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Antagonism of dsRNA-Induced Innate Immune Pathways by NS4a and NS4b Accessory Proteins during MERS Coronavirus Infection.

ABSTRACT: Middle East respiratory syndrome coronavirus (MERS-CoV) was first identified in 2012 as a novel etiological agent of severe respiratory disease in humans. As during infection by other viruses, host sensing of viral double-stranded RNA (dsRNA) induces several antiviral pathways. These include interferon (IFN), oligoadenylate synthetase (OAS)-RNase L, and protein kinase R (PKR). Coronaviruses, including MERS-CoV, potently suppress the activation of these pathways, inducing only modest host responses. Our study describes the functions of two accessory proteins unique to MERS-CoV and related viruses, NS4a and NS4b, during infection in human airway epithelium-derived A549 cells. NS4a has been previously characterized as a dsRNA binding protein, while NS4b is a 2',5'-phosphodiesterase with structural and enzymatic similarity to NS2 encoded by mouse hepatitis virus (MHV). We found that deletion of NS4a results in increased interferon lambda (IFNL1) expression, as does mutation of either the catalytic site or nuclear localization sequence of NS4b. All of the mutant viruses we tested exhibited slight decreases in replication. We previously reported that, like MHV NS2, NS4b antagonizes OAS-RNase L, but suppression of IFN is a previously unidentified function for viral phosphodiesterases. Unexpectedly, deletion of NS4a does not result in robust activation of the PKR or OAS-RNase L pathways. Therefore, MERS-CoV likely encodes other proteins that contribute to suppression or evasion of these antiviral innate immune pathways that should be an important focus of future work. This study provides additional insight into the complex interactions between MERS-CoV and the host immune response.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is the second novel zoonotic coronavirus to emerge in the 21st century and cause outbreaks of severe respiratory disease. More than 2,200 cases and 800 deaths have been reported to date, yet there are no licensed vaccines or treatments. Coronaviruses encode unique accessory proteins that are not required for replication but most likely play roles in immune antagonism and/or pathogenesis. Our study describes the functions of MERS-CoV accessory proteins NS4a and NS4b during infection of a human airway-derived cell line. Loss of these accessory proteins during MERS-CoV infection leads to host antiviral activation and modestly attenuates replication. In the case of both NS4a and NS4b, we have identified roles during infection not previously described, yet the lack of robust activation suggests much remains to be learned about the interactions between MERS-CoV and the infected host.

SUBMITTER: Comar CE

PROVIDER: S-EPMC6437052 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Antagonism of dsRNA-Induced Innate Immune Pathways by NS4a and NS4b Accessory Proteins during MERS Coronavirus Infection.

Comar Courtney E CE Goldstein Stephen A SA Li Yize Y Yount Boyd B Baric Ralph S RS Weiss Susan R SR

mBio 20190326 2

Middle East respiratory syndrome coronavirus (MERS-CoV) was first identified in 2012 as a novel etiological agent of severe respiratory disease in humans. As during infection by other viruses, host sensing of viral double-stranded RNA (dsRNA) induces several antiviral pathways. These include interferon (IFN), oligoadenylate synthetase (OAS)-RNase L, and protein kinase R (PKR). Coronaviruses, including MERS-CoV, potently suppress the activation of these pathways, inducing only modest host respons ...[more]

PMID: 30914508

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Project description:UnlabelledFlavivirus replication is mediated by a membrane-associated replication complex where viral membrane proteins NS2A, NS2B, NS4A, and NS4B serve as the scaffold for the replication complex formation. Here, we used dengue virus serotype 2 (DENV-2) as a model to characterize viral NS4A-NS4B interaction. NS4A interacts with NS4B in virus-infected cells and in cells transiently expressing NS4A and NS4B in the absence of other viral proteins. Recombinant NS4A and NS4B proteins directly bind to each other with an estimated Kd (dissociation constant) of 50 nM. Amino acids 40 to 76 (spanning the first transmembrane domain, consisting of amino acids 50 to 73) of NS4A and amino acids 84 to 146 (also spanning the first transmembrane domain, consisting of amino acids 101 to 129) of NS4B are the determinants for NS4A-NS4B interaction. Nuclear magnetic resonance (NMR) analysis suggests that NS4A residues 17 to 80 form two amphipathic helices (helix α1, comprised of residues 17 to 32, and helix α2, comprised of residues 40 to 47) that associate with the cytosolic side of endoplasmic reticulum (ER) membrane and helix α3 (residues 52 to 75) that transverses the ER membrane. In addition, NMR analysis identified NS4A residues that may participate in the NS4A-NS4B interaction. Amino acid substitution of these NS4A residues exhibited distinct effects on viral replication. Three of the four NS4A mutations (L48A, T54A, and L60A) that affected the NS4A-NS4B interaction abolished or severely reduced viral replication; in contrast, two NS4A mutations (F71A and G75A) that did not affect NS4A-NS4B interaction had marginal effects on viral replication, demonstrating the biological relevance of the NS4A-NS4B interaction to DENV-2 replication. Taken together, the study has provided experimental evidence to argue that blocking the NS4A-NS4B interaction could be a potential antiviral approach.ImportanceFlavivirus NS4A and NS4B proteins are essential components of the ER membrane-associated replication complex. The current study systematically characterizes the interaction between flavivirus NS4A and NS4B. Using DENV-2 as a model, we show that NS4A interacts with NS4B in virus-infected cells, in cells transiently expressing NS4A and NS4B proteins, or in vitro with recombinant NS4A and NS4B proteins. We mapped the minimal regions required for the NS4A-NS4B interaction to be amino acids 40 to 76 of NS4A and amino acids 84 to 146 of NS4B. NMR analysis revealed the secondary structure of amino acids 17 to 80 of NS4A and the NS4A amino acids that may participate in the NS4A-NS4B interaction. Functional analysis showed a correlation between viral replication and NS4A-NS4B interaction, demonstrating the biological importance of the NS4A-NS4B interaction. The study has advanced our knowledge of the molecular function of flavivirus NS4A and NS4B proteins. The results also suggest that inhibitors of the NS4A-NS4B interaction could be pursued for flavivirus antiviral development.

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Dataset Information

Antagonism of dsRNA-Induced Innate Immune Pathways by NS4a and NS4b Accessory Proteins during MERS Coronavirus Infection.

Publications

Antagonism of dsRNA-Induced Innate Immune Pathways by NS4a and NS4b Accessory Proteins during MERS Coronavirus Infection.

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