Rasputin/G3BP mediates subversion of antiviral immunity by o'nyong-nyong virus in Anopheles coluzzii.

Cellular G3BP proteins are essential for alphavirus infection in both vertebrate and mosquito hosts, but the underlying mechanism of their proviral activity is poorly understood in any host. Whether the mosquito G3BP ortholog, Rasputin (Rin), interacts with host immunity to influence alphavirus infection has not been investigated, and anopheline mosquito interactions with arboviruses have been little studied. Here, we find that Rin silencing in Anopheles mosquitoes results in decreased ONNV infection levels, indicating a proviral activity for Anopheles Rin. We find that Rin function is required to maintain basal activity of the antiviral Imd and JAK/STAT pathways in uninfected mosquitoes. However, during ONNV infection, the control of the Imd pathway by Rin activity appears corrupted because Rin silencing leads to overexpression of the Imd positive regulator, Rel2. Thus, silencing of Rin both augments Rel2 transcript abundance and decreases ONNV load. Co-silencing of Rel2 with Rin restores normal ONNV infection levels, indicating that Rin activity is required to inhibit Imd function during ONNV infection, and which explains most of the Rin proviral phenotype. In addition, we show that the ONNV non-structural protein 3 (nsP3), which binds to Rin, strongly alters the pattern of Anopheles cellular protein partners interacting with Rin. In the presence of ONNV nsP3, 48 Rin-binding host proteins are unchanged but seven binding proteins are excluded and eight new cellular proteins bind Rin. The altered cellular protein partners are candidate host factors involved in viral subversion of Rin control over Imd activity. Overall, these results reveal a molecular mechanism in which ONNV, probably through nsP3, co-opts the normal Rin function for basal cellular immune activity by subverting the Imd antiviral pathway to promote infection. These results may be generalizable for Rin function during alphavirus infection of other mosquitoes, as well as for G3BP function in the mammalian host, and could offer a target for development of vector-based genetic control tools against arbovirus transmission.