SUMMARYHigh multiplicity infection of mouse fibroblast L-2 cells with mouse hepatitis virus (MHV) resulted, within 6 h, in a decline in total protein synthesis to about 7~ of that observed in uninfected cells. The amount of intracellular total translatable RNA, however, increased approximately threefold, as a result of the accumulation of virusencoded mRNAs. MHV-infected cells could be superinfected with vesicular stomatitis virus, demonstrating that MHV infection did not irreversibly alter the cellular translational machinery to the exclusion of non-MHV mRNAs. Comparative polysome analysis from MHV-infected and uninfected L-2 cells showed that MHV infection resulted in an increase in single 80S ribosomes and in a shift from longer to shorter polysomes. These observations suggest first, that MHV infection inhibits total protein synthesis at a very early stage, as evidenced by the increase in 80S ribosomes, and, second, that the increased number of viral mRNAs produced after infection compete with cellular mRNAs for cellular ribosomes. In vitro translation of RNA extracted from MHV-infected and mock-infected cells suggested that levels of cellular mRNAs were decreased after infection. This suggestion was confirmed by demonstrating the loss of cellular actin mRNA, using a radiolabelled cDNA probe, as a consequence of MHV infection.

Ammonium chloride at a concentration of 20 mM delayed by 4-5 hr the production of virus progeny in mouse L-2 cells infected at high multiplicity with mouse hepatitis virus (MHV). This delay was seen in the production of both intracellular and extracellular virus. However, the final titers were similar to those produced by MHV-infected cells maintained in normal medium. The manifestation of virus-induced cell fusion was similarly found to be delayed, but not otherwise decreased in severity, when ammonium chloride was present in the culture medium. Ammonium chloride caused similar delays in production of virus-specific, positive-sense RNAs and of viral polypeptides. The relative proportions and apparent molecular weights of viral RNAs and polypeptides were similar to those found in MHV-infected cells cultured in normal medium. In vitro translation of endogenously produced viral RNAs in cell extracts, prepared from MHV-infected cells, was not inhibited by ammonium chloride. Thus, ammonium chloride has no specific, inhibitory effect on viral protein synthesis. Ammonium chloride did not reduce the number of virus-infected cells in culture, as monitored by infectious center assay. Analysis of early events in MHV infection showed that ammonium chloride did not affect adsorption or internalization of MHV by L-2 cells. However, the subsequent eclipse phase, as monitored by decline in infectivity of internalized virus inoculum proceeded less efficiently in the presence of ammonium chloride. On the basis of the known inhibitory effects of ammonium chloride on lysosomal/endosomal functions, the results suggest an endosomal mechanism of MHV uncoating. Thus the primary effect of ammonium chloride on MHV infection of L-2 cells is to attenuate virus uncoating, thereby chronologically displacing all subsequent virus-encoded functions.