The overall aim of this research program is to define the mechanism by which cellular mRNA translation is inhibited in vesicular stomatitis virus (VSV) and mengovirus infected L cells. Studies on the defect in the process of initiation of protein synthesis will be carried out. Both initiation factor eIF-2 and a fraction containing a mixture of eIF-4B and eIF-4F are required for restoring protein synthesis activity in lysates from VSV infected cells. It is proposed that the catalytic function of eIF-2 is altered after infection. To test this hypothesis the state of phosphorylation of eIF-2 in infected cells and in extracts from infected cells will be determined. The ability of the purified eIF-2.eIF-2B complex to rescue cellular mRNA translation will also be tested. The fraction containing eIF-4B and eIF-4F will be fractionated and the isolated components tested individually to ascertain which is the actual rescue factor. The two regions of the VSV genome which must be transcribed for the inhibition of cellular protein synthesis will be further characterized using RNA-DNA hybridization techniques with recombinant plasmid DNAs containing cDNAs to VSV mRNAs and the exact 3' end of the virus genome as probes. Temperature sensitive VSV mutants will be screened at the permissive and restrictive temperatures to determine if there is a correlation between the presence of the transcripts and the inhibition of protein synthesis. Cell killing and protein synthesis inhibition are strongly related. The less cytopathic the virus stock, the less severe the immediate inhibition of protein synthesis. VSV stocks will be fractionated using sucrose gradient analysis in an attempt to isolate the putative protein synthesis inhibitory particles. These particles will be tested for their ability to kill cells. The S-l00 fraction of mouse L cells will be fractionated by conventional means in order to characterize the factor necessary for restoring protein synthesis activity in lysates from mengovirus infected cells. VSV and mengovirus are prototypes of such extremely virulent viruses as rabies and poliovirus, respectively. Therefore, it is hoped that these studies will help us understand the basis for the cytopathogenicity of certain virulent viruses that affect mankind.
