A rapid RIG-I signaling relay mediates efficient antiviral response.

RIG-I is essential for host defense against viral pathogens, as it triggers the release of type I interferons upon encounter with viral RNA molecules. In this study, we show that RIG-I is rapidly and efficiently activated by small quantities of incoming viral RNA and that it relies exclusively on the constitutively expressed resident pool of RIG-I receptors for a strong antiviral response. Live-cell imaging of RIG-I following stimulation with viral or synthetic dsRNA reveals that RIG-I signaling occurs without mass aggregation at the mitochondrial membrane. By contrast, interferon-induced RIG-I protein becomes embedded in cytosolic aggregates that are functionally unrelated to signaling. These findings suggest that endogenous RIG-I efficiently recognizes viral RNA and rapidly relays an antiviral signal to MAVS via a transient signaling complex and that cellular aggregates of RIG-I have a function that is distinct from signaling.

Sensitivity of Alphaviruses to G3BP Deletion Correlates with Efficiency of Replicase Polyprotein Processing.

We present a comprehensive overview of the dependency of several Old World alphaviruses for the host protein G3BP. Based on their replication ability in G3BP-deleted cells, Old World alphaviruses can be categorized into two groups, being either resistant or sensitive to G3BP deletion. We observed that all sensitive viruses have an Arg residue at the P4 position of the cleavage site between the nonstructural protein P1 (nsP1) and nsP2 regions of the replicase precursor polyprotein (1/2 site), while a different residue is found at this site in viruses resistant to G3BP deletion. Swapping this residue between resistant and sensitive viruses also switches the G3BP deletion sensitivity. In the absence of G3BP, chikungunya virus (CHIKV) replication is at the limit of detection. The P4 Arg-to-His substitution partially rescues this defect. The P4 residue of the 1/2 site is known to play a regulatory role during processing at this site, and we found that if processing is blocked, the influence of the P4 residue on the sensitivity to G3BP deletion is abolished. Immunofluorescence experiments with CHIKV replicase with manipulated processing indicate that the synthesis of double-stranded RNA is defective in the absence of G3BP and suggest a role of G3BP during negative-strand RNA synthesis. This study provides a functional link between the host protein G3BP and the P4 residue of the 1/2 site for viral RNA replication of Old World alphaviruses. While this suggests a link between G3BP proteins and viral replicase polyprotein processing, we propose that G3BP proteins do not have a regulatory role during polyprotein processing.IMPORTANCE Old World alphaviruses comprise several medically relevant viruses, including chikungunya virus and Ross River virus. Recurrent outbreaks and the lack of antivirals and vaccines demand ongoing research to fight the emergence of these infectious diseases. In this context, a thorough investigation of virus-host interactions is critical. Here, we highlight the importance of the host protein G3BP for several Old World alphaviruses. Our data strongly suggest that G3BP plays a crucial role for the activity of the viral replicase and, thus, the amplification of the viral RNA genome. To our knowledge, the present work is the first to provide a functional link between the regulation of viral polyprotein processing and RNA replication and a host factor for alphaviruses. Moreover, the results of this study raise several questions about the fundamental regulatory mechanisms that dictate the activity of the viral replicase, thereby paving the way for future studies.

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.

Arabidopsis thaliana G3BP Ortholog Rescues Mammalian Stress Granule Phenotype across Kingdoms.

Stress granules (SGs) are dynamic RNA-protein complexes localized in the cytoplasm that rapidly form under stress conditions and disperse when normal conditions are restored. The formation of SGs depends on the Ras-GAP SH3 domain-binding protein (G3BP). Formations, interactions and functions of plant and human SGs are strikingly similar, suggesting a conserved mechanism. However, functional analyses of plant G3BPs are missing. Thus, members of the Arabidopsis thaliana G3BP (AtG3BP) protein family were investigated in a complementation assay in a human G3BP knock-out cell line. It was shown that two out of seven AtG3BPs were able to complement the function of their human homolog. GFP-AtG3BP fusion proteins co-localized with human SG marker proteins Caprin-1 and eIF4G1 and restored SG formation in G3BP double KO cells. Interaction between AtG3BP-1 and -7 and known human G3BP interaction partners such as Caprin-1 and USP10 was also demonstrated by co-immunoprecipitation. In addition, an RG/RGG domain exchange from Arabidopsis G3BP into the human G3BP background showed the ability for complementation. In summary, our results support a conserved mechanism of SG function over the kingdoms, which will help to further elucidate the biological function of the Arabidopsis G3BP protein family.

Functional homo- and heterodimeric actin capping proteins from the malaria parasite.

Actin capping proteins belong to the core set of proteins minimally required for actin-based motility and are present in virtually all eukaryotic cells. They bind to the fast-growing barbed end of an actin filament, preventing addition and loss of monomers, thus restricting growth to the slow-growing pointed end. Actin capping proteins are usually heterodimers of two subunits. The Plasmodium orthologs are an exception, as their α subunits are able to form homodimers. We show here that, while the ß subunit alone is unstable, the α subunit of the Plasmodium actin capping protein forms functional homo- and heterodimers. This implies independent functions for the αα homo- and αß heterodimers in certain stages of the parasite life cycle. Structurally, the homodimers resemble canonical αß heterodimers, although certain rearrangements at the interface must be required. Both homo- and heterodimers bind to actin filaments in a roughly equimolar ratio, indicating they may also bind other sites than barbed ends.

RNA processing bodies are disassembled during Old World alphavirus infection.

RNA processing bodies (P-bodies) are non-membranous cytoplasmic aggregates of mRNA and proteins involved in mRNA decay and translation repression. P-bodies actively respond to environmental stresses, associated with another type of RNA granules, known as stress granules (SGs). Alphaviruses were previously shown to block SG induction at late stages of infection, which is important for efficient viral growth. In this study, we found that P-bodies were disassembled or reduced in number very early in infection with Semliki Forest virus (SFV) or chikungunya virus (CHIKV) in a panel of cell lines. Similar to SGs, reinduction of P-bodies by a second stress (sodium arsenite) was also blocked in infected cells. The disassembly of P-bodies still occurred in non-phosphorylatable eIF2α mouse embryonal fibroblasts (MEFs) that are impaired in SG assembly. Studies of translation status by ribopuromycylation showed that P-body disassembly is independent of host translation shutoff, which requires the phosphorylation of eIF2α in the SFV- or CHIKV-infected cells. Labelling of newly synthesized RNA with bromo-UTP showed that host transcription shutoff correlated with P-body disassembly at the same early stage (3-4 h) after infection. However, inhibition of global transcription with actinomycin D (ActD) failed to disassemble P-bodies as effectively as the viruses did. Interestingly, blocking nuclear import with importazole led to an efficient P-bodies loss. Our data reveal that P-bodies are disassembled independently from SG formation at early stages of Old World alphavirus infection and that nuclear import is involved in the dynamic of P-bodies.

Differential Phosphatidylinositol-3-Kinase-Akt-mTOR Activation by Semliki Forest and Chikungunya Viruses Is Dependent on nsP3 and Connected to Replication Complex Internalization.

UNLABELLED: Many viruses affect or exploit the phosphatidylinositol-3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway, a crucial prosurvival signaling cascade. We report that this pathway was strongly activated in cells upon infection with the Old World alphavirus Semliki Forest virus (SFV), even under conditions of complete nutrient starvation. We mapped this activation to the hyperphosphorylated/acidic domain in the C-terminal tail of SFV nonstructural protein nsP3. Viruses with a deletion of this domain (SFV-Δ50) but not of other regions in nsP3 displayed a clearly delayed and reduced capacity of Akt stimulation. Ectopic expression of the nsP3 of SFV wild type (nsP3-wt), but not nsP3-Δ50, equipped with a membrane anchor was sufficient to activate Akt. We linked PI3K-Akt-mTOR stimulation to the intracellular dynamics of viral replication complexes, which are formed at the plasma membrane and subsequently internalized in a process blocked by the PI3K inhibitor wortmannin. Replication complex internalization was observed upon infection of cells with SFV-wt and SFV mutants with deletions in nsP3 but not with SFV-Δ50, where replication complexes were typically accumulated at the cell periphery. In cells infected with the closely related chikungunya virus (CHIKV), the PI3K-Akt-mTOR pathway was only moderately activated. Replication complexes of CHIKV were predominantly located at the cell periphery. Exchanging the hypervariable C-terminal tail of nsP3 between SFV and CHIKV induced the phenotype of strong PI3K-Akt-mTOR activation and replication complex internalization in CHIKV. In conclusion, infection with SFV but not CHIKV boosts PI3K-Akt-mTOR through the hyperphosphorylated/acidic domain of nsP3 to drive replication complex internalization. IMPORTANCE: SFV and CHIKV are very similar in terms of molecular and cell biology, e.g., regarding replication and molecular interactions, but are strikingly different regarding pathology: CHIKV is a relevant human pathogen, causing high fever and joint pain, while SFV is a low-pathogenic model virus, albeit neuropathogenic in mice. We show that both SFV and CHIKV activate the prosurvival PI3K-Akt-mTOR pathway in cells but greatly differ in their capacities to do so: Akt is strongly and persistently activated by SFV infection but only moderately activated by CHIKV. We mapped this activation capacity to a region in nonstructural protein 3 (nsP3) of SFV and could functionally transfer this region to CHIKV. Akt activation is linked to the subcellular dynamics of replication complexes, which are efficiently internalized from the cell periphery for SFV but not CHIKV. This difference in signal pathway stimulation and replication complex localization may have implications for pathology.

The E3 ligase Riplet promotes RIG-I signaling independent of RIG-I oligomerization.

RIG-I is an essential innate immune receptor that responds to infection by RNA viruses. The RIG-I signaling cascade is mediated by a series of post-translational modifications, the most important of which is ubiquitination of the RIG-I Caspase Recruitment Domains (CARDs) by E3 ligase Riplet. This is required for interaction between RIG-I and its downstream adapter protein MAVS, but the mechanism of action remains unclear. Here we show that Riplet is required for RIG-I signaling in the presence of both short and long dsRNAs, establishing that Riplet activation does not depend upon RIG-I filament formation on long dsRNAs. Likewise, quantitative Riplet-RIG-I affinity measurements establish that Riplet interacts with RIG-I regardless of whether the receptor is bound to RNA. To understand this, we solved high-resolution cryo-EM structures of RIG-I/RNA/Riplet complexes, revealing molecular interfaces that control Riplet-mediated activation and enabling the formulation of a unified model for the role of Riplet in signaling.

The Enigmatic Alphavirus Non-Structural Protein 3 (nsP3) Revealing Its Secrets at Last.

Alphaviruses encode 4 non-structural proteins (nsPs), most of which have well-understood functions in capping and membrane association (nsP1), polyprotein processing and RNA helicase activity (nsP2) and as RNA-dependent RNA polymerase (nsP4). The function of nsP3 has been more difficult to pin down and it has long been referred to as the more enigmatic of the nsPs. The protein comprises three domains, an N-terminal macro domain, a central zinc-binding domain and a C-terminal hypervariable domain (HVD). In this article, we review old and new literature about the functions of the three domains. Much progress in recent years has contributed to a picture of nsP3, particularly through its HVD as a hub for interactions with host cell molecules, with multiple effects on the biology of the host cell at early points in infection. These and many future discoveries will provide targets for anti-viral therapies as well as strategies for modification of vectors for vaccine and oncolytic interventions.

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.