Mosquito Rasputin interacts with chikungunya virus nsP3 and determines the infection rate in Aedes albopictus

Abstract

BackgroundChikungunya virus (CHIKV) is an arthritogenic alphavirus (family Togaviridae), transmitted by Aedes species mosquitoes. CHIKV re-emerged in 2004 with multiple outbreaks worldwide and recently reached the Americas where it has infected over a million individuals in a rapidly expanding epidemic. While alphavirus replication is well understood in general, the specific function (s) of non-structural protein nsP3 remain elusive. CHIKV nsP3 modulates the mammalian stress response by preventing stress granule formation through sequestration of G3BP. In mosquitoes, nsP3 is a determinant of vector specificity, but its functional interaction with mosquito proteins is unclear.MethodsIn this research we studied the domains required for localization of CHIKV nsP3 in insect cells and demonstrated its molecular interaction with Rasputin (Rin), the mosquito homologue of G3BP. The biological involvement of Rin in CHIKV infection was investigated in live Ae. albopictus mosquitoes.ResultsIn insect cells, nsP3 localized as cytoplasmic granules, which was dependent on the central domain and the C-terminal variable region but independent of the N-terminal macrodomain. Ae. albopictus Rin displayed a diffuse, cytoplasmic localization, but was effectively sequestered into nsP3-granules upon nsP3 co-expression. Site-directed mutagenesis showed that the Rin-nsP3 interaction involved the NTF2-like domain of Rin and two conserved TFGD repeats in the C-terminal variable domain of nsP3. Although in vitro silencing of Rin did not impact nsP3 localization or CHIKV replication in cell culture, Rin depletion in vivo significantly decreased the CHIKV infection rate and transmissibility in Ae.albopictus.ConclusionsWe identified the nsP3 hypervariable C-terminal domain as a critical factor for granular localization and sequestration of mosquito Rin. Our study offers novel insight into a conserved virus-mosquito interaction at the molecular level, and reveals a strong proviral role for G3BP homologue Rin in live mosquitoes, making the nsP3-Rin interaction a putative target to interfere with the CHIKV transmission cycle.

Complete Genome Sequencing and Phylogenetic Analysis of a Getah Virus Strain (GenusAlphavirus, FamilyTogaviridae) Isolated fromCulex tritaeniorhynchusMosquitoes in Nagasaki, Japan in 2012

“…Nemoto et al (2016) suggested that the mutations in the nsP3 region of the strains 14-I-605-C1 and 14-I-605-C2 might result in altered interactions between GETV and its vectors because carboxylterminal domain of nsP3 has been reported to be involved in replication and vector specificity of the genus Alphavirus. In this regard, a recent study revealed that the nsP3 protein of Chikungunya virus (CHIKV; genus Alphavirus, family Togaviridae) interacts with a protein of the host mosquito, which is implicated in the infection efficiency of the virus to the host mosquito (Fros et al 2015). Therefore, like CHIKV, aa substitutions in the nsP3 of the recent Japansese GETV strains might alter their virological properties.…”

Rasputin a decade on and more promiscuous than ever? A review of G3BPs

“…Albopictus) shows that the normally diffuse cytoplasmic localization of Rin, is effectively sequestered to granules, containing nsP3, during coexpression studies. The interaction of Rin and nsP3 is moderated through an interaction of the C-terminal domain of nsP3 and the NTF2like domain of G3BPs [114]. More recently it has been shown that viral and cellular proteins containing FGDF motifs also bind with G3BPs and inhibit SGs assembly by G3BPs.…”

ADP-ribosylhydrolase activity of Chikungunya virus macrodomain is critical for virus replication and virulence

“…It induces the membrane remodeling necessary for the formation of cytoplasmic vacuoles and regulates RNA synthesis in a cell type-dependent manner (5). In vivo, nsP3 is a determinant of neurovirulence in mice (6)(7)(8)(9)(10). nsP3 has three major domains, a highly conserved N-terminal macrodomain, a central zinc-binding region, and a poorly conserved, unstructured, acidic, and highly phosphorylated C-terminal domain.…”