Virion-associated RNA Polymerase Research Articles

Messenger RNA species synthesized in vitro by the virion-associated RNA polymerase of vesicular stomatitis virus

The virion-associated RNA-dependent RNA polymerase of vesicular stomatitis virus (VSV) synthesizes in vitro two size classes of RNA products similar to those observed in VSV-infected cells. One RNA product sediments at 31S with an approximate molecular weight of 2.1 × 10 6. The smaller products consist of at least three classes of RNA sedimenting at 17S, 14.5S, and 12S with molecular weights of 0.7 × 10 6, 0.52 × 10 6, and 0.37 × 10 6, respectively. Hybridization experiments show that both the 31S and 12–18S RNA products are complementary to the genome RNA, and that each class is transcribed from different nucleotide sequences. From the molecular weights of the RNA species and the hybridization experiments, it seems that almost the entire VSV genome RNA is transcribed in vitro.

Reovirus messenger RNA contains a methylated, blocked 5'-terminal structure: m-7G(5')ppp(5')G-MpCp-.

Reovirus mRNA synthesized in vitro by the virus-associated RNA polymerase in the presence of S-adenosylmethionine contains blocked, methylated 5'-termini with the structure, m-7G(5')ppp(5')G-MpCp. The functional significance and possible mechanism of formation of this novel 5'-5' terminal nucleotide linkage are discussed.

In vitro synthesis of methylated messenger RNA by the virion-associated RNA polymerase of vesicular stomatitis virus

Purified vesicular stomatitis virus contains an enzyme activity that incorporates the methyl group of S-adenosyl-L-methionine into the messenger RNA products during their synthesis in vitro by the virion-associated transcriptase. The methylation reaction is subject to inhibition by S-adenosyl-L-homocysteine, suggesting the transfer of only the methyl group of S-adenosyl-L-methionine to RNA products. The methyl-labeled RNA products are sensitive to RNAase and alkaline hydrolysis and bind efficiently to oligo-(dT)-cellulose. The methylase activity is further shown to be associated only with the VSV ribonucleoprotein core isolated from virions. The methylation occurs at the 5′ terminal base of the RNA chains with approximately 2 methyl groups incorporated per RNA molecule. Alkaline hydrolysis of the methyl-labeled RNA released a 5′ terminal dinucleotide, consistent with some methylation occurring at the 2′ position of the terminal nucleotide.

Ribonucleic acid synthesis by isolated viruses of Penicillium stoloniferum.

SUMMARY Purified Penicillium stoloniferum viruses PsV-F and PsV-S both possess virus-associated RNA polymerase activity. The PsV-F polymerase was dependent upon GTP, ATP and magnesium chloride. It was not activated by Triton X-100. No DNA synthesis was observed when deoxyribonucleoside triphosphates were substituted for RNA precursors. The RNA reaction product for both viruses remained associated with the virus in sedimentation and electrophoresis and was resistant to digestion by pancreatic ribonuclease both before and after extraction from the virus. Extracted PsV-F RNA reaction product co-electrophoresed with parental double-stranded RNA.

Poly(A) polymerase activity in reovirus.

An enzymatic activity which synthesized oligo(A) in vitro was found in highly purified reovirus. The poly(A) polymerase activity was dependent on Mn(2+) and utilized only ATP, whereas the virion-associated RNA polymerase required all four ribonucleoside triphosphates and Mg(2+). Oligo(A) synthesis was demonstrated with complete virions and infectious subviral particles derived from virus by limited chymotrypsin digestion but not with cores, a product of extensive chymotrypsin digestion of virus. The enzymatic product and the oligo(A) from purified virions were isolated by binding to oligo(dT)-cellulose columns. Most of the in vitro product was similar in size and structure to the oligo(A) from purified virions by the criteria of gel electrophoresis, DEAE-cellulose chromatography, end-group analysis, and sensitivity to RNase. The evidence suggests that oligo(A) synthesis is mediated by the poly(A) polymerase during a late step in viral morphogenesis and may result from an alternative activity of the virion-associated transcriptase.

Inhibition of the particle-associated RNA-dependent RNA polymerase activity of influenza viruses by chelating agents.

SUMMARY Approximately 0.05 mm concentrations of the chelating agents bathocuproine and bathophenanthroline disulphonic acid disodium salts, which form very stable complexes with ‘soft’ heavy-metal ions such as zinc, inhibited in vitro the RNA-dependent RNA polymerase activity associated with influenza B/LEE/40 and A/RI-5+ (H2N2) viruses in the presence of a large molar excess of Mn(II) and Mg(II). Certain heterocyclic thiosemicarbazones also inhibited influenza RNA polymerase activity, although complete inhibition was not detected even with high concentrations (0.1 mm) of the compounds. Analogues of the active heterocyclic thiosemicarbazones, such as isatin 3-semicarbazone, which have reduced chelating activities for zinc, also had reduced inhibitory effects on influenza virus associated RNA polymerase activity. However, inhibition of influenza RNA polymerase activity by 0.1 mm-bathocuproine or 0.1 mm-isatin 3-thiosemicarbazone was not reversed by the addition of molar equivalent or excess concentrations of zinc. Relatively high concentrations of the chelating agents had no detectable effect on the haemagglutinin, neuraminidase or infectivity titres of influenza B/LEE/40 virus. The chelating agents may inhibit influenza virus-associated RNA polymerase activity by the formation of a ternary complex of enzyme-metal-ligand.

"Methylation-coupled" transcription by virus-associated transcriptase of cytoplasmic polyhedrosis virus containing double-stranded RNA.

S-adenosyl-L-methionine (SAM) activated the virus-associated RNA polymerase of cytoplasmic polyhedrosis virus in vitro. Synthesis of single-stranded viral RNA (mRNA) proceeded depending on the presence of SAM.A methyl residue of SAM was incorporated into an RNA molecule. A ribose moiety of adenylic acid in the 5'-terminal region of the nascent RNA was methylated in the very early stage of the transcription. The dependence of the viral transcription on the presence of SAM and the methylation of terminal nucleotide suggests that the transcription of CPV is a "methylation-coupled" reaction.

In vitro synthesis of RNA that contains polyadenylate by virion-associated RNA polymerase of vesicular stomatitis virus.

The RNA synthesized in vitro by the virion-associated RNA-instructed RNA polymerase of purified vesicular stomatitis virus contains polyadenylate sequences. These have been demonstrated by their partial resistance to pancreatic and T(1) ribonucleases and their capacity to bind to poly(U) filters and oligo(dT)-cellulose. The polyadenylate sequences range in apparent size from 50 to 200 bases, similar to the size of the poly(A) in mRNA from vesicular stomatitis virus-infected cells. Possible mechanisms of polyadenylylation of the in vitro RNA product are discussed.

An identical 3′-terminal sequence in the ten reovirus genome RNA segments

An identical oligonucleotide, PyrpApApC, is released from the 3′-ends of all ten reovirus genome double-stranded RNA segments by digestion with pancreatic RNase. This sequence may be the recognition site for the virion-associated RNA polymerase or involved in linking the genome segments within the virion.

Guanosine-5′-diphosphate at the 5′ termini of reovirus RNA: Evidence for a segmented genome within the virion

All ten double-stranded RNA fragments isolated from purified reovirus contain ppGp at the 5′ termini. The presence of a unique 5′-terminal nucleotide indicates that the viral genome in situ consists of segments which are synthesized as discrete units in infected cells. The penultimate base is a pyrimidine. This 5′ sequence, ppGpPyp, is identical to that reported previously for the ten reovirus messenger RNA species synthesized in vitro. The results indicate that the double-stranded RNA segments are perfect duplexes which are transcribed end-to-end by the virion-associated RNA polymerase.

Cytosine at the 3'-termini of reovirus genome and in vitro mRNA.

DOUBLE stranded RNA isolated from purified reoviruses consists of a mixture of ten different genome fragments1. We have shown that a unique 5′-terminal sequence, ppGpYp (Y = pyrimidine), is present in all ten segments, indicating that they exist as distinct entities within the virion (A. K. S. and A. J. S., manuscript submitted for publication). The duplex segments are transcribed in vitro by a virion-associated RNA polymerase and the resulting single stranded mRNAs also terminate with 5′-ppGpYp2. In view of the similarity between the 5′ ends of reovirus genome RNA and in vitro mRNA, it was of interest to compare their 3′-termini. We now report that both types of RNA have cytosine as the 3′-terminal base.

Vaccinia virus directed RNA and protein synthesis in the presence of rifampicin

The effects of rifampicin, an inhibitor of bacterial DNA transcription, on the formation of infectious vaccinia virus and viral particles, RNA and proteins were studied under single step growth conditions. Although virion formation was inhibited by more than 99%, the synthesis of early and late viral directed RNA species, as characterized by their sedimentation properties, was unaffected. Rifampicin did not prevent the synthesis of early and late viral proteins identified by immunodiffusion, disc gel electrophoresis and disc gel immunoelectrophoresis. The drug also did not inhibit the in - vitro activity of the virion associated RNA polymerase.