Alphavirus Minus Strand RNA Synthesis and RNase L

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DESCRIPTION (provided by applicant): We are submitting a revision of a new application that took advantage of possibilities suggested by findings that we believe will begin a new research direction for this family of viruses. Alphaviruses are of general interest as they produce disease in animals and humans, and replicate in invertebrates and vertebrates. Synthesis of infectious progeny starts with translation and copying of the infecting genome into a minus strand RNA template. Of note, alphavirus minus strand synthesis ceases by an as yet unknown mechanism at the end of the early phase, ~4 h post-infection (pi). Previous models argued cessation was a virus property, with saturation of host factors or assembly sites, or possibly premature and improper replicase processing playing a role. We now know the host cell activates a latent, antivirus response leading to cessation during the 4 h period after entry. In support of a role for host functions, minus strand cessation did not occur in C7/10 mosquito cells that naturally lack RNase L and form persistent infections or in RNase L knockout (ko) mouse embryo fibroblasts (MEF) that are deficient in this latent endonuclease mediator of the dsRNA-2'-5' oligoA synthetase pathway; reconstitution of RNaseL in ko cells restored cessation. Moreover, BHK cells persistently infected with mutant Sindbis nsP2 replicons also continued minus strand synthesis, unlike wildtype nsP2 replicons. Unexpectedly, in cells lacking RNase L or expressing mutant nsP2 proteins, the Sindbis replication complex (RC) was now transcriptionally shortlived. Thus, RNase L and nsP2 play role(s) in cessation and RC stability. Cessation was normal in PKR ko and wt MEF, eliminating roles for Mx1 and PKR. Four aims are proposed. Aim 1 will map the region of RNase L required for cessation. Aim 2 will use gene microarrays to identify changes in host mRNAs during infection and with cessation. Aim 3 will analyze mutant nsP2 proteins, whose presence in BHK cells led to loss of the same virus replication features as seen in RNase L-/- MEF and Aedes mosquito cells. Aim 4 will probe the molecular basis of the loss (turnover) of Sindbis RC in RNase L deficient and mutant nsP2 cell environments. Together, the results will elucidate the innate response to alphavirus infection and the role of the host RNase L and the viral nsP2 protein in its outcome.