Characterization of the infections of permissive and nonpermissive cells by host range mutants of vesicular stomatitis virus defective in RNA methylation

Abstract

Two host range mutants of VSV, hr 1 and hr 8, which, unlike the wild-type virus, have a mRNA methylation defect and direct the in vitro synthesis of full-length capped but unmethylated viral mRNAs have been described previously ( S. M. Horikami and S. A. Moyer, 1982, Proc. Natl. Acad. Sci. USA 79, 7694–7698). It is shown that the in vivo nonpermissive infection of HEp-2 cells by either of these two mutants is characterized by the reduced synthesis of full-length mRNAs at levels characteristic of primary transcription and the total lack of synthesis of genome-length RNA. The VSV mRNAs synthesized by either mutant in HEp-2 cells are not translated either in vivo or in vitro in mRNA-dependent rabbit reticulocyte lysates. Subsequent isolation and analysis of the mRNAs from infected HEp-2 cells has shown that the 5′ termini of the messages contain a cap structure which is guanylylated, but unmethylated (GpppA), a finding that might account for the lack of translatability. Hence these mutants are unable to properly methylate mRNAs whether they are synthesized in vitro or in vivo within nonpermissively infected cells. It is also shown that unlike hr 1, the undermethylation of mRNA synthesized by hr 8 is partially reversible by the addition of high levels of AdoMet in vitro. It is interesting to note, therefore, that permissive baby hamster kidney (BHK) cells have a 10-fold higher level of endogenous AdoMet than the nonpermissive HEp-2 cells. Unlike singly infected cells, the coinfection of HEp-2 cells with either hr mutant and a poxvirus yields a permissive infection for these two host range mutants. Analysis of the VSV mRNAs produced in vivo under the conditions of rescue reveals the presence of fully methylated caps (7mGppp(m)A m), suggesting that poxvirus may rescue the mutants by converting the VSV mRNAs to a translationally active form due to methylation by the cytoplasmic poxvirus mRNA methyltransferase enzymes. Both mutants are, however, able to grow normally in permissive BHK cells. An analysis of the translationally active mRNAs from infected permissive cells shows the presence primarily of a 5′-monomethylated cap, 7mGpppA. Finally, we have examined the nonpermissive infections of two other host range mutants of VSV ( hr 5 and hr 7). Unlike mutants hr 1 and hr 8 described above, these two mutants synthesize mRNA in HEp-2 cells which is translated both in vivo and in vitro. However, hr 5 and hr 7 also fail to synthesize genome-length RNA in vivo in HEp-2 cells and, hence, appear to be blocked directly at the level of the replication of the viral genome RNA.