Role of viral and host factors in regulation of translation during sindbis virus infection

Sindbis Virus (SINV) causes an acute cytopathic infection in vertebrate cells and a persistent non cytopathic infection in mosquito cells. Unlike mosquito cells, there is translation shut off in vertebrate cells few hours post infection. Using a system to study SINV replication in Drosophila melanogaster, I determined that overexpression of Akt enhanced SINV replication. Moreover, SINV replication complex formation led to activation of the PI3K-Akt-TOR pathway early in infection in mosquito cells. This led to a downstream increase in p4E-BP1 and in the translation of a capped reporter mRNA in mosquito but not in mammalian cells. We propose that this virus induced increase in translation allows the efficient translation of viral mRNA while minimizing the burden on the cell; hence enabling the establishment of persistence. The second part of my study was aimed at looking at the role of the 5' untranslated region (5'UTR) of the subgenomic (SG) RNA in inhibiting host translation. Mutations were made in the 5'UTR region where nucleotides 1-10 and 11-20 were deleted. Analysis of the effect of these mutations and a revertant of 11-20 mutant on translation efficiency of the SG mRNA indicated that the ability to shut-off host cell translation directly correlated with the efficiency of translation of the SG mRNA. This suggests a novel mechanism where the translational quality and quantity of the SG mRNA play a role in the inhibition of host cell translation independent of viral proteins. Lastly, a comparative proteomic analysis of the infected membrane fractions of mosquito and chicken embryonic fibroblasts was performed. A number of signaling molecules and translation factors were identified detailed analysis of which would add to our current knowledge of SINV replication in different hosts.