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 co-evolved 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, post-translational 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 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 post-translational modification of pp71, protein S-nitrosylation. Using site-directed mutagenesis, we mutated the specific sites of this modification thereby blocking this pp71post-translational modification. In contexts where pp71 is not protein S-nitrosylated, host antiviral response was inhibited. The net result of this post-translational 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.

Human Cytomegalovirus UL138 Protein Inhibits the STING Pathway and Reduces Interferon Beta mRNA Accumulation during Lytic and Latent Infections

“…Many viruses encode one or more proteins that inactivate innate immunity pathways ( 17 ), including the cGAS/STING/TBK1 pathway. Indeed, HCMV encodes 9 proteins known to inactivate the cGAS/STING/TBK1 pathway that are among the ∼200 proteins expressed during productive infections of highly differentiated cells: UL31 ( 13 ), UL35 ( 11 ), UL42 ( 12 ), UL48 ( 18 ), UL82 (pp71) ( 19 , 20 ), UL83 (pp65) ( 21 ), UL94 ( 22 ), UL122 (IE2) ( 14 ), and Us9 ( 10 ).…”

Nitric Oxide Circumvents Virus-Mediated Metabolic Regulation during Human Cytomegalovirus Infection

“…HCMV infection of endothelial and smooth muscle cells induces NOS2 mRNA in an IE2-dependent manner ( 28 ). More recent work by Nukui et al ( 29 ) provides insight into potential mechanisms by identifying S-nitrosation, a modification involving nitric oxide, of several HCMV proteins including pp71, which disrupts its activity against the STING pathway. In contrast to lytic infection, nitric oxide produced during latent HCMV infection via NOS2 was demonstrated to be essential for viral latency by silencing of immediate early gene expression through an unknown mechanism ( 30 ).…”

Innate Immune Responses to Herpesvirus Infection

“…Nukui and colleagues identified a post-translational modification, called protein S-nitrosylation, which specifically modifies pp71 at cysteines 34, 94, and 218. While mutations of each of these cysteines to serines did not impact incorporation of pp71 into the viral particle or viral growth, the C218S mutant increased pp71 s interaction with STING and IFNβ production [75]. Thus, pp71 protein S-nitrosylation decreases this tegument protein's interaction with STING, leading to upregulation of antiviral cytokines.…”