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

Date Published: June 14, 2019

Publisher: Public Library of Science

Author(s): Benjamin Götte, Marc D. Panas, Kirsi Hellström, Lifeng Liu, Baila Samreen, Ola Larsson, Tero Ahola, Gerald M. McInerney, Mark T. Heise.


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.

Partial Text

Viral infections are inevitably accompanied by a competitive crosstalk between the host and the pathogen, engaging a complex network of protein-protein interactions. Since exploitation of host resources is crucial for the viral replication cycle, host responses aim to interfere with such measures and to clear the threat. Consequently, viruses have evolved to target host proteins involved in cellular defence mechanisms. G3BP-1 and -2 (hereafter jointly referred to as G3BP) are homologous proteins with critical roles in the assembly of cellular stress granules (SGs), dynamic assemblies of stalled translation initiation complexes and RNAs [1, 2]. The proteins contain an N-terminal NTF2-like domain, which is involved in dimerization and interaction with other proteins, long stretches of intrinsically disordered protein sequence as well as RNA-recognition motifs (RRMs) and arginine-glycine rich RGG motifs at the C terminus [2]. SG formation requires the NTF2-like and RGG domains and likely involves G3BP-driven condensation of stalled mRNP complexes as well as numerous SG nucleator proteins including TIA-1. SG induction is triggered by inhibition of translation initiation caused by cellular stresses including virus infection. As viruses strictly depend on the host translation machinery it is not surprising that many viruses from diverse virus families have developed mechanisms to disrupt SG assembly [3]. For Old World alphaviruses, including Semliki Forest virus (SFV) and chikungunya virus (CHIKV), interaction between FGDF motifs in the C-terminal region of viral non-structural protein 3 (nsP3) and G3BP disrupts SGs and blocks their formation [4, 5].

The host translation machinery plays an indispensable role in viral life cycles. Despite their often very limited coding capacity, viruses have developed remarkable strategies to manipulate cellular translation to ensure synthesis of viral proteins [44, 45]. Here we describe a novel strategy by which Old World alphaviruses utilize the cellular ribosome-associated protein G3BP1 to enrich components of the translation machinery at the sites of viral RNA replication. We show that this enrichment depends on the NTF2-like and RGG regions of G3BP1. These are the same domains that are necessary for the formation of SGs under conditions of cellular stress, the NTF2-like domain is needed for homodimerisation as well as binding to caprin-1 and USP10, the positive and negative regulators of SGs, while the RGG region mediates binding to the 40S ribosomal subunits [2]. Under infection conditions, nsP3 binds to the N-terminal NTF2-like domain of G3BP1 and recruits it to viral replication complexes. We demonstrate that 40S ribosomal subunits remain associated with nsP3:G3BP1 complexes (Fig 5), promoting the condensation of electron-dense patches around viral spherules at the PM and around internal CPV clusters (Fig 3B and S3B Fig). These electron-dense patches show noticeable similarity to bona fide SGs under the electron microscope (S3D Fig and [33]) and are enriched for translation initiation factors that are also found in SGs [35]. In contrast to stress-induced SGs however, the nsP3:G3BP1:40S complex-dependent protein accumulations are sites of enhanced translation (Fig 6). We also show that SFV-infected cells, in which the SFV nsP3:G3BP1:40S complex can form (Fig 5), engage significantly more ribosomes in translating polysomes than in cells where this complex cannot form (Fig 7A). The sequestration of G3BP upon infection with Old World alphaviruses thus not only subverts the cellular stress response, but also more explicitly exploits a host mechanism to condense the translation machinery and target it for production of viral proteins. Our data suggest that recruitment of components of the host translation machinery to CPVs is important for the efficient synthesis of CHIKV proteins, particularly at early stages of replication and subsequently influencing all steps of the viral life cycle.




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