Stress Granule Components G3BP1 and G3BP2 Play a Proviral Role Early in Chikungunya Virus Replication

Self Cite

111

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-⌬50) 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-⌬50, 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-⌬50, 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. IMPORTANCESFV 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.

Section: Methodsmentioning

confidence: 99%

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

Thaa

Biasiotto

Eng

et al. 2015

Self Cite

111

“…Other viruses, including chikungunya virus, vaccinia virus, Semliki Forest virus, and vesicular stomatitis virus, have been shown to redistribute SG-associated proteins into distinct cytoplasmic aggregates with altered protein composition or function compared to those of canonical SGs (31, 33, 60Ϫ62, 79). The IB granules we observed during EBOV infection were devoid of the canonical SG marker protein TIA-1 and, similar to the antiviral granules observed in vaccinia virus infection and the proviral G3BP aggregates induced by chikungunya virus, did not dissolve upon CHX treatment (33,61,62,79). CHX-mediated dissolution of SGs is driven by the stabilization of polysomes, indicating that SG formation requires a pool of free messenger ribonucleoprotein (mRNP) complexes (25).…”

Section: Discussionmentioning

confidence: 70%

“…3C), suggesting that these structures are distinct from canonical SGs. Other viruses, including chikungunya virus, Semliki Forest virus, and vaccinia virus, induce the formation of granules containing various SG components that are morphologically, structurally, or functionally different from canonical SGs (31,33,60,61). To further characterize the IB granules in EBOV-infected cells and determine potential differences from canonical SGs, we used cycloheximide (CHX), an inhibitor of protein synthesis known to block polysome disassembly and dissolve SGs.…”

Section: Stress Granules Do Not Form During Ebov Infectionmentioning

confidence: 99%

See 1 more Smart Citation

Ebola Virus Does Not Induce Stress Granule Formation during Infection and Sequesters Stress Granule Proteins within Viral Inclusions

Nelson

Schmidt

Deflubé

et al. 2016

A hallmark of Ebola virus (EBOV) infection is the formation of viral inclusions in the cytoplasm of infected cells. These viral in-clusions contain the EBOV nucleocapsids and are sites of viral replication and nucleocapsid maturation. Although there is growing evidence that viral inclusions create a protected environment that fosters EBOV replication, little is known about their role in the host response to infection. The cellular stress response is an effective antiviral strategy that leads to stress granule (SG) formation and translational arrest mediated by the phosphorylation of a translation initiation factor, the ␣ subunit of eukaryotic initiation factor 2 (eIF2␣). Here, we show that selected SG proteins are sequestered within EBOV inclusions, where they form distinct granules that colocalize with viral RNA. These inclusion-bound (IB) granules are functionally and structurally different from canonical SGs. Formation of IB granules does not indicate translational arrest in the infected cells. We further show that EBOV does not induce formation of canonical SGs or eIF2␣ phosphorylation at any time postinfection but is unable to fully inhibit SG formation induced by different exogenous stressors, including sodium arsenite, heat, and hippuristanol. Despite the sequestration of SG marker proteins into IB granules, canonical SGs are unable to form within inclusions, which we propose might be mediated by a novel function of VP35, which disrupts SG formation. This function is independent of VP35's RNA binding activity. Further studies aim to reveal the mechanism for SG protein sequestration and precise function within inclusions. IMPORTANCEAlthough progress has been made developing antiviral therapeutics and vaccines against the highly pathogenic Ebola virus (EBOV), the cellular mechanisms involved in EBOV infection are still largely unknown. To better understand these intracellular events, we investigated the cellular stress response, an antiviral pathway manipulated by many viruses. We show that EBOV does not induce formation of stress granules (SGs) in infected cells and is therefore unrestricted by their concomitant translational arrest. We identified SG proteins sequestered within viral inclusions, which did not impair protein translation. We further show that EBOV is unable to block SG formation triggered by exogenous stress early in infection. These findings provide insight into potential targets of therapeutic intervention. Additionally, we identified a novel function of the interferon antagonist VP35, which is able to disrupt SG formation.

“…The CHIKV and SINV nsP3 HVDs were found to interact with both G3BP1 and G3BP2 but not with FXRs, while the VEEV nsP3 HVD binds all three FXRs but not G3BPs. Accordingly, G3BPs were found to function in the replication of the OW arthritogenic alphaviruses through binding to the carboxy-terminal repeating amino acid sequences of the nsP3 HVD, resulting in assembly of viral pre-vRCs at the plasma membrane (23,27,35). Interference with HVD's ability to interact with G3BPs, caused either by deletion of the identified carboxy-terminal repeating peptides or by KO of both G3bp genes in NIH 3T3 cells, strongly affected the rates of SINV replication and made CHIKV essentially nonviable (23).…”

Section: Discussionmentioning

confidence: 99%

Hypervariable Domain of Eastern Equine Encephalitis Virus nsP3 Redundantly Utilizes Multiple Cellular Proteins for Replication Complex Assembly

Frolov

Kim

Akhrymuk

et al. 2017

109

Eastern equine encephalitis virus (EEEV) is a representative member of the New World alphaviruses. It is pathogenic for a variety of vertebrate hosts, in which EEEV induces a highly debilitating disease, and the outcomes are frequently lethal. Despite a significant public health threat, the molecular mechanism of EEEV replication and interaction with hosts is poorly understood. Our previously published data and those of other teams have demonstrated that hypervariable domains (HVDs) of the alphavirus nsP3 protein interact with virus-specific host factors and play critical roles in assembly of viral replication complexes (vRCs). The most abundantly represented HVD-binding proteins are the FXR and G3BP family members. FXR proteins drive the assembly of vRCs of Venezuelan equine encephalitis virus (VEEV), and G3BPs were shown to function in vRC assembly in the replication of chikungunya and Sindbis viruses. Our new study demonstrates that EEEV exhibits a unique level of redundancy in the use of host factors in RNA replication. EEEV efficiently utilizes both the VEEV-specific FXR protein family and the Old World alphavirusspecific G3BP protein family. A lack of interaction with either FXRs or G3BPs does not affect vRC formation; however, removal of EEEV's ability to interact with both protein families has a deleterious effect on virus growth. Other identified EEEV nsP3 HVDinteracting host proteins are also capable of supporting EEEV replication, albeit with a dramatically lower efficiency. The ability to use a wide range of host factors with redundant functions in vRC assembly and function provides a plausible explanation for the efficient replication of EEEV and may contribute to its highly pathogenic phenotype.IMPORTANCE Eastern equine encephalitis virus (EEEV) is one of the most pathogenic New World alphaviruses. Despite the continuous public health threat, to date, the molecular mechanisms of its very efficient replication and high virulence are not sufficiently understood. The results of this new study demonstrate that North American EEEV exhibits a high level of redundancy in using host factors in replication complex assembly and virus replication. The hypervariable domain of the EEEV nsP3 protein interacts with all of the members of the FXR and G3BP protein families, and only a lack of interaction with both protein families strongly affects virus replication rates. Other identified HVD-binding factors are also involved in EEEV replication, but their roles are not as critical as those of FXRs and G3BPs. The new data present a plausible explanation for the exceptionally high replication rates of EEEV and suggest a new means of its attenuation and new targets for screening of antiviral drugs.KEYWORDS eastern equine encephalitis virus, FMR1, FXR1, FXR2, G3BP1, G3BP2, chikungunya virus, nsP3, replication complex, virus-host interactions