Translation and identification of the mRNA species synthesized in vitro by the virion-associated RNA polymerase of vesicular stomatitis virus.

Abstract

Vesicular stomatitis virus messenger RNA has been transcribed in vitro from the viral genome by the virion-associated RNA polymerase in quantities suitable for translation. Wheat germ cell-free extracts programmed with the isolated in vitro 12-18S RNA fraction synthesize polypeptides similar to the viral N, NS, and M proteins, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic peptide mapping of the in vitro products and the viral marker polypeptides. In addition, the RNA synthesized in vitro also codes for a protein of molecular weight 63,000 which may be a nonglycosylated form of the viral glycoprotein G. The 12-18S RNA has been partially separated into individual messenger species and these have been identified by the proteins for which they code. There are four monocistronic messenger species in the in vitro 12-18 S RNA and the coding capacity of three of these molecules agrees with the estimated molecular weight of the polypeptide assigned to it. Vesicular stomatitis virus (VSV) is a membrane-enveloped rhabdovirus (1) with a single-stranded RNA genome (2, 3) of molecular weight 3.6 to 4 X 10O (1,3). The virion also contains five structural polypeptides (4-6); the glycoprotein G (7, 8) is located in the virion surface projections, the matrix protein M is found in the virion envelope and the nucleoprotein N, together with the genome RNA and two minor proteins, NS and L, constitutes the ribonucleoprotein core (9).Polysomes of VSV-infected cells contain virus-specific messenger RNA (mRNA) which is complementary to the viral genome (10, 11). The mRNA can be resolved into two size classes by sucrose gradient sedimentation; a homogeneous species sedimenting at 28 S and heterogeneous RNAs sedimenting at 13-15 S (10). Further analysis of mRNA prepared from VSV-infected cells resolved the 13-15S component into at least three species (12-14) while the 28S RNA migrated as a homogeneous band (12). It was suggested, based on the molecular weights of these RNA species, that the 13-15S RNA could code for the G, N, NS, and M proteins (10) and that the 28S RNA could code for the L protein (6, 15). Indeed, subsequent work has shown that reticulocyte lysates programmed with 28S RNA isolated from VSV-infected cells can synthesize a protein which co-migrates with the viral L protein on sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels (16). Similarly, it has also been shown that proteins which co-migrate with the viral N, NS, and M polypeptides can be synthesized in several different cell-free extracts programmed with 13-15S RNA, but it was not clear whether authentic G protein was made.Abbreviations: VSV, vesicular stomatitis virus; NaDodSO4, sodium dodecyl sulfate.VSV has a virion-associated RNA polymerase which is capable of synthesizing, in vitro, RNA complementary to the viral genome (17,18). Recent work in this laboratory (19) has shown that RNA species of two size classes, 31 S and 12-18 S, can be synthesized in vitro by the virion-associated RNA polymerase of VSV. The in vitro ...

In vitro translation of mRNA species from cells infected with Machupo virus

“…Perhaps this protein is a nonglycosylated G protein precursor precipitated with anti-VSV IgG. This result is in agreement with previous data that there was little or no production of virus glycoproteins in cell-free translation systems (4,5,12).…”

“…The translation products were immunopreeipitated and the amount of immunoprecipitable radioactivity [~S~] was counted. B SDS-polyaci3damide gel electrophoresis ofimmunoprecipitable polypeptides from the cellfree systems programmed by RNA fractions (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11) kilodaltons. I t is in correlation with a molecular weight of N P protein (7,10).…”

A Vesiculovirus Showing a Steepened Transcription Gradient and Dominant trans -Repression of Virus Transcription

“…This small plaque isolate was shown to be 1,000 times less lethal than wild-type virus in animal models but still able to induce a robust interferon response (39). Further characterization showed that S 2 VSV transcribes more small viral mRNA species (12S to 18S; N, P, M and G mRNA) but less of the largest viral RNA species (40S; replication products) than wild-type VSV (5,12,23,32).For our studies both wild-type VSV-San Juan and S 2 VSV were obtained and plaque purified and the growth attenuation earlier reported was verified. To quantify the observed attenuation, we carried out a time course of infection with wild-type and S 2 VSV and measured the titer of virus released into supernatant fluid by plaque assay on BHK-21 cells.…”

“…This small plaque isolate was shown to be 1,000 times less lethal than wild-type virus in animal models but still able to induce a robust interferon response (39). Further characterization showed that S 2 VSV transcribes more small viral mRNA species (12S to 18S; N, P, M and G mRNA) but less of the largest viral RNA species (40S; replication products) than wild-type VSV (5,12,23,32).…”

“…The large polymerase subunit (L) carries out the enzymatic functions of the polymerase, including transcription, capping, methylation, and polyadenylation (5,23,33). The phosphoprotein (P) is a required cofactor for the polymerase and interacts with both L and the nucleocapsid protein (N).…”