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

Schulte, Tim; Liu, Lifeng; Panas, Marc D.; Thaa, Bastian; Dickson, Nicole M.; Götte, Benjamin; Achour, Adnane; McInerney, Gerald M.

doi:10.1098/rsob.160078

Cited by 68 publications

(157 citation statements)

References 61 publications

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“…G3BP1 interacts with the 5 0 end of the minus strand of HCV RNA and several non-structural proteins associated to replication complexes [28]. This process takes advantage of the intrinsically disordered domains of the viral nonstructural protein nsP3 [33]. Consistent with these findings, HCV infection triggers PKR phosphorylation, thereby down-regulating synthesis of interferon-stimulated genes [29,30].…”

Section: Discussionmentioning

confidence: 68%

G3BP1 interacts directly with the FMDV IRES and negatively regulates translation

et al. 2017

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RNA-protein interactions play a pivotal role in the function of picornavirus internal ribosome entry site (IRES) elements. Here we analysed the impact of Ras GTPase SH3 domain binding protein 1 (G3BP1) in the IRES activity of foot-and-mouth disease virus (FMDV). We found that G3BP1 interacts directly with three distinct sequences of the IRES element using RNA electrophoretic mobility-shift assays. Analysis of the interaction with domain 5 indicated that the G3BP1 binding-site is placed at the single-stranded region although it allows large sequence heterogeneity and the hairpin located upstream of this region enhances retarded complex formation. In addition, G3BP1 interacts directly with the polypyrimidine tract-binding protein and the translation initiation factor 4B (eIF4B) through the C-terminal region. Moreover, G3BP1 is cleaved during FMDV infection yielding two fragments, Ct-G3BP1 and Nt-G3BP1. Both fragments inhibit cap- and IRES-dependent translation, but the Ct-G3BP1 fragment shows a stronger effect on IRES-dependent translation. Assembly of complexes with G3BP1 results in a significantly reduced local flexibility of the IRES element, consistent with the negative effect of this protein. Our results highlight the IRES-binding capacity of G3BP1 and illustrate its function as a translation inhibitor.

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Section: Discussionmentioning

confidence: 68%

G3BP1 interacts directly with the FMDV IRES and negatively regulates translation

et al. 2017

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“…In addition, despite demonstrating very low similarity in the G3BP-binding peptide, EEEV-and CHIKV-specific HVDs interact with the same NTF2-like domain of G3BP (Fig. 8C) (26).…”

Section: Resultsmentioning

confidence: 99%

“…In contrast to HVD interactions with G3BPs and FXRs, described for CHIKV and VEEV, the EEEV HVD contains single binding sites for both FXRs and G3BPs, and interaction with the proteins belonging to one family does not depend on binding to those belonging to another family. This independent binding is another factor differentiating EEEV and CHIKV, in which two G3BP molecules bind to the peptide repeats with different affinities (26).…”

Section: Discussionmentioning

confidence: 99%

“…Replication of the OW alphaviruses, such as Semliki Forest virus (SFV), Sindbis virus (SINV), and chikungunya virus (CHIKV), is critically dependent on interaction of their HVDs with both members of the cellular G3BP protein family, G3BP1 and G3BP2 (23,26). The HVD-G3BP interaction is determined by short repeating amino acid sequences, located at the carboxy terminus of the HVD (27).…”

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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

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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“…Therefore, in addition to the replication complexes, nsP3 is prominently localized in granular cytoplasmic structures (Neuvonen et al 2011). G3BPs have a proviral function (presumably facilitating the switch from nspolyprotein translation to RNA replication), as depletion of these proteins results in reduced CHIKV replication (Scholte et al 2015;Kim et al 2016;Schulte et al 2016). Additionally, alphavirus nsP3 can interact with several other host proteins, but the significance of these interactions is as yet poorly understood.…”

Section: Macro Domainmentioning

confidence: 96%

Functions of Chikungunya Virus Nonstructural Proteins

Ahola

Merits

2016

Chikungunya Virus

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IntroductionThe nonstructural proteins (nsPs) of chikungunya virus (CHIKV) and other alphaviruses are encoded at the 5′ region of the positive-strand viral RNA genome, and thus translated directly after the entry of the RNA into the cytoplasm, to enable the immediate start of the RNA replication process. Many of the replicative functions remain poorly studied for CHIKV nsPs, but have been earlier characterized for proteins from other alphaviruses, primarily Semliki Forest virus (SFV) and Sindbis virus (SINV; Kaariainen and Ahola 2002). It is expected that the basic biochemical functions are conserved in CHIKV proteins, the amino acid (aa) sequence of which are on average 71 % identical with the more closely related SFV nsPs. Therefore we review data from CHIKV whenever available, but also draw heavily on other alphaviruses, and point out the uncertainties regarding the functions of CHIKV proteins.Secondly, the nsPs have many functions in host-virus interactions, including the evasion of antiviral responses. Clearly, these functions are more virus-(and/or celltype) specific in nature, so care needs to be taken in making inferences regarding CHIKV. For instance, it has been shown that hnRNP K, a cellular protein identified as a pro-viral (essential) factor for CHIKV replication (Bourai et al. 2012) acts as a negative effector of replication for SFV (Varjak et al. 2013). A primary division within the mammalian/avian alphaviruses lies between the Old World viruses (e.g., CHIKV, SFV, and SINV) and the New World viruses (e.g., Venezuelan equine encephalitis virus, VEEV), and these two groups display quite different modes of T. Ahola

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

Cited by 68 publications

References 61 publications

G3BP1 interacts directly with the FMDV IRES and negatively regulates translation

G3BP1 interacts directly with the FMDV IRES and negatively regulates translation

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

Functions of Chikungunya Virus Nonstructural Proteins

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