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 3xFlag 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.