Panas, M. D. | bioRxiv Science

bioRxiv2019

Revisiting G3BP-S149 phosphorylation and its impact on stress granule assembly

Stress granules (SGs) are cytoplasmic, non-membranous RNA/protein structures that assemble in response to environmental stress. G3BP is a critical SG-nucleating protein, and its ability to regulate SGs has been reported to be regulated by serine 149 phosphorylation. We now report that the constructs engineered to contain non-phosphorylatable and phosphomimetic (G3BP1-S149A and G3BP1-S149E, respectively) mutations used in many studies include additional unintended mutations (A54T/S149A and S99P/S149E) one of which (S99P) is responsible for the effects on SG assembly attributed to S149E. Specifically, the S99P mutation alone reduces SG nucleation and impairs the ability to rescue SG assembly in {Delta}{Delta}G3BP1/2 U2OS KO cells, challenging the widely-stated conclusion that de-phosphorylation of serine 149 in G3BP1 promotes SG assembly. We used comparative mass spectrometry analysis of both (1) ectopically expressed GFP-G3BP1 in {Delta}{Delta}G3BP1/2 U2OS KO and (2) endogenous G3BP1 in wild-type U2OS, with and without sodium arsenite treatment, in an attempt to reproduce earlier findings, but found no significant changes in S149 phosphorylation that correlate with arsenite-induced SG formation.

cell biology↗

bioRxiv2019

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

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.

microbiology↗