Formation of Antiviral Cytoplasmic Granules during

Vaccinia virus (VV) mutants lacking the double-stranded RNA (dsRNA)-binding E3L protein (E3Lmutant VV) show restricted replication in most cell types, as dsRNA produced by VV activates protein kinase R (PKR), leading to eIF2 phosphorylation and impaired translation initiation. Here we show that cells infected with E3L mutant VV assemble cytoplasmic granular structures which surround the VV replication factories at an early stage of the nonproductive infection. These structures contain the stress granule-associated proteins G3BP, TIA-1, and USP10, as well as poly(A)-containing RNA. These structures lack large ribosomal subunit proteins, suggesting that they are translationally inactive. Formation of these punctate structures correlates with restricted replication, as they occur in>80 % of cells infected with E3L mutant VV but in only 10 % of cells infected with wild-type VV. We therefore refer to these structures as antiviral granules (AVGs). Formation of AVGs requires PKR and phosphorylated eIF2, as mouse embryonic fibroblasts (MEFs) lacking PKR displayed reduced granule formation and MEFs lacking phosphorylatable eIF2 showed no granule formation. In both cases, these decreased levels of AVG formation correlated with increased E3L mutant VV replication. Surprisingly, MEFs lacking the AVG component protein TIA-1 supported increased replication of E3L mutant VV, despite increased eIF2 phosphorylation and the assembly of AVGs that lacked TIA-1. These data indicate that the effective PKR-mediated restriction of E3L mutant VV replication requires AVG formatio