absence of l-glutamine.

Isolation and characterization of Sendai virus temperature-sensitive mutants.

Ten temperature-sensitive mutants of Sendai virus, a paramyxovirus, were isolated and partially characterized. The mutants replicated in chicken embryo lung cells at 30 C, but not at 38 C; wild-type virus grew equally well at both temperatures. Complementation tests divided the mutants into seven groups. Six groups synthesized neither infectious virus nor RNA when incubated at 38 C from the beginning of infection. Temperature shift-up experiments demonstrated that three of these complementation groups were blocked in early steps required for RNA synthesis, but these gene functions were not needed throughout the replicative cycle. In contrast, the other three RNA-negative complementation groups were defective throughout the replicative cycle in functions required for virus-specific RNA synthesis. Only one mutant, which complemented all of the above, synthesized RNA but not infectious virus when placed at 38 C; the hemagglutinin of this mutant functioned only at the permissive temperature.

Conditional lethal mutants of vesicular stomatitis virus: I. Phenotypic characterization of single and double mutants exhibiting host restriction and temperature sensitivity

Chemically mutagenized populations of vesicular stomatitis virus (VSV) were screened for two general classes of conditional lethal viruses. Host-restricted ( hr) clones that were unable to elicit productive infections in nonpermissive HeLa or HEp-2 cells were selected from small plaques formed on mixed indicator cultures of chick embryo fibroblasts and HeLa cells. Most of these hr mutants also displayed a temperature-sensitive ( ts) phenotype in permissive CEF cells. Evidence for the existence of spontaneous VSV hr mutants was obtained. The majority of the ts clones selected in CEF cells by conventional methods lacked the hr marker. Specific hr and ts mutants could be distinguished from wild-type virus on the basis of increased thermolability of their infectivity or diminished in vitro activity of their virion-associated transcriptase. Tests for the ability of these mutants to promote virus-specific RNA synthesis under nonpermissive conditions in cells treated with actinomycin D established the following relationships: (a) both “RNA +” and “RNA −” classes of mutants were found when infections were carried out in permissive cells incubated at temperatures restrictive for growth of ts phenotypes; (b) all clones possessing the hr marker failed to induce significant uridine incorporation (“RNA −”) in nonpermissive HEp-2 cells incubated at high or low temperatures; (c) all mutants scored as “RNA −” types when infections were carried out in nonpermissive HEp-2 cells at restrictive temperature irregardless of their phenotype for the hr and ts markers.

RNA synthesis of vesicular stomatitis virus. V. Interactions between transcription and replication.

The temperature-sensitive mutant ts114 of vesicular stomatitis virus is temperature-sensitive in both primary and secondary transcription, but not in the replication of 40S RNA. The synthesis of 40S RNA is specifically inhibited when protein synthesis is shut off. The addition of cycloheximide to cells infected by ts114, rapidly inhibits RNA replication at the permissive and nonpermissive temperatures. However, the addition of cycloheximide at the nonpermissive temperature also results in almost complete recovery of transcription to the level found at the permissive temperature. Other inhibitors of protein synthesis applied at the nonpermissive temperature result in the same recoverability of the apparent temperature-sensitive lesion. Ribonucleic acid products synthesized by ts114 at the nonpermissive temperature in the presence of cycloheximide are identical to virus-specific messenger RNA by the criteria of size and complementarity to virion RNA. When mutant-infected cells are shifted to the nonpermissive temperature, incubated for a period of time, and then treated with cycloheximide, the ratio of transcriptive to replicative activity varies depending on the time at which radioactive precursor is added to cells. This suggests an interdependence between replication and transcription during virus-specific RNA synthesis.

Early synthesis of virus-specific RNA and DNA in cells rapidly transformed with Rous sarcoma virus

Chick embryo fibroblasts (CEF) were infected with the Prague strain (subgroup C) of RSV (PrC-RSV) under conditions of rapid transformation and the synthesis of RNA and DNA capable of hybridizing with single-stranded DNA transcripts of 70 S PrC-RSV RNA (PrC-RSV DNA) was measured by hybridization kinetics analysis. PrC-RSV DNA was synthesized in the presence of high concentrations of actinomycin D and was freed from double-stranded DNA by chromatography; it protected over 90% of 70 S PrC-RSV RNA from digestion by ribonuclease at DNA:RNA ratios greater than 9. Virus-specific RNA sequences were measured with cellular RNA in vast excess, using the hybridization kinetics of the PrC-RSV DNA with 70 S PrC-RSV RNA as the reference. Virus-specific DNA sequences were measured with cellular DNA in vast excess, using the kinetics of self-annealing of the unique sequences of cellular DNA as the reference. Uninfected gs − CEF contained on the average one viral genome copy of DNA per cell and no detectable virus-specific RNA. DNA capable of hybridizing with the probe began to be synthesized within 3 hr after infection, and by 24 hr 2 viral genome copies had been synthesized. No additional virus-specific DNA was synthesized thereafter. During the first 6 hr after infection, CEF contained about 40 viral genome equivalents of RNA per cell, about 85% of which were in the cytoplasm. This represented most probably RNA in inoculum virus. Between 6 and 24 hr the number of viral genome equivalents per cell increased to 100. This newly synthesized RNA was equally distributed between the nucleus and polyribosomes. Between 24 and 96 hr the amount of virus-specific RNA in the nucleus and in polyribosomes increased by just under 2-fold, but in the remainder of the cytoplasm it rose by over 8-fold. By 96 hr cells contained about 340 genome equivalents of RNA per cell, which was about one-half of the number present in cells 14 days after infection. The size of the virus-specific RNA present in polyribosomes was predominantly 35 S, but some 10–30 S material was also present.

Virus-specific RNA synthesis in the midgut of silkworm, Bombyx mori, infected with cytoplasmic-polyhedrosis virus

Virus-specific RNA synthesis in the midgut of silkworm infected with cytoplasmic-polyhedrosis virus was investigated under the condition inhibiting host RNA synthesis by actinomycin D injection. Two species of virus-induced RNA were formed; one was sensitive to ribonuclease (RNase) but the other was resistant. The resistant RNA had a sedimentation coefficient of 15 S and was considered as viral progeny with doublestranded RNA. The sensitive RNA, presumably single-stranded RNA, consisted of two classes with 15 S and 22 S sedimentation coefficients. Annealing the single-stranded RNA with heat-denatured CPV-RNA indicated that the single-stranded RNA was transcribed from viral genome RNA. The function of 22 S and 15 S single-stranded RNAs was discussed from the viewpoint of virus multiplication.

Sedimentational pattern of virus-specific RNA synthesized in Newcastle disease virus-infected cells treated with amino acid analogues.

The synthesis of virus-specific RNA in Newcastle disease virus-infected cells is strongly suppressed by amino acid analogues (p-fluorophenylalanine, ethionine and canavanine) if they are present throughout the reproduction cycle or added 2–4 hours p.i. The analogues exert only a partial inhibitory effect when added 6 hours p.i. Virus-specific RNA synthesized in cells exposed to ethionine or canavanine has essentially the same sedimentational pattern as RNA formed in the absence of the analogues, whereas p-fluorophenylalanine preferentially inhibits 35S RNA accumulation. The content of “minus” strands in different RNA classes is not changed in p-fluorophenylalanine-treated cells. 50S RNA formation, although readily suppressed by cycloheximide was not inhibited by the amino acid analogues tested.

Temperature-sensitive defect of mutants isolated from L cells persistently infected with Newcastle disease virus.

The temperature-sensitive defects of virus mutants isolated from L cells persistently infected with Newcastle disease virus (NDV) were analyzed. Genetic grouping of the mutants by complementation tests was attempted by using several different methods, including yield analysis, RNA synthesis, and heterozygote formation at 42 to 43 C, the nonpermissive temperature. In each case, specific interference prevented detection of complementation. This interference was shown to occur prior to or at the level of virus RNA synthesis. Temperature-shift experiments with five different NDV(pi) clones showed that virus replication begun at 37 C could not be completed at the nonpermissive temperature. The activity of the NDV-specific RNA-dependent RNA polymerase in the cytoplasm of infected chicken embryo cells was not stable and could not be demonstrated directly. However, indirect measurement of RNA polymerase activity at the nonpermissive temperature was accomplished by studying the kinetics of virus-specific RNA synthesis in infected cells after temperature shift. Two types of response were obtained: with three NDV(pi) clones, virus-specific RNA synthesis ceased immediately upon transfer of infected cells to 42 to 43 C, whereas in cells infected with two other NDV(pi) clones, RNA synthesis continued for several hours at this temperature. These results suggested that there may be two types of ts defects in NDV(pi), both associated with virus-specific RNA polymerase activity.