Suppression of the translation defect phenotype specific for a virus-associated RNA-deficient adenovirus mutant in monkey cells by simian virus 40

S Subramanian, R A Bhat, M K Rundell, B Thimmappaya
1986 Journal of Virology  
Human cells infected with adenovirus type 2 (Ad2) or AdS require VAI RNA for efficient translation of viral mRNAs at late times after infection. The Ad5 mutant dl-sub720 synthesized neither virus-associated I (VAI) nor VAII RNAs, and infection of human cells with this mutant resulted in reduced virion polypeptide synthesis. Infection of monkey cells with this mutant also resulted in drastic reduction of polypeptide synthesis compared with wild-type (WT) adenovirus infections. Steady-state
more » ... of hexon-specific mRNA were found to be comparable in WTand mutant-infected monkey cells. The in vitro translation experiments showed that double-mutantand WT-infected cells contained comparable levels of translatable hexon mRNA (and other adenovirus late mRNAs), suggesting that the severe inhibition of hexon protein synthesis in the VA mutant involves a translation block. Preinfection of monkey cells with simian virus 40 fully restored the efficient translation of this mRNA in the VA mutant infections to the level observed in WT-infected cultures. These results raise the possibility that simian virus 40 may encode or induce factors that suppress the translation block that occurs during adenovirus infections in the absence of the VA RNAs. labeled with [35S]methionine (50 uCi/ml, specific activity, >800 Ci/mmol; Amersham Corp.) for 1 h. The cells were washed twice with phosphate-buffered saline (pH 7.4) and lysed in cold RIPA buffer (0.15 M NaCl, 0.1% sodium dodecyl sulfate [SDS], 1.0% sodium deoxycholate, 1.0% Triton X-100, 1 mM EDTA, 20 mM Tris, pH 7.4) at a concentration of 2.0 x 106 cells per ml as described by Cepko and Sharp (9). Cell extracts derived from equal numbers of cells were diluted into SDS gel sample buffer and loaded directly onto 20% SDS-polyacrylamide gels (acrylamide-bisacrylamide, 20.0:0.1) to analyze late polypeptides. Hexon polypeptide was immunoprecipitated with a mono-363 Vol. 60, No. 2 on May 8, 2020 by guest
doi:10.1128/jvi.60.2.363-368.1986 fatcat:bciq7todbvaj7cdxeoooee37le