Mutational analysis of the functional sites in porcine reproductive and respiratory syndrome virus non-structural protein 10

Zhang, Yumeng; Li, Huan; Peng, Guiqing; Zhang, Yong; Gao, Xiao; Xiao, Shaobo; Cao, Shengbo; Chen, Huanchun; Song, Yinglin

doi:10.1099/jgv.0.000004

Cited by 11 publications

(13 citation statements)

References 24 publications

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“…The integrity of nsp10 is essential for its helicase properties and this was in line with the previous studies of Arterivirus nsp10, which showed that the ZBD was required for its helicase activity and involved in genome replication (Seybert et al 2000(Seybert et al , 2005. PRRSV nsp10 has ATPase activity and can unwind dsRNA and dsDNA in a 5´ to 3´ direction (Bautista et al 2002;Zhang et al 2015).…”

Section: Discussionsupporting

confidence: 88%

Identification of the strain-specifically truncated nonstructural protein 10 of porcine reproductive and respiratory syndrome virus in infected cells

Zhang

Wen

et al. 2018

Journal of Integrative Agriculture

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The nonstructural protein 10 (nsp10) of porcine reproductive and respiratory syndrome virus (PRRSV) encodes for helicase which plays a vital role in viral replication. In the present study, a truncated form of nsp10, termed nsp10a, was found in PRRSV-infected cells and the production of nsp10a was strain-specific. Mass spectrometric analysis and deletion mutagenesis indicated that nsp10a may be short of about 70 amino acids in the N terminus of nsp10. Further studies by rescuing recombinant viruses showed that the Glu-69 in nsp10 was the key amino acid for nsp10a production. Finally, we demonstrated that nsp10a exerted little influence on the growth kinetics of PRRSV in vitro.

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Section: Discussionsupporting

confidence: 88%

Identification of the strain-specifically truncated nonstructural protein 10 of porcine reproductive and respiratory syndrome virus in infected cells

Zhang

Wen

et al. 2018

Journal of Integrative Agriculture

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“…Secondly, the C-terminal treble-clef zinc finger of EAV nsp10 is stabilized by multiple hydrogen-bonding interactions, while the corresponding zinc finger of PRRSV nsp10 is more flexible without such an extensive array of hydrogen bonds, suggesting that the C-terminal zinc-binding motif may have less effect on the enzymatic activities in PRRSV nsp10 than in EAV nsp10. This speculation is confirmed by previous mutagenesis studies that H44A of EAV nsp10 (ligand for Zn3) retained a limited level of ATPase and helicase activities while the mutations in the mutant variant H43A had no obvious influence on the enzymatic activities of PRRSV nsp10 [55,56]. Thirdly, the resolution of the crystals of full-length EAV nsp10 was poor after extensive optimization and diffracting crystals could only be obtained for a truncated form lacking the CTD.…”

Section: Discussionsupporting

confidence: 62%

Helicase of Type 2 Porcine Reproductive and Respiratory Syndrome Virus Strain HV Reveals a Unique Structure

Tang

Deng

et al. 2020

Viruses

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Porcine reproductive and respiratory syndrome virus (PRRSV) is prevalent throughout the world and has caused great economic losses to the swine industry. Nonstructural protein 10 (nsp10) is a superfamily 1 helicase participating in multiple processes of virus replication and one of the three most conserved proteins in nidoviruses. Here we report three high resolution crystal structures of highly pathogenic PRRSV nsp10. PRRSV nsp10 has multiple domains, including an N-terminal zinc-binding domain (ZBD), a β-barrel domain, a helicase core with two RecA-like domains, and a C-terminal domain (CTD). The CTD adopts a novel fold and is required for the overall structure and enzymatic activities. Although each domain except the CTD aligns well with its homologs, PRRSV nsp10 adopts an unexpected extended overall structure in crystals and solution. Moreover, structural and functional analyses of PRRSV nsp10 versus its closest homolog, equine arteritis virus nsp10, suggest that DNA binding might induce a profound conformational change of PRRSV nsp10 to exert functions, thus shedding light on the mechanisms of activity regulation of this helicase.In 2006, a large-scale atypical PRRS outbreak caused by the highly pathogenic PRRSV (HP-PRRSV, belonging to PRRSV-2) emerged in China [13,14]. Yan Li and her colleagues demonstrated that nsp9 and nsp10 together contributed to the replication efficiency and the high virulence of HP-PRRSV [15]. Nsp9 contains an RNA-dependent RNA-polymerase (RdRp) domain [16]. Nsp10 belongs to the superfamily 1B (SF1B) Upf1-like family of helicases, which could unwind both DNA and RNA duplexes [17][18][19]. In addition, this family of helicases contains an N-terminal predicted zinc-binding domain (ZBD) which is conserved in all nidovirus helicases, including 12 or 13 conserved Cys and His residues [20]. However, the C-terminal domain (CTD) is variable among nidoviruses. Besides, nsp10 can vary up to approximately 42% on the amino acid level between the two genotypes, and the strains within PRRSV-2 also vary considerably with amino acid differences as high as 8% [21,22]. We previously reported the first structure of the nidovirus helicase, equine arteritis virus (EAV; family Arteriviridae) nsp10, and demonstrated that the CTD perhaps exerts a regulatory function on the helicase core, facilitating coupling between NTPase and polynucleotide binding activities [23]. Another solved structure of nidovirus helicase is Middle East respiratory syndrome coronavirus (MERS-CoV; family Coronaviridae) nsp13 [24]. While the domain organization of nsp13 is similar to EAV nsp10, structural comparisons of the individual domains showed that nsp13 is closely related to Upf1. The N-terminal Cys-His-rich domain (CH domain) of nsp13 is more related to the CH of Upf1 than to the ZBD of EAV nsp10. The helicase core of EAV nsp10 is more compact than that of MERS-CoV nsp13 which has a similar size as Upf1. Meanwhile, MERS-CoV nsp13 does not contain a C-terminal regulatory domain homologous to the CTD of EAV nsp10.Reflecting its ...

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“…The helicase domain of PRRSV nsp10 participates in multiple processes during virus replication (23). Cys25 and His32 in nsp10 are critical for nucleic acid binding and unwinding, and Ala227 plays an important role in helicase activity (24). nsp10 also contains a putative zinc finger region which is implicated in subgenomic mRNA synthesis and genome replication.…”

mentioning

confidence: 99%

The Nucleocapsid Protein and Nonstructural Protein 10 of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Enhance CD83 Production via NF-κB and Sp1 Signaling Pathways

Chen

Zhang

Bai

et al. 2017

J Virol

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Porcine reproductive and respiratory syndrome, caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a panzootic disease that is one of the most economically costly diseases to the swine industry. A key aspect of PRRSV virulence is that the virus suppresses the innate immune response and induces persistent infection, although the underlying mechanisms are not well understood. The dendritic cell (DC) marker CD83 belongs to the immunoglobulin superfamily and is associated with DC activation and immunosuppression of T cell proliferation when expressed as soluble CD83 (sCD83). In this study, we show that PRRSV infection strongly stimulates CD83 expression in porcine monocyte-derived DCs (MoDCs) and that the nucleocapsid (N) protein and nonstructural protein 10 (nsp10) of PRRSV enhance CD83 promoter activity via the NF-κB and Sp1 signaling pathways. R43A and K44A amino acid substitution mutants of the N protein suppress the N protein-mediated increase of CD83 promoter activity. Similarly, P192-5A and G214-3A mutants of nsp10 (with 5 and 3 alanine substitutions beginning at residues P192 and G214, respectively) abolish the nsp10-mediated induction of the CD83 promoter. Using reverse genetics, four mutant viruses (rR43A, rK44A, rP192-5A, and rG214-3A) and four revertants [rR43A(R), rK44A(R), rP192-5A(R), and rG214-3A(R)] were generated. Decreased induction of CD83 in MoDCs was observed after infection by mutants rR43A, rK44A, rP192-5A, and rG214-3A, in contrast to the results obtained using rR43A(R), rK44A(R), rP192-5A(R), and rG214-3A(R). These findings suggest that PRRSV N and nsp10 play important roles in modulating CD83 signaling and shed light on the mechanism by which PRRSV modulates host immunity. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically costly pathogens affecting the swine industry. It is unclear how PRRSV inhibits the host's immune response and induces persistent infection. The dendritic cell (DC) marker CD83 belongs to the immunoglobulin superfamily and has previously been associated with DC activation and immunosuppression of T cell proliferation and differentiation when expressed as soluble CD83 (sCD83). In this study, we found that PRRSV infection induces sCD83 expression in porcine MoDCs via the NF-κB and Sp1 signaling pathways. The viral nucleocapsid protein, nonstructural protein 1 (nsp1), and nsp10 were shown to enhance CD83 promoter activity. Amino acids R43 and K44 of the N protein, as well as residues 192 to 196 (P192-5) and 214 to 216 (G214-3) of nsp10, play important roles in CD83 promoter activation. These findings provide new insights into the molecular mechanism of immune suppression by PRRSV.

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Mutational analysis of the functional sites in porcine reproductive and respiratory syndrome virus non-structural protein 10

Cited by 11 publications

References 24 publications

Identification of the strain-specifically truncated nonstructural protein 10 of porcine reproductive and respiratory syndrome virus in infected cells

Identification of the strain-specifically truncated nonstructural protein 10 of porcine reproductive and respiratory syndrome virus in infected cells

Helicase of Type 2 Porcine Reproductive and Respiratory Syndrome Virus Strain HV Reveals a Unique Structure

The Nucleocapsid Protein and Nonstructural Protein 10 of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Enhance CD83 Production via NF-κB and Sp1 Signaling Pathways

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