Novel dsRNA-dependent activation of a cellular antiviral response to vesicular stomatitis virus

Vesicular Stomatitus Virus (VSV) is the prototypic member of the Rhabdoviridae family and has played an important role in our understanding of host-cell responses to infections by nonsegmented negative-strand RNA viruses. VSV polR mutants, which display subtle alterations in viral RNA synthesis, are also growth restricted in a number of established cell lines. Both mouse L-929 and rat 3Y1 cells (but not BHK cells) display strong growth restriction of the polR virus (∼100-fold) which differs from the wild-type (wt) virus by a single amino Arg179 to His substitution in the nucleocapsid protein. The block in viral multiplication takes place at two levels: viral genome replication (∼6-fold less) and infectivity of released virus particles (∼10-fold less). PolR transcription, protein synthesis, and released particle protein composition are not significantly affected in non-permissive cell infections.; Furthermore, vaccinia virus co-infection rescues polR virus growth in mouse L-929 cells. A VSV minigenome system to express Vaccinia-encoded dsRNA-binding E3L protein in polR-infected cells was developed. Expression of E3L or antiviral antagonist proteins from influenza virus (NS1A and NS1B) cells rescues polR virus growth in L-929. Antiviral antagonists that do not bind dsRNA, like Sendai virus C, are unable to rescue polR. In addition we show that the N-terminal dsRNA-binding domain of NS1 alone is sufficient for polR rescue.; This cell-type specific growth restriction appears to be due to enhanced dsRNA production in polR-infected L-929 cells virus as evidenced by a ∼3-fold increase in PKR-mediated eIF2alpha phosphorylation compared to wt PKR activation is also not required for polR restriction since the potent PKR inhibitor 2-aminopurine had only a slight rescuing effect in L-929 cells and none in 3Y1 cells. Conditioned media from restricted cell types (L-929 and 3Y1) failed to induce polR restriction in permissive BHK cells, strongly arguing against the involvement of secreted antiviral factors. Furthermore, production of non-viral dsRNA (GFP reporter gene sequences) during wt virus infection leads to the same growth restriction as polR virus. These findings show that certain cell types possess a currently undescribed antiviral mechanism that depends on dsRNA signaling but is independent of gene induction and relies on novel antiviral effector(s) activities.