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

• DOI: 10.1371/journal.ppat.1007842
• Publication Date: 2019-06-14T17:43:09Z
• Authors: Benjamin Götte; Marc D. Panas; Kirsi Hellström; Lifeng Liu; Baila Samreen; Ola Larsson; Tero Ahola; Gerald M. McInerney
• Keywords: Semliki Forest virus

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