Dissection of virus-host cell interactions in the early response to infection

Stress granules (SG) are dynamic RNA/protein assemblies in the cytoplasm of the cell, formed under conditions of oxidative stress, heat shock or viral infections. These stress conditions trigger a sudden translational arrest, leading to a rapid switch of translation from housekeeping genes to stress-related factors. SGs fulfil multiple roles in the cell one of which is acting as triage centres for mRNA, where the mRNA is stored pending either degradation or reinitiation of translation. Many proteins are sequestered to SGs, among them signalling molecules, which make SGs signal centres to communicate a “state of emergency”. The importance of SGs is also underlined by the fact that they restrict viral propagation. The assembly of SGs is dependent on many RNA-binding proteins, one of which is G3BP (Ras-GAP SH3 domain binding protein). Semliki Forest virus (SFV) belongs to the alphaviruses, a large group of arthropod-borne animal viruses including several relevant human pathogens such as the re-emerging Chikungunya virus (CHIKV). Alphavirus infections lead to fever, rashes, arthralgia and can be lethal. Recent CHIKV outbreaks in the Caribbean area and the US, brings alphavirus research back on the agenda. Therefore there is a need to understand the molecular mechanisms how alphaviruses interact with their host. The aim of this thesis was to dissect virus-host cell interactions in the early response to alphavirus infection. Alphavirus infection leads to the formation of SGs at very early time points. Interestingly, they dissolve in the vicinity of viral replication complexes at later time points. In paper I, we showed that the non-structural protein nsP3 of SFV is responsible for sequestration of G3BP to replication complexes, by doing so, actively disassembling SGs and blocking their reformation. We mapped the binding site for G3BP to two C-terminal repeat domains of nsP3. A recombinant virus mutant lacking these repeats showed a longer and more persistent stress response and was attenuated in growth. In paper II, we extended this finding to the closely related CHIKV. Our results show that nsP3 of both SFV and CHIKV interact with G3BP via two C-terminal repeat domains and that the proline-rich region of nsP3 is dispensable for this interaction. In paper III we investigated the interaction between nsP3 and G3BP in molecular detail and determined that the residues FGDF in the C-terminal repeats of nsP3 are the G3BP binding motif. We further asked whether other proteins use the same mechanism as nsP3 to bind G3BP and whether this interaction inhibits the formation of SGs. We revealed that the phenylalanines and the glycine in the FGDF are essential for binding G3BP. We further demonstrated that the cellular ubiquitin-specific protease 10 (USP10) and the herpes simplex virus (HSV) protein ICP8 (infected cell protein 8) also bind G3BP via an FGDF motif. In addition we show that the FGDFmediated binding to G3BP leads to a negative regulation of G3BP’s SG-nucleating function. Lastly we present a 3D-model of G3BP bound to an FGDF-containing peptide, which we validated by site-directed mutagenesis. Our findings present a common FGDF motif to bind G3BP, which has a negative regulatory effect on the SG-nucleating function of G3BP. This molecular mechanism and the presented 3Dmodel demonstrate the therapeutic potential of targeting this interaction

Sequestration of G3BP coupled with efficient translation inhibits stress granules in Semliki Forest virus infection

Dynamic, mRNA-containing stress granules (SGs) form in the cytoplasm of cells under environmental stresses, including viral infection. Many viruses appear to employ mechanisms to disrupt the formation of SGs on their mRNAs, suggesting that they represent a cellular defense against infection. Here, we report that early in Semliki Forest virus infection, the C-terminal domain of the viral nonstructural protein 3 (nsP3) forms a complex with Ras-GAP SH3-domain–binding protein (G3BP) and sequesters it into viral RNA replication complexes in a manner that inhibits the formation of SGs on viral mRNAs. A viral mutant carrying a C-terminal truncation of nsP3 induces more persistent SGs and is attenuated for propagation in cell culture. Of importance, we also show that the efficient translation of viral mRNAs containing a translation enhancer sequence also contributes to the disassembly of SGs in infected cells. Furthermore, we show that the nsP3/G3BP interaction also blocks SGs induced by other stresses than virus infection. This is one of few described viral mechanisms for SG disruption and underlines the role of SGs in antiviral defense

Mechanistic insights into mammalian stress granule dynamics

The accumulation of stalled translation preinitiation complexes (PICs) mediates the condensation of stress granules (SGs). Interactions between prion-related domains and intrinsically disordered protein regions found in SG-nucleating proteins promote the condensation of ribonucleoproteins into SGs. We propose that PIC components, especially 40S ribosomes and mRNA, recruit nucleators that trigger SG condensation. With resolution of stress, translation reinitiation reverses this process and SGs disassemble. By cooperatively modulating the assembly and disassembly of SGs, ribonucleoprotein condensation can influence the survival and recovery of cells exposed to unfavorable environmental conditions

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

Author summary In order to repel viral infections, cells activate stress responses. One such response involves inhibition of translation and restricted availability of the translation machinery via the formation of stress granules. However, the host translation machinery is absolutely essential for synthesis of viral proteins and consequently viruses have developed a broad spectrum of strategies to circumvent this restriction. Old World alphaviruses, such as Semliki Forest virus (SFV) and chikungunya virus (CHIKV), interfere with stress granule formation by sequestration of G3BP, a stress granule nucleating protein, mediated by the viral non-structural protein 3 (nsP3). Here we show that nsP3:G3BP complexes engage factors of the host translation machinery, which during the course of infection accumulate in the vicinity of viral replication complexes. Accordingly, we demonstrate that the nsP3:G3BP interaction is required for high localized translational activity around viral replication complexes. We find the RGG domain of G3BP to be essential for the recruitment of the host translation machinery. In cells expressing mutant G3BP lacking the RGG domain, SFV replication was attenuated, but detectable, while CHIKV was essentially non-viable. Our data demonstrate a novel mechanism by which viruses can recruit factors of the translation machinery in a G3BP-dependent manner.Peer reviewe

Combined structural, biochemical and cellular evidence demonstrates that both FGDF motifs in alphavirus nsP3 are required for efficient replication

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

Viral and Cellular Proteins Containing FGDF Motifs Bind G3BP to Block Stress Granule Formation

<div><p>The Ras-GAP SH3 domain–binding proteins (G3BP) are essential regulators of the formation of stress granules (SG), cytosolic aggregates of proteins and RNA that are induced upon cellular stress, such as virus infection. Many viruses, including Semliki Forest virus (SFV), block SG induction by targeting G3BP. In this work, we demonstrate that the G3BP-binding motif of SFV nsP3 consists of two FGDF motifs, in which both phenylalanine and the glycine residue are essential for binding. In addition, we show that binding of the cellular G3BP-binding partner USP10 is also mediated by an FGDF motif. Overexpression of wt USP10, but not a mutant lacking the FGDF-motif, blocks SG assembly. Further, we identified FGDF-mediated G3BP binding site in herpes simplex virus (HSV) protein ICP8, and show that ICP8 binding to G3BP also inhibits SG formation, which is a novel function of HSV ICP8. We present a model of the three-dimensional structure of G3BP bound to an FGDF-containing peptide, likely representing a binding mode shared by many proteins to target G3BP.</p></div

G3BP–Caprin1–USP10 complexes mediate stress granule condensation and associate with 40S subunits

Mammalian stress granules (SGs) contain stalled translation preinitiation complexes that are assembled into discrete granules by specific RNA-binding proteins such as G3BP. We now show that cells lacking both G3BP1 and G3BP2 cannot form SGs in response to eukaryotic initiation factor 2α phosphorylation or eIF4A inhibition, but are still SG-competent when challenged with severe heat or osmotic stress. Rescue experiments using G3BP1 mutants show that phosphomimetic G3BP1-S149E fails to rescue SG formation, whereas G3BP1-F33W, a mutant unable to bind G3BP partner proteins Caprin1 or USP10, rescues SG formation. Caprin1/USP10 binding to G3BP is mutually exclusive: Caprin binding promotes, but USP10 binding inhibits, SG formation. G3BP interacts with 40S ribosomal subunits through its RGG motif, which is also required for G3BP-mediated SG formation. We propose that G3BP mediates the condensation of SGs by shifting between two different states that are controlled by the phosphorylation of S149 and by binding to Caprin1 or USP10

An FGDF motif in HSV-1 ICP8 binds G3BP and inhibits SGs.

<p>(A) HEK293T cells were mock transfected or cotransfected with pE29 (ICP8) and either pEGFP-C1, pEGFP-G3BP-wt, -F33W or -F124W. Cell lysates were prepared 24 h after transfection and immunoprecipitated with anti-GFP and separated by SDS–PAGE. Lysates and IPs were probed for ICP8, GFP or actin. (B) BHK cells were mock transfected or transfected with pE29 (ICP8). After 23 h the transfected cells were mock treated or treated with sodium arsenite for 1 h fixed and stained for ICP8, G3BP-1 and TIA1. ICP8+ cells were categorized as cytoplasmic or nuclear, and scored as SG+ based on G3BP1 and TIA1 colocalization. Open bars, mock treated; closed bars, sodium arsenite. Data are presented as mean +/- SD from three independent experiments in which 50 cells per treatment were counted. Unpaired Student’s t-test, * p < 0.05, *** p < 0.001.</p

Mutagenesis of the G3BP-binding domain in SFV-nsP3 reveals a core binding motif of FGDF.

<p>(A) C-terminal sequences of pEGFP-C1, pEGFP-nsP3-31-wt, -T2A, -F3A, -G4A, -D5A, -F6A or -D7A. Alanine mutations are shown in bold. (B) BHK cells were mock transfected (M) or transfected with pEGFP-nsP3-31-wt, -T2A, -F3A, -G4A, -D5A, -F6A or -D7A or pEGFP-C1. Cell lysates were prepared 16 h after transfection and immunoprecipitated with anti-GFP or G3BP-1 antisera and separated by SDS–PAGE and transferred to nitrocellulose. Blots of total lysates and IPs were probed for G3BP-1, GFP or actin.</p

Molecular model of G3BP/FGDF interaction.

<p>(A) G3BP-NTF2 (PDB ID: 4FCM) with modelled peptide LTFGDFDE (yellow sticks). The protein is shown as the surface colored according to the electrostatic potential (red: acidic, blue: basic). (B) Details of the interaction of the peptide with G3BP-NTF2. G3BP-NTF2 is shown in the cartoon representation. The N-terminal 11 residues as well as several residues lining the peptide-binding groove are displayed as grey sticks. The LTFGDFDE peptide is displayed as yellow sticks, peptide amino acid residues are labeled in red and in bold font. G3BP-1 residues mentioned in the text are labeled in black.</p