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The dimer interface of the SARS coronavirus nucleocapsid protein adapts a porcine respiratory and reproductive syndrome virus-like structure.

ABSTRACT: We have employed NMR to investigate the structure of SARS coronavirus nucleocapsid protein dimer. We found that the secondary structure of the dimerization domain consists of five alpha helices and a beta-hairpin. The dimer interface consists of a continuous four-stranded beta-sheet superposed by two long alpha helices, reminiscent of that found in the nucleocapsid protein of porcine respiratory and reproductive syndrome virus. Extensive hydrogen bond formation between the two hairpins and hydrophobic interactions between the beta-sheet and the alpha helices render the interface highly stable. Sequence alignment suggests that other coronavirus may share the same structural topology.

SUBMITTER: Chang CK

PROVIDER: S-EPMC7094587 | biostudies-literature | 2005 Oct

REPOSITORIES: biostudies-literature

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The dimer interface of the SARS coronavirus nucleocapsid protein adapts a porcine respiratory and reproductive syndrome virus-like structure.

Chang Chung-ke CK Sue Shih-Che SC Yu Tsan-hung TH Hsieh Chiu-Min CM Tsai Cheng-Kun CK Chiang Yen-Chieh YC Lee Shin-jye SJ Hsiao Hsin-Hao HH Wu Wen-Jin WJ Chang Chi-Fon CF Huang Tai-huang TH

FEBS letters 20050930 25

We have employed NMR to investigate the structure of SARS coronavirus nucleocapsid protein dimer. We found that the secondary structure of the dimerization domain consists of five alpha helices and a beta-hairpin. The dimer interface consists of a continuous four-stranded beta-sheet superposed by two long alpha helices, reminiscent of that found in the nucleocapsid protein of porcine respiratory and reproductive syndrome virus. Extensive hydrogen bond formation between the two hairpins and hydro ...[more]

PMID: 16214138

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Project description:To fulfill virus replication and persistent infection in hosts, viruses have to find ways to compromise innate immunity, including timely impedance on antiviral RNases and inflammatory responses. Porcine reproductive and respiratory syndrome virus (PRRSV) is a major swine pathogen causing immune suppression. MALT1 is a central immune regulator in both innate and adaptive immunity. In this study, MALT1 was confirmed to be induced rapidly upon PRRSV infection and mediate the degradation of two anti-PRRSV RNases, MCPIP1 and N4BP1, relying on its proteolytic activity, consequently facilitating PRRSV replication. Multiple PRRSV nsps, including nsp11, nsp7β, and nsp4, contributed to MALT1 elicitation. Interestingly, the elevated expression of MALT1 began to decrease once intracellular viral expression reached a high enough level. Higher infection dose brought earlier MALT1 inflection. Further, PRRSV nsp6 mediated significant MALT1 degradation via ubiquitination-proteasome pathway. Downregulation of MALT1 suppressed NF-κB signals, leading to the decrease in proinflammatory cytokine expression. In conclusion, MALT1 expression was manipulated by PRRSV in an elaborate manner to antagonize precisely the antiviral effects of host RNases without excessive and continuous activation of inflammatory responses. These findings throw light on the machinery of PRRSV to build homeostasis in infected immune system for viral settlement. IMPORTANCE PRRSV is a major swine pathogen, suppresses innate immunity, and causes persistent infection and coinfection with other pathogens. As a central immune mediator, MALT1 plays essential roles in regulating immunity and inflammation. Here, PRRSV was confirmed to manipulate MALT1 expression in an accurate way to moderate the antiviral immunity. Briefly, multiple PRRSV nsps induced MALT1 protease to antagonize anti-PRRSV RNases N4BP1 and MCPIP1 upon infection, thereby facilitating viral replication. In contrast, PRRSV nsp6 downregulated MALT1 expression via ubiquitination-proteasome pathway to suppress the inflammatory responses upon infection aggravation, contributing to immune defense alleviation and virus survival. These findings revealed the precise expression control on MALT1 by PRRSV for antagonizing antiviral RNases, along with recovering immune homeostasis. For the first time, this study enlightens a new mechanism of PRRSV adapting antiviral innate immunity by modulating MALT1 expression.

Project description:Porcine reproductive and respiratory syndrome (PRRS) is a serious global pig industry disease. Understanding the mechanism of viral replication and developing efficient antiviral strategies are necessary for combating with PRRS virus (PRRSV) infection. Recently, nanobody is considered to be a promising antiviral drug, especially for respiratory viruses. The present study evaluated two nanobodies against PRRSV nucleocapsid (N) protein (PRRSV-N-Nb1 and -Nb2) for their anti-PRRSV activity in vitro and in vivo. The results showed that intracellularly expressed PRRSV-N-Nb1 significantly inhibited PRRSV-2 replication in MARC-145 cells (approximately 100%). Then, the PRRSV-N-Nb1 fused with porcine IgG Fc (Nb1-pFc) as a delivering tag was produced and used to determine its effect on PRRSV-2 replication in porcine alveolar macrophages (PAMs) and pigs. The inhibition rate of Nb1-pFc against PRRSV-2 in PAMs could reach >90%, and it can also inhibit viral replication in vivo. Epitope mapping showed that the motif Serine 105 (S105) in PRRSV-2 N protein was the key amino acid binding to PRRSV-N-Nb1, which is also pivotal for the self-interaction of N protein via binding to Arginine 97. Moreover, viral particles were not successfully rescued when the S105 motif was mutated to Alanine (S105A). Attachment, entry, genome replication, release, docking model analysis, and blocking enzyme-linked immunosorbent assay (ELISA) indicated that the binding of PRRSV-N-Nb1 to N protein could block its self-binding, which prevents the viral replication of PRRSV. PRRSV-N-Nb1 may be a promising drug to counter PRRSV-2 infection. We also provided some new insights into the molecular basis of PRRSV N protein self-binding and assembly of viral particles.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) causes serious economic losses to the swine industry worldwide, and there are no highly effective strategies for prevention. Nanobodies are considered a promising novel approach for treating diseases because of their ease of production and low costing. Here, we showed that PRRSV-N-Nb1 against PRRSV-N protein significantly inhibited PRRSV-2 replication in vitro and in vivo. Furthermore, we demonstrated that the motif Serine 105 (S105) in PRRSV-N protein was the key amino acid to interact with PRRSV-N-Nb1 and bond to its motif R97, which is important for the self-binding of N protein. The PRRSV-N-Nb1 could block the self-interaction of N protein following viral assembly. These findings not only provide insights into the molecular basis of PRRSV N protein self-binding as a key factor for viral replication for the first time but also highlight a novel target for the development of anti-PRRSV replication drugs.

Dataset Information

The dimer interface of the SARS coronavirus nucleocapsid protein adapts a porcine respiratory and reproductive syndrome virus-like structure.

Publications

The dimer interface of the SARS coronavirus nucleocapsid protein adapts a porcine respiratory and reproductive syndrome virus-like structure.

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