Hypervariable Domain of Nonstructural Protein nsP3 of Venezuelan Equine Encephalitis Virus Determines Cell-Specific Mode of Virus Replication

Foy, Niall J.; Akhrymuk, Maryna; Shustov, Alexandr V.; Frolova, Elena I.; Frolov, Ilya

doi:10.1128/jvi.00720-13

Cited by 44 publications

(79 citation statements)

References 71 publications

(84 reference statements)

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“…Supporting our findings, recently published work with chimeric Sindbis virus and Venezuelan equine encephalitis virus (VEEV) revealed that the HVD of VEEV, which contains a proline-rich sequence potentially binding SH3 domains but lacks repeat motifs with homology to the SFV L/ITF GDFD repeats, does not form a complex with G3BP (17,18). We conclude, therefore, that for the Old World but not the New World alphaviruses, the G3BP binding site resides in the C-terminal repeat motifs of nsP3.…”

supporting

confidence: 69%

The C-Terminal Repeat Domains of nsP3 from the Old World Alphaviruses Bind Directly to G3BP

Panas

Ahola

McInerney

2014

J Virol

129

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bThe Old World alphaviruses block stress granule assembly by sequestration of RasGAP SH3-domain binding protein (G3BP). Here, we show that the proline-rich sequences in the hypervariable domain of nonstructural protein 3 (nsP3) of both Semliki Forest virus and Chikungunya virus were dispensable for binding to G3BP. nsP3 variants with or without this domain colocalized with G3BP. Furthermore, we show that the C-terminal repeat motifs of nsP3 were sufficient for G3BP binding. Stress granules (SGs) are foci of accumulation of translationally silent mRNP complexes, which are induced during cellular stress (1). Their assembly is dependent on the proteins TIA-1/R (2) and G3BP (3). Many diverse virus infections employ mechanisms to restrict the formation of SGs (4, 5), which are induced by early events in the replication of many viruses, including the alphaviruses (6, 7). We recently showed that in cells infected with the alphavirus Semliki Forest virus (SFV), nonstructural protein 3 (nsP3) binds RasGAP SH3-domain binding proteins 1 and 2 (G3BP-1 and G3BP-2, respectively), recruits them to foci containing other viral proteins and often double-stranded RNA (dsRNA), and, in doing so, inhibits SG assembly (8). Fros and colleagues (9) also showed that the expression of the closely related Chikungunya virus (CHIKV) nsP3 blocks SG assembly by recruitment of G3BP-1 into cytoplasmic foci. These reports therefore describe a function for Old World alphavirus nsP3 in inhibition of the stress response. A surprising difference in the findings of the two groups was that the work of Fros and colleagues suggested that the CHIKV nsP3 sequence binding the SH3 domain of amphiphysins ) was essential for colocalization of nsP3 and G3BP (9). In contrast to that work, our study showed that the SFV nsP3 C-terminal L/ITFGDFD repeat motifs at positions 449 to 455 and 466 to 472, both well conserved in the Old World alphaviruses (11), were necessary and sufficient for formation of the nsP3/G3BP complex in infected cells (8). Both these regions, although situated within the hypervariable domain (HVD) of nsP3 (12-15), are highly conserved between both CHIKV and SFV, and it was therefore surprising that the viruses would differ in the region used to bind and recruit G3BP.Recently, a variant of SFV (SFV-⌬P1ϩ2) was described, lacking the proline-rich SH3-domain binding sequences of nsP3 (10). It was shown that the deletion impairs the recruitment of amphiphysin proteins to foci of nsP3 accumulation and delays replication complex formation (10). To determine if this domain of SFV nsP3 was important for the recruitment of G3BP-1, we infected mouse embryonic fibroblasts (MEFs) maintained as described previously (16) with SFV wild type (wt) or SFV-⌬P1ϩ2 at a multiplicity of infection (MOI) of 1, fixed the cells at 8 h postinfection (hpi), and stained them for nsP3 and G3BP-1. Single-plane images were captured using a supercontinuum confocal TCS SP5 X microscope (Leica, Wetzlar, Germany) with a pulsed white light laser. No deficiency in G3BP-1 recruitment...

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supporting

confidence: 69%

The C-Terminal Repeat Domains of nsP3 from the Old World Alphaviruses Bind Directly to G3BP

Panas

Ahola

McInerney

2014

J Virol

129

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“…However, a detailed analysis of the amino acid sequence of EEEV's HVD revealed that no such repeats are present in its carboxy terminus. We also failed to identify peptides with clear levels of identity with G3BP-and FXR-binding sequences which had been previously identified in CHIKV or SINV and VEEV, respectively (23,25,27). Therefore, in the next experiments, we performed a preliminary mapping of the FXR-and G3BP-binding fragments in the EEEV HVD.…”

Section: Resultsmentioning

confidence: 99%

“…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). Similarly, the VEEV nsP3 HVD interaction with FXRs is also determined by a unique carboxy-terminal amino acid repeat (23,25). This interaction is a critical determinant of VEEV RC assembly, and its alteration by deletion of FXR-binding sites in the VEEV HVD or by KO of all three FXR genes has a strong negative effect on both the infectivity of VEEV and its replication rates.…”

Section: Discussionmentioning

confidence: 99%

“…The results of our previous study demonstrated that the intrinsically disordered, hypervariable domains of alphavirus nsP3 proteins are critical players in the formation of viral vRCs and viral replication (23)(24)(25). HVDs interact with sets of host factors which are specific for each alphavirus representative.…”

Section: Discussionmentioning

confidence: 99%

“…The nsP3 HVD of the latter representative member of the NW alphaviruses does not interact with G3BPs. However, its HVD also contains a repeating peptide which demonstrates no similarity to the repeating amino acid sequence of the OW alphaviruses and binds all of the members of the fragile X syndrome (FXR) protein family (23,25). Each of these proteins can independently support VEEV replication but not replication of the OW alphaviruses.…”

mentioning

confidence: 99%

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

Frolov

Kim

Akhrymuk

et al. 2017

J Virol

Self Cite

109

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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

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Molecular Virology of Chikungunya Virus

Frolov

Frolova

2018

Current Topics in Microbiology and Immunology

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Hypervariable Domain of Nonstructural Protein nsP3 of Venezuelan Equine Encephalitis Virus Determines Cell-Specific Mode of Virus Replication

Cited by 44 publications

References 71 publications

The C-Terminal Repeat Domains of nsP3 from the Old World Alphaviruses Bind Directly to G3BP

The C-Terminal Repeat Domains of nsP3 from the Old World Alphaviruses Bind Directly to G3BP

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

Molecular Virology of Chikungunya Virus

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