| Alphaviruses can cause encephalitis, arthritis and rash in humans and are one of the main sources of mosquito-borne encephalitis in the United States. Sindbis virus (SINV) is the prototype alphavirus and is used as a model system for alphavirus encephalitis. Susceptibility to SINV infection depends on both viral factors and host factors, but how virus and host interact to affect the outcome of infection is not known. Among SINV nonstructural proteins (nsPs), the function of nsP3 has been elusive although the main role is speculated to be formation of virus replication complexes by interacting with cellular proteins. At the N-terminus, nsP3 contains an evolutionarily conserved macro domain that can mediate protein-protein interactions. To prove the importance of the nsP3 macro domain, we introduced mutations to the nsP3 macro domain and characterized the phenotypes of SINV with these mutations. Mutations impaired SINV replication in neurons, but not in BHK21 cells. In neurons, the stability of nsP3 was decreased by mutations, resulting in diminished virus replication complexes. To understand the mechanism, we determined the interaction of nsP3 with poly(ADP-ribose) polymerase (PARP)-1, a transcription regulator that can bind macro domains. PARP-1 was transiently recruited to virus replication complexes during active viral RNA synthesis, but this recruitment was mainly mediated by C-terminus of nsP3 and was not affected by mutations in the macro domain. However, interaction of nsP3 with a 30 KD cellular protein was affected by mutations, suggesting its involvement in SINV replication via the nsP3 macro domain. In addition, mutations reduced SINV virulence for mature neurons and 2 week old mice. Furthermore, the level of SINV-induced nuclear factor kappa B (NFkappa3) activation was reduced by mutations only in mature neurons. Inhibition of NFkappaB activation impaired early virus replication, late virus production and virus-induced neuronal death, indicating a pro-viral function of NFkappaB in SINV infection. We conclude that nsP3 interacts functionally and/or physically with host proteins such as PARP-1 and NFkappaB, and that these interactions play a role in SINV replication and virus-induced cell death in a cell type-dependent fashion. The nsP3 macro domain may be important for mediating virus-host interaction. |
