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.     

Reovirus: Effect of Noninfective Viral Components on Cellular Deoxyribonucleic Acid Synthesis

We determined the effects of noninfective reovirus components on cellular deoxyribonucleic acid (DNA) synthesis. Reovirus inactivated by ultraviolet light inhibited cellular DNA synthesis, whereas reovirus cores and empty capsids did not. Both cores and empty capsids were adsorbed to cells. Adenine-rich ribonucleic acid (RNA) from reovirus, adsorbed to cells in the presence of diethyl-aminoethyl-dextran, produced a partial inhibition of DNA synthesis. RNA was synthesized in the presence of actinomycin D after infection with ultraviolet light-irradiated reovirus, and this RNA synthesis was not due to multiplicity reactivation of virus infectivity. These data suggest that viral structural proteins do not inhibit DNA synthesis and that the inhibition produced by ultraviolet-irradiated virus may be mediated in part or in toto by a newly synthesized viral product.     

Inhibition of arbovirus assembly by cycloheximide

Addition of cycloheximide (100 μg/ml) to cultures of chick cells infected with Semliki Forest virus (SFV) halted subsequent increase in virus titers. When added after 4 hr of infection, the drug had no effect on the rate of viral ribonucleic acid (RNA) synthesis, although marked inhibition of protein synthesis was seen. All of the previously identified forms of SFV RNA were seen in the drug-treated cells at higher concentrations than were present in untreated controls. The latter observation appeared to result from a failure to form viral “cores” or nucleocapsids in the cycloheximide-treated cells, resulting in sequestration of viral RNA intracellularly. The failure to form new virus cores was correlated with the failure of type II cytopathic vacuoles to appear in thin sections. Virus budding from the cell surface and the formation of type I cytopathic vacuoles persisted in cycloheximide-treated cells. The cellular pool of the major protein present in the virus core appeared to be small. None of this protein was found in a free pool in cytoplasm. The results indicated that, in the presence of cycloheximide, virus assembly was impaired because of the small size of the cellular pool of the major protein required for virus core formation.     

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.     

Interaction of viral RNA with Escherichia coli. 3. Synthesis of poliovirus-specific RNA directed by isolated poliovirus RNA

Poliovirus RNA directs the synthesis of virus-specific RNA in E. coli as reported previously for poliovirus-induced double-stranded RNA. Synthesis of viral RNA can be followed by conversion of viral RNA into a double-stranded RNase-resistant state, by increase in infectivity and by hybridization of newly synthesized RNA to viral RNA. Virus-specific RNA synthesis occurs also in the presence of inhibitors of protein synthesis indicating that an enzyme is present in E. coli which can use RNA as a template.     

Effects of Elevated Temperatures on Mengovirus Ribonucleic Acid Synthesis and Virus Production in Novikoff Rat Hepatoma Cells

The production of mengovirus in Novikoff rat hepatoma cells is progressively reduced with an increase in incubation temperature of the cells from 34 to 40 C, in spite of the fact that about the same amounts of single-stranded and double-stranded viral ribonucleic acid (RNA) are synthesized at 34, 37, and 40 C; the rate of overall protein synthesis is as high at 40 C as at 37 C. At 40 C, progeny viral RNA accumulates in an undegraded form without being incorporated into virus particles. The results suggest that virus maturation is preferentially inhibited at supraoptimal temperatures. At 42 C, on the other hand, no viral RNA is produced and no viral RNA polymerase activity is detectable in cell lysates. Failure of infected cells to form viral RNA polymerase at 42 C is probably due to an impairment of protein synthesis since most of the polyribosomes are rapidly lost during incubation at 42 C and the rate of amino acid incorporation into protein is 70% lower at 42 C than at 37 C. When infected cells are shifted from 37 to 42 C during the period of active viral RNA synthesis, viral RNA polymerase activity is rapidly lost from the cells, and viral RNA synthesis ceases within 45 min. In contrast, the RNA polymerase is as active in vitro at 42 C as at 37 C, and the activity is relatively stable at 42 C.     

Encephalomyocarditis virus replication complexes preferentially utilizing nucleoside diphosphates as substrates for viral RNA synthesis. Nucleotide kinases specifically associated with the complex channel RNA precursor

Replication complexes (RC) of the encephalomyocarditis (EMC) virus were shown previously to contain components that exhibit marked preference for nucleoside diphosphates over nucleoside triphosphates (NTP) as substrates for viral RNA synthesis [Koonin and Agol (1983), Virology 129, 309-318]. These NDP-preferring components have now been found to posses the following properties. When RC preparations were fractionated by sucrose density gradient centrifugation, the fractions containing NDP-preferring components exhibited a considerably higher nucleotide kinase activity as compared to either the fractions containing NTP-preferring components or corresponding fractions from mock-infected cells. When NDP-preferring RC were incubated with ADP and three other NTP, very low concentrations of endogenously generated ATP ensured a greater rate of RNA synthesis than did much higher concentrations of exogenous ATP. When an equimolar mixture of differently labelled UDP and UTP was used as a substrate for NDP-preferring RC, the label from UDP predominated in the newly synthesized RNA, even though the UDP-derived UTP constituted a minor portion of the total UTP pool. When labelled UDP was diluted with unlabelled uridine nucleotides, unlabelled UTP proved to be far less efficient than unlabelled UDP in diminishing the specific radioactivity of UMP incorporated into RNA by NDP-preferring RC. These data are interpreted in the sense that the NTP generated by the built-in nucleotide kinase system are not freed into the external milieu but rather form a separate pool preferentially used for synthesis of viral RNA by NDP-preferring RC. It is suggested that this functional compartmentation of NTP may be significant for the replication of viral RNA in vivo.     

Purification of influenza viral complementary RNA: its genetic content and activity in wheat germ cell-free extracts.

Influenza viral complementary RNA (cRNA) was purified free from any detectable virion-type RNA (vRNA), and its genetic content and activity in wheat germ cell-free extracts were examined. After phenol-chloroform extraction of cytoplasmic fractions from infected cells, poly(A)-containing viral cRNA is found in two forms: in single-stranded RNA and associated with vRNA in partially and fully double-stranded RNA. To purify single-stranded cRNA free of these double-stranded forms, it was necessary to employ, as starting material, RNA fractions in which cRNA was predominantly single stranded. Two RNA fractions were successfully employed as starting material: polyribosomal RNA and the total cytoplasmic RNA from infected cells treated with 100 mug of cycloheximide (CM) per ml at 3 h after infection. In WSN virus-infected canine kidney (MDCK) cells, the addition of CM at 3 h after infection stimulates the production of cRNA threefold and causes a very large increase in the proportion of the cytoplasmic cRNA which is single stranded; double-stranded RNA forms are greatly reduced in amount. Total cRNA was obtained by oligo(dT)-cellulose chromatography, and single-stranded cRNA was separated from double-stranded forms by Sepharose 4B chromatography. The cRNA preparation purified from polyribosomes consists of 95% single-stranded cRNA, with the remaining 5% apparently being double-stranded RNA forms. The cRNA preparation purified from CM-treated cells (CM cRNA) is even more pure: 100% of the radiolabeled RNA is single-stranded cRNA. Annealing experiments, in which a limited amount of 32P-labeled genome RNA was annealed to the cRNA, indicate that the purified cRNA contains at least 84 to 90% of the genetic information in the vRNA genome. Purified viral cRNA (CM cRNA) is very active in directing the synthesis of virus-specific proteins in wheat germ cell-free extracts.     

Template/primer requirements and single nucleotide incorporation by hepatitis C virus nonstructural protein 5B polymerase

Nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) possesses an RNA-dependent RNA polymerase activity responsible for viral genome RNA replication. Despite several reports on the characterization of this essential viral enzyme, little is known about the reaction pathway of NS5B-catalyzed nucleotide incorporation due to the lack of a kinetic system offering efficient assembly of a catalytically competent polymerase/template/primer/nucleotide quaternary complex. In this report, specific template/primer requirements for efficient RNA synthesis by HCV NS5B were investigated. For intramolecular copy-back RNA synthesis, NS5B utilizes templates with an unstable stem-loop at the 3' terminus which exists as a single-stranded molecule in solution. A template with a stable tetraloop at the 3' terminus failed to support RNA synthesis by HCV NS5B. Based on these observations, a number of single-stranded RNA templates were synthesized and tested along with short RNA primers ranging from two to five nucleotides. It was found that HCV NS5B utilized di- or trinucleotides efficiently to initiate RNA replication. Furthermore, the polymerase, template, and primer assembled initiation-competent complexes at the 3' terminus of the template RNA where the template and primer base paired within the active site cavity of the polymerase. The minimum length of the template is five nucleotides, consistent with a structural model of the NS5B/RNA complex in which a pentanucleotide single-stranded RNA template occupies a groove located along the fingers subdomain of the polymerase. This observation suggests that the initial docking of RNA on NS5B polymerase requires a single-stranded RNA molecule. A unique beta-hairpin loop in the thumb subdomain may play an important role in properly positioning the single-stranded template for initiation of RNA synthesis. Identification of the template/primer requirements will facilitate the mechanistic characterization of HCV NS5B and its inhibitors.     

De novo synthesis of minus strand RNA by the rotavirus RNA polymerase in a cell-free system involves a novel mechanism of initiation

The replicase activity of rotavirus open cores has been used to study the synthesis of (-) strand RNA from viral (+) strand RNA in a cell-free replication system. The last 7 nt of the (+) strand RNA, 5'-UGUGACC-3', are highly conserved and are necessary for efficient (-) strand synthesis in vitro. Characterization of the cell-free replication system revealed that the addition of NaCl inhibited (-) strand synthesis. By preincubating open cores with (+) strand RNA and ATP, CTP, and GTP prior to the addition of NaCl and UTP, the salt-sensitive step was overcome. Thus, (-) strand initiation, but not elongation, was a salt-sensitive process in the cell-free system. Further analysis of the requirements for initiation showed that preincubating open cores and the (+) strand RNA with GTP or UTP, but not with ATP or CTP, allowed (-) strand synthesis to occur in the presence of NaCl. Mutagenesis suggested that in the presence of GTP, (-) strand synthesis initiated at the 3'-terminal C residue of the (+) strand template, whereas in the absence of GTP, an aberrant initiation event occurred at the third residue upstream from the 3' end of the (+) strand RNA. During preincubation with GTP, formation of the dinucleotides pGpG and ppGpG was detected; however, no such products were made during preincubation with ATP, CTP, or UTP. Replication assays showed that pGpG, but not GpG, pApG, or ApG, served as a specific primer for (-) strand synthesis and that the synthesis of pGpG may occur by a template-independent process. From these data, we conclude that initiation of rotavirus (-) strand synthesis involves the formation of a ternary complex consisting of the viral RNA-dependent RNA polymerase, viral (+) strand RNA, and possibly a 5'-phosphorylated dinucleotide, that is, pGpG or ppGpG.     

Semiconservative synthesis of single-stranded RNA by bacteriophage phi 6 RNA polymerase.

The RNA polymerase in the nucleocapsid of Pseudomonas phaseolicola bacteriophage phi 6 transcribed large, medium, and small single-stranded RNA from the viral double-stranded RNA genome by a semiconservative (displacement) mechanism. Approximately 23%, 63%, and 65% of the nucleocapsid particles in the assay mixture synthesized at least one round of large, medium, and small single-stranded RNA molecules, respectively. Some of these particles reinitiated synthesis such that an average of 1.5 large, 33 medium, and 24 small single-stranded RNAs were synthesized from each double-stranded RNA.     

Role of sindbis virus capsid protein region II in nucleocapsid core assembly and encapsidation of genomic RNA

Sindbis virus is an enveloped positive-sense RNA virus in the alphavirus genus. The nucleocapsid core contains the genomic RNA surrounded by 240 copies of a single capsid protein. The capsid protein is multifunctional, and its roles include acting as a protease, controlling the specificity of RNA that is encapsidated into nucleocapsid cores, and interacting with viral glycoproteins to promote the budding of mature virus and the release of the genomic RNA into the newly infected cell. The region comprising amino acids 81 to 113 was previously implicated in two processes, the encapsidation of the viral genomic RNA and the stable accumulation of nucleocapsid cores in the cytoplasm of infected cells. In the present study, specific amino acids within this region responsible for the encapsidation of the genomic RNA have been identified. The region that is responsible for nucleocapsid core accumulation has considerable overlap with the region that controls encapsidation specificity.     

Hepatitis C virus translation preferentially depends on active RNA replication

Hepatitis C virus (HCV) RNA initiates its replication on a detergent-resistant membrane structure derived from the endoplasmic reticulum (ER) in the HCV replicon cells. By performing a pulse-chase study of BrU-labeled HCV RNA, we found that the newly-synthesized HCV RNA traveled along the anterograde-membrane traffic and moved away from the ER. Presumably, the RNA moved to the site of translation or virion assembly in the later steps of viral life cycle. In this study, we further addressed how HCV RNA translation was regulated by HCV RNA trafficking. When the movement of HCV RNA from the site of RNA synthesis to the Golgi complex was blocked by nocodazole, an inhibitor of ER-Golgi transport, HCV protein translation was surprisingly enhanced, suggesting that the translation of viral proteins occurred near the site of RNA synthesis. We also found that the translation of HCV proteins was dependent on active RNA synthesis: inhibition of viral RNA synthesis by an NS5B inhibitor resulted in decreased HCV viral protein synthesis even when the total amount of intracellular HCV RNA remained unchanged. Furthermore, the translation activity of the replication-defective HCV replicons or viral RNA with an NS5B mutation was greatly reduced as compared to that of the corresponding wildtype RNA. By performing live cell labeling of newly synthesized HCV RNA and proteins, we further showed that the newly synthesized HCV proteins colocalized with the newly synthesized viral RNA, suggesting that HCV RNA replication and protein translation take place at or near the same site. Our findings together indicate that the translation of HCV RNA is coupled to RNA replication and that the both processes may occur at the same subcellular membrane compartments, which we term the replicasome.     

Regulation of influenza virus RNA polymerase activity by cellular and viral factors.

An in vitro RNA synthesis system mimicking replication of genomic influenza virus RNA was developed with nuclear extracts prepared from influenza virus-infected HeLa cells using exogenously added RNA templates. The RNA synthesizing activity was divided into two complementing fractions, i.e. the ribonucleoprotein (RNP) complexes and the fraction free of RNP, which could be replaced with RNP cores isolated from virions and nuclear extracts from uninfected cells, respectively. When nuclear extracts from uninfected cells were fractionated by phosphocellulose column chromatography, the stimulatory activity for RNA synthesis was further separated into two distinct fractions. One of them, tentatively designated RAF (RNA polymerase activating factor), stimulated RNA synthesis with either RNP cores or RNA polymerase and nucleocapsid protein purified from RNP cores as the enzyme source. In contrast, the other, designated PRF (polymerase regulating factor), functioned as an activator only when RNP cores were used as the enzyme source. Biochemical analyses revealed that PRF facilitates dissociation of RNA polymerase from RNP cores. Of interest is that virus-coded non-structural protein 1 (NS1), which has been thought to be involved in regulation of replication, counteracted PRF function. Roles of cellular factors and viral proteins, NS1 in particular, are discussed in terms of regulation of influenza virus RNA genome replication.     

Deoxyribonucleic Acid Polymerase of Rous Sarcoma Virus: Studies on the Mechanism of Double-Stranded Deoxyribonucleic Acid Synthesis

The deoxyribonucleic acid (DNA) polymerase of Rous sarcoma virus synthesizes both single- and double-stranded DNA, utilizing the ribonucleic acid (RNA) of the viral genome as the initial template. Results of pulse-chase experiments indicate that the single-stranded DNA serves as unconserved template and precursor for the synthesis of double-stranded DNA. The latter reaction is apparently initiated in association with the viral RNA and may involve a partially double-stranded intermediate form.     

In vitro RNA replication directed by replicase complexes isolated from the subgenomic replicon cells of hepatitis C virus

Replication of hepatitis C virus (HCV) RNA in virus-infected cells is believed to be catalyzed by viral replicase complexes (RCs), which may consist of various virally encoded nonstructural proteins and host factors. In this study, we characterized the RC activity of a crude membrane fraction isolated from HCV subgenomic replicon cells. The RC preparation was able to use endogenous replicon RNA as a template to synthesize both single-stranded (ss) and double-stranded (ds) RNA products. Divalent cations (Mg2+ and Mn2+) showed different effects on RNA synthesis. Mg2+ ions stimulated the synthesis of ss RNA but had little effect on the synthesis of ds RNA. In contrast, Mn2+ ions enhanced primarily the synthesis of ds RNA. Interestingly, ss RNA could be synthesized under certain conditions in the absence of ds RNA, and vice versa, suggesting that the ss and ds RNA were derived either from different forms of replicative intermediates or from different RCs. Pulse-chase analysis showed that radioactivity incorporated into the ss RNA was chased into the ds RNA and other larger RNA species. This observation indicated that the newly synthesized ss RNA could serve as a template for a further round of RNA synthesis. Finally, 3' deoxyribonucleoside triphosphates were able to inhibit RNA synthesis in this cell-free system, presumably through chain termination, with 3' dGTP having the highest potency. Establishment of the replicase assay will facilitate the identification and evaluation of potential inhibitors that would act against the entire RC of HCV.