A Tale of 20 Alphaviruses; Inter-species Diversity and Conserved Interactions Between Viral Non-structural Protein 3 and Stress Granule Proteins

Abstract

Alphaviruses infect a diverse range of host organisms including mosquitoes, mammals, and birds. The enigmatic alphavirus non-structural protein 3 (nsP3) has an intrinsically disordered, C-terminal hypervariable domain (HVD) that can interact with a variety of host proteins associated with stress granules (SGs). The HVD displays the highest variability across the more than 30 known alphaviruses, yet it also contains several motifs that are conserved amongst different subgroups of alphaviruses. For some alphaviruses, specific nsP3–SG protein interactions are essential for virus replication. However, it remains difficult to attribute general roles to these virus-host interactions, as multiple amino acid motifs in the HDV display a degree of redundancy and previous studies were performed with a limited number of alphaviruses. To better understand nsP3-host protein interactions we conducted comprehensive co-localization experiments with the nsP3s of 20 diverse alphaviruses: chikungunya, Semliki Forest, Sindbis, Bebaru, Barmah Forest, Getah, Mayaro, Middelburg, O'nyong-nyong, Ross River QML and T48, Una, Whataroa, Southern Elephant Seal, Eilat, Tai Forest (TAFV), Venezuelan/Eastern/Western equine encephalitis (V/E/WEEV) and the aquatic Salmonid alphavirus (SAV), with three different SG proteins (G3BP and its insect homolog Rasputin, FMRP) and BIN1 in mammalian and mosquito cell lines. Despite that all terrestrial alphavirus nsP3s contained at least one BIN1-binding motif (PxPxPR), not all nsP3s co-localized with BIN1. Further, all alphaviruses except SAV, TAFV and VEEV displayed co-localization with G3BP. Although viruses lacking FGxF-like motifs contained Agenet-like domain binding motifs to facilitate interaction with FMRP, cytoplasmic nsP3 granules of all tested alphaviruses co-localized with FMRP. Crispr-Cas9 knockout of G3BP in mammalian cells abolished nsP3-FMRP co-localization for all alphaviruses except V/E/WEEV nsP3s that bind FMRP directly. G3BP knockout also changed nsP3 subcellular localization of Bebaru, Barmah Forest, Getah, and Sindbis viruses. Taken together this study paints a more detailed picture of the diverse interactions between alphavirus nsP3 and SG-associated host proteins. The interaction between nsP3 and G3BP clearly plays a central role and results in recruitment of additional host proteins such as FMRP. However, direct binding of FMRP can make the interaction with G3BP redundant which exemplifies the alternate evolutionary paths of alphavirus subgroups.

Intracellular mono-ADP-ribosyltransferases at the host–virus interphase

“…Early on it was found that SINV nsP3 is in complexes containing G3BP1 and G3BP2 [ 221 ]. The nsP3 proteins of other viruses were also found to colocalize with G3BP [ 222 ]. Of note is that several of the over 30 alphaviruses, which include a large number of mosquito-borne vertebrate pathogens [ 223 , 224 ], possess two FGxF-like motifs in nsP3.…”

The Putative Roles and Functions of Indel, Repetition and Duplication Events in Alphavirus Non-Structural Protein 3 Hypervariable Domain (nsP3 HVD) in Evolution, Viability and Re-Emergence

“…The ability of nsP3 to interact with various cellular proteins and functioning as gathering hubs also has allowed the formation of large cytoplasmic complexes [9,10,14,87,[129][130][131], which were suggested to regulate the host stress response during viral infection [108,129]. The OW alphaviruses SFV, SINV, CHIKV, and RRV and the NW alphavirus EEEV interact with mammalian G3BP family members and its mosquito homolog, Rasputin (Rin), forming the HVD-G3BP and HVD-Rin granule complexes in the cytoplasm of mammalian and mosquito hosts, respectively [32,132,133].…”

“…The FMRP was suggested to suppress viral RNA translation by redirecting it into the host's stress granules. The interaction site is known as the Agenet-like domain binding motif [9,87,131,134]. Subsequently, the alphavirus nsP3 HVD is suggested to modulate the host stress response by interacting with stress granules' components and preventing host stress granule formation [32,[131][132][133].…”

“…Despite being highly variable and having non-conserved genetic characteristics, nsP3 HVD from different species of alphavirus still consist of several conserved identified linear motifs that have been proven to interact with specific sets of host cellular factors from at least three protein families. These include several protein families with SH3 domains, either the G3BP, FXR, or both family proteins, and also two members of the NAP1 family [2,9,10,12,20,79,86,87,131]. Interestingly, particular HVD linear motifs are short, repetitive, and located at the very end of the HVD C-terminal [9,129] and demonstrate a high redundancy in functions during RC assembly and alphavirus replication [2,87].…”

Metagenomic Analysis of Togaviridae in Mosquito Viromes Isolated From Yunnan Province in China Reveals Genes from Chikungunya and Ross River Viruses

“…Mosquitoes are important biological vectors of many infectious viruses, including Getah virus (GETV), Ross River virus (RRV), chikungunya virus (CHIKV), Zika virus (ZIKV), and dengue virus (DENV), which were a menace to global public health and security (Sinclair and Asgari, 2020;Nowee et al, 2021;Pyke et al, 2021). Mosquitoes were considered as an intermediate host of numerous viruses that infect humans, animals, insects, plants, and other species (de Boer et al, 2021).…”