Abstract
Recent findings have highlighted the role of the Old World alphavirus non-structural protein 3 (nsP3) as a host defence modulator that functions by disrupting stress granules, subcellular phase-dense RNA/protein structures formed upon environmental stress. This disruption mechanism was largely explained through nsP3-mediated recruitment of the host G3BP protein via two tandem FGDF motifs. Here, we present the 1.9 Å resolution crystal structure of the NTF2-like domain of G3BP-1 in complex with a 25-residue peptide derived from Semliki Forest virus nsP3 (nsP3-25). The structure reveals a poly-complex of G3BP-1 dimers interconnected through the FGDF motifs in nsP3-25. Although in vitro and in vivo binding studies revealed a hierarchical interaction of the two FGDF motifs with G3BP-1, viral growth curves clearly demonstrated that two intact FGDF motifs are required for efficient viral replication. Chikungunya virus nsP3 also binds G3BP dimers via a hierarchical interaction, which was found to be critical for viral replication. These results highlight a conserved molecular mechanism in host cell modulation.
Highlights
Alphaviruses are a genus of enveloped RNA viruses in the family Togaviridae that are further divided into Old World and New World alphaviruses
We have described the structural organization of the contact areas between Old World alphavirus nonstructural protein 3 (nsP3) and the cellular stress granule protein G3BP
The region between FGDF motif of the nsP3 peptide (FGDFN) and FGDFC adopts a helical conformation with the two FGDF motifs at the Nand C-termini of the helix, excluding the possibility that both FGDF motifs bind to the same G3BP-1 dimer due to the short length of the helical region
Summary
Alphaviruses are a genus of enveloped RNA viruses in the family Togaviridae that are further divided into Old World and New World alphaviruses. Old World alphaviruses comprise severe human pathogens such as chikungunya virus (CHIKV) and low-pathogenic model viruses such as Semliki Forest virus (SFV). The alphaviral positive-strand RNA genome is translated immediately after host cell entry to yield the viral non-structural proteins (nsP) 1–4, comprising the viral replicase complex [1,2]. Together with a number of cellular proteins, interact with the plasma membrane by targeting sequences on nsP1 [3] and begin to replicate the viral template RNA via a negative strand intermediate in membrane invaginations termed spherules. SFV in particular, the spherules are internalized from the plasma membrane to form cytopathic vacuoles in the body of the cell in a process that correlates with the activation of the PI3K-Akt-mTOR pathway [4,5]
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