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Protein S-Nitrosylation of Human Cytomegalovirus pp71 Inhibits Its Ability To Limit STING Antiviral Responses.

ABSTRACT: Human Cytomegalovirus (HCMV) is a ubiquitous pathogen that has coevolved with its host and, in doing so, is highly efficient in undermining antiviral responses that limit successful infections. As a result, HCMV infections are highly problematic in individuals with weakened or underdeveloped immune systems, including transplant recipients and newborns. Understanding how HCMV controls the microenvironment of an infected cell so as to favor productive replication is of critical importance. To this end, we took an unbiased proteomics approach to identify the highly reversible, stress-induced, posttranslational modification (PTM) protein S-nitrosylation on viral proteins to determine the biological impact on viral replication. We identified protein S-nitrosylation of 13 viral proteins during infection of highly permissive fibroblasts. One of these proteins, pp71, is critical for efficient viral replication, as it undermines host antiviral responses, including stimulator of interferon genes (STING) activation. By exploiting site-directed mutagenesis of the specific amino acids we identified in pp71 as protein S-nitrosylated, we found this pp71 PTM diminishes its ability to undermine antiviral responses induced by the STING pathway. Our results suggest a model in which protein S-nitrosylation may function as a host response to viral infection that limits viral spread.IMPORTANCE In order for a pathogen to establish a successful infection, it must undermine the host cell responses inhibitory to the pathogen. As such, herpesviruses encode multiple viral proteins that antagonize each host antiviral response, thereby allowing for efficient viral replication. Human Cytomegalovirus encodes several factors that limit host countermeasures to infection, including pp71. Herein, we identified a previously unreported posttranslational modification of pp71, protein S-nitrosylation. Using site-directed mutagenesis, we mutated the specific sites of this modification thereby blocking this pp71 posttranslational modification. In contexts where pp71 is not protein S-nitrosylated, host antiviral response was inhibited. The net result of this posttranslational modification is to render a viral protein with diminished abilities to block host responses to infection. This novel work supports a model in which protein S-nitrosylation may be an additional mechanism in which a cell inhibits a pathogen during the course of infection.

SUBMITTER: Nukui M

PROVIDER: S-EPMC7431796 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Protein S-Nitrosylation of Human Cytomegalovirus pp71 Inhibits Its Ability To Limit STING Antiviral Responses.

Nukui Masatoshi M Roche Kathryn L KL Jia Jie J Fox Paul L PL Murphy Eain A EA

Journal of virology 20200817 17

Human <i>Cytomegalovirus</i> (HCMV) is a ubiquitous pathogen that has coevolved with its host and, in doing so, is highly efficient in undermining antiviral responses that limit successful infections. As a result, HCMV infections are highly problematic in individuals with weakened or underdeveloped immune systems, including transplant recipients and newborns. Understanding how HCMV controls the microenvironment of an infected cell so as to favor productive replication is of critical importance. ...[more]

PMID: 32581105

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Project description:Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory infections that can be life-threatening. To establish an infection and spread, MERS-CoV, like most other viruses, must navigate through an intricate network of antiviral host responses. Besides the well-known type I interferon (IFN-α/β) response, the protein kinase R (PKR)-mediated stress response is being recognized as an important innate response pathway. Upon detecting viral dsRNA, PKR phosphorylates eIF2α, leading to the inhibition of cellular and viral translation and the formation of stress granules (SGs), which are increasingly recognized as platforms for antiviral signaling pathways. It is unknown whether cellular infection by MERS-CoV activates the stress response pathway or whether the virus has evolved strategies to suppress this infection-limiting pathway. Here, we show that cellular infection with MERS-CoV does not lead to the formation of SGs. By transiently expressing the MERS-CoV accessory proteins individually, we identified a role of protein 4a (p4a) in preventing activation of the stress response pathway. Expression of MERS-CoV p4a impeded dsRNA-mediated PKR activation, thereby rescuing translation inhibition and preventing SG formation. In contrast, p4a failed to suppress stress response pathway activation that is independent of PKR and dsRNA. MERS-CoV p4a is a dsRNA binding protein. Mutation of the dsRNA binding motif in p4a disrupted its PKR antagonistic activity. By inserting p4a in a picornavirus lacking its natural PKR antagonist, we showed that p4a exerts PKR antagonistic activity also under infection conditions. However, a recombinant MERS-CoV deficient in p4a expression still suppressed SG formation, indicating the expression of at least one other stress response antagonist. This virus also suppressed the dsRNA-independent stress response pathway. Thus, MERS-CoV interferes with antiviral stress responses using at least two different mechanisms, with p4a suppressing the PKR-dependent stress response pathway, probably by sequestering dsRNA. MERS-CoV p4a represents the first coronavirus stress response antagonist described.

Dataset Information

Protein S-Nitrosylation of Human Cytomegalovirus pp71 Inhibits Its Ability To Limit STING Antiviral Responses.

Publications

Protein S-Nitrosylation of Human Cytomegalovirus pp71 Inhibits Its Ability To Limit STING Antiviral Responses.

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