ABSTRACT
Sindbis virus (SINV) is the prototype member of the Alphavirus genus, whose members cause severe human diseases for which there is no specific treatment. To ascertain host factors important in the replication of the SINV RNA genome, we generated a SINV expressing nsP4, the viral RNA-dependent RNA polymerase, with an in-frame 3×Flag epitope tag. Proteomic analysis of nsP4-containing complexes isolated from cells infected with the tagged virus revealed 29 associated host proteins. Of these, 10 proteins were associated only at a later time of infection (12 h), 14 were associated both early and late, and five were isolated only at the earlier time (6 h postinfection). These results demonstrate the dynamic nature of the virus-host interaction that occurs over the course of infection and suggest that different host proteins may be required for the multiple functions carried out by nsP4. Two related proteins found in association with nsP4 at both times of infection, GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1) and G3BP2 were also previously identified as associated with SINV nsP2 and nsP3. We demonstrate a likely overlapping role for these host factors in limiting SINV replication events. The present study also identifies 10 host factors associated with nsP4 6 h after infection that were not found to be associated with nsP2 or nsP3. These factors are candidates for playing important roles in the RNA replication process. Identifying host factors essential for replication should lead to new strategies to interrupt alphavirus replication.
Host factors associated to false negative and indeterminate results in an interferon‐γ release assay in patients with active tuberculosis
