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

摘要

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.