Separate domains of G3BP promote efficient clustering of alphavirus replication complexes and recruitment of the translation initiation machinery

Abstract

G3BP-1 and -2 (hereafter referred to as G3BP) are multifunctional RNA-binding proteins involved in stress granule (SG) assembly. Viruses from diverse families target G3BP for recruitment to replication or transcription complexes in order to block SG assembly but also to acquire pro-viral effects via other unknown functions of G3BP. The Old World alphaviruses, including Semliki Forest virus (SFV) and chikungunya virus (CHIKV) recruit G3BP into viral replication complexes, via an interaction between FGDF motifs in the C-terminus of the viral non-structural protein 3 (nsP3) and the NTF2-like domain of G3BP. To study potential proviral roles of G3BP, we used human osteosarcoma (U2OS) cell lines lacking endogenous G3BP generated using CRISPR-Cas9 and reconstituted with a panel of G3BP1 mutants and truncation variants. While SFV replicated with varying efficiency in all cell lines, CHIKV could only replicate in cells expressing G3BP1 variants containing both the NTF2-like and the RGG domains. The ability of SFV to replicate in the absence of G3BP allowed us to study effects of different domains of the protein. We used immunoprecipitation to demonstrate that that both NTF2-like and RGG domains are necessary for the formation a complex between nsP3, G3BP1 and the 40S ribosomal subunit. Electron microscopy of SFV-infected cells revealed that formation of nsP3:G3BP1 complexes via the NTF2-like domain was necessary for clustering of cytopathic vacuoles (CPVs) and that the presence of the RGG domain was necessary for accumulation of electron dense material containing G3BP1 and nsP3 surrounding the CPV clusters. Clustered CPVs also exhibited localised high levels of translation of viral mRNAs as detected by ribopuromycylation staining. These data confirm that G3BP is a ribosomal binding protein and reveal that alphaviral nsP3 uses G3BP to concentrate viral replication complexes and to recruit the translation initiation machinery, promoting the efficient translation of viral mRNAs.

Sensitivity of Alphaviruses to G3BP Deletion Correlates with Efficiency of Replicase Polyprotein Processing

“…The results confirmed sustained higher levels of nsP expression in the U2OS ΔΔ than in WT U2OS cells. This is consistent with a previous observation made for transient transfection of these cells lacking the ability to form stress granules (SGs) (15,17). The introduction of RH, F3A NC , or F3A NC -RH mutations had no effect on the observed difference of expression in the U2OS ΔΔ compared to WT cells (data not shown).…”

“…Alphaviruses differ in their sensitivity to G3BP deletion. Initially, we aimed to determine if different SFV strains share a resistance to G3BP deletion as previously observed for SFV4 (17). We infected human U2OS wild-type (WT) cells or G3BP1/2 double-knockout cells (U2OS ΔΔ [15]) with either SFV4 or SFV A774 at a multiplicity of infection (MOI) of 0.1 and determined virus titers over a period of 48 hpi (Fig.…”

“…G3BP1 and -2 (here combinedly referred to as G3BP) are homologous multifunctional RNA-binding proteins, best characterized for their stress granule-nucleating function in response to environmental stress, including virus infection (14,15). Whereas New World alphaviruses do not interact with G3BP, with Eastern equine encephalitis virus (EEEV) being the only reported exception to this (16), Old World alphaviruses recruit G3BP to viral replication complexes (17,18). This recruitment is mediated by FGDF motifs, located in the hypervariable domain (HVD) of nsP3, which bind to the N-terminal NTF2-like domain of G3BP (19).…”

“…The two FGDF motifs thereby interlink G3BP dimers into an nsP3-G3BP polycomplex. We have previously shown that the NTF2-like domain of G3BP1 promotes clustering of SFV4 (molecular clone derived from prototype SFV [22]) replication complexes, likely via the formation of these nsP3-G3BP polycomplexes, and is sufficient to facilitate efficient replication of SFV4, suggesting a predominantly structural proviral role of G3BP for SFV4 (17). In the absence of G3BP, SFV4 is attenuated but continues to produce progeny viruses to detectable levels.…”

Activation of the PI3K-AKT Pathway by Old World Alphaviruses

“…All nsPs fulfil a role in viral RNA synthesis [8]. Those roles are quite well understood although the function of nsP3 remained obscure for a long time but is starting to be more understood [9,10]. nsP3 has three domains: the macrodomain, the alphavirus unique or zinc-binding domain (AUD/ZBD) and the hypervariable region (HVD).…”