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

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.

Selective 4-Thiouracil Labeling of RNA Transcripts within Latently Infected Cells after Infection with Human Cytomegalovirus Expressing Functional Uracil Phosphoribosyltransferase.

Infections with human cytomegalovirus (HCMV) are highly prevalent in the general population as the virus has evolved the capacity to undergo distinct replication strategies resulting in lytic, persistent, and latent infections. During the latent life cycle, HCMV resides in subsets of cells within the hematopoietic cell compartment, including hematopoietic progenitor cells (HPCs) and peripheral blood monocytes. Since only a small fraction of these cell types harbor viral genomes during natural latency, identification and analysis of distinct changes mediated by viral infection are difficult to assess. In order to characterize latent infections of HPCs, we used an approach that involves complementation of deficiencies within the human pyrimidine salvage pathway, thus allowing for conversion of labeled uracil into rUTP. Here, we report the development of a recombinant HCMV that complements the defective human pyrimidine salvage pathway, allowing incorporation of thiol containing UTP into all RNA species that are synthesized within an infected cell. This virus grows to wild-type kinetics and can establish a latent infection within two distinct culture models of HCMV latency. Using this recombinant HCMV, we report the specific labeling of transcripts only within infected cells. These transcripts reveal a transcriptional landscape during HCMV latency that is distinct from uninfected cells. The utility of this labeling system allows for the identification of distinct changes within host transcripts and will shed light on characterizing how HCMV establishes and maintains latency.IMPORTANCE HCMV is a significant pathogen that accounts for a substantial amount of complications within the immunosuppressed and immunocompromised. Of particular significance is the capacity of HCMV to reactivate within solid tissue and bone marrow transplant recipients. While it is known that HCMV latency resides within a fraction of HPCs and monocytes, the exact subset of cells that harbor latent viral genomes during natural infections remain uncharacterized. The capacity to identify changes within the host transcriptome during latent infections is critical for developing approaches that therapeutically or physically eliminate latent viral genome containing cells and will represent a major breakthrough for reducing complications due to HCMV reactivation posttransplant. In this report, we describe the generation and use of a recombinant HCMV that allows specific and distinct labeling of RNA species that are produced within virally infected cells. This is a critical first step in identifying how HCMV affects the host cell during latency and more importantly, allows one to characterize cells that harbor latent HCMV.

An allosteric inhibitor of sirtuin 2 deacetylase activity exhibits broad-spectrum antiviral activity.

Most drugs used to treat viral disease target a virus-coded product. They inhibit a single virus or virus family, and the pathogen can readily evolve resistance. Host-targeted antivirals can overcome these limitations. The broad-spectrum activity achieved by host targeting can be especially useful in combating emerging viruses and for treatment of diseases caused by multiple viral pathogens, such as opportunistic agents in immunosuppressed patients. We have developed a family of compounds that modulate sirtuin 2, an NAD+-dependent deacylase, and now report the properties of a member of that family, FLS-359. Biochemical and x-ray structural studies show that the drug binds to sirtuin 2 and allosterically inhibits its deacetylase activity. FLS-359 inhibits the growth of RNA and DNA viruses, including members of the coronavirus, orthomyxovirus, flavivirus, hepadnavirus, and herpesvirus families. FLS-359 acts at multiple levels to antagonize cytomegalovirus replication in fibroblasts, causing modest reductions in viral RNAs and DNA, together with a much greater reduction in infectious progeny, and it exhibits antiviral activity in humanized mouse models of infection. Our results highlight the potential of sirtuin 2 inhibitors as broad-spectrum antivirals and set the stage for further understanding of how host epigenetic mechanisms impact the growth and spread of viral pathogens.

A Viral Long Non-Coding RNA Protects against Cell Death during Human Cytomegalovirus Infection of CD14+ Monocytes.

Long non-coding RNA ß2.7 is the most highly transcribed viral gene during latent human cytomegalovirus (HCMV) infection. However, as yet, no function has ever been ascribed to ß2.7 during HCMV latency. Here we show that ß2.7 protects against apoptosis induced by high levels of reactive oxygen species (ROS) in infected monocytes, which routinely support latent HCMV infection. Monocytes infected with a wild-type (WT) virus, but not virus deleted for the ß2.7 gene (Δß2.7), are protected against mitochondrial stress and subsequent apoptosis. Protected monocytes display lower levels of ROS and additionally, stress-induced death in the absence of ß2.7 can be reversed by an antioxidant which reduces ROS levels. Furthermore, we show that infection with WT but not Δß2.7 virus results in strong upregulation of a cellular antioxidant enzyme, superoxide dismutase 2 (SOD2) in CD14+ monocytes. These observations identify a role for the ß2.7 viral transcript, the most abundantly expressed viral RNA during latency but for which no latency-associated function has ever been ascribed, and demonstrate a novel way in which HCMV protects infected monocytes from pro-death signals to optimise latent carriage.

SAMHD1 Modulates Early Steps during Human Cytomegalovirus Infection by Limiting NF-κB Activation.

Cellular SAMHD1 inhibits replication of many viruses by limiting intracellular deoxynucleoside triphosphate (dNTP) pools. We investigate the influence of SAMHD1 on human cytomegalovirus (HCMV). During HCMV infection, we observe SAMHD1 induction, accompanied by phosphorylation via viral kinase UL97. SAMHD1 depletion increases HCMV replication in permissive fibroblasts and conditionally permissive myeloid cells. We show this is due to enhanced gene expression from the major immediate-early (MIE) promoter and is independent of dNTP levels. SAMHD1 suppresses innate immune responses by inhibiting nuclear factor κB (NF-κB) activation. We show that SAMHD1 regulates the HCMV MIE promoter through NF-κB activation. Chromatin immunoprecipitation reveals increased RELA and RNA polymerase II on the HCMV MIE promoter in the absence of SAMHD1. Our studies reveal a mechanism of HCMV virus restriction by SAMHD1 and show how SAMHD1 deficiency activates an innate immune pathway that paradoxically results in increased viral replication through transcriptional activation of the HCMV MIE gene promoter.

Aging impairs induction of redox factor-1 after heat stress: a potential mechanism for heat-induced liver injury.

Aging is associated with reduced tolerance to physiological stressors such as hyperthermia. In animal models, heat stress is associated with increased oxidative damage in the livers of old rats. In this study, we evaluated the expression of redox factor-1 (Ref-1), a DNA repair enzyme, and thioredoxin-1 (Trx-1), an antioxidant protein. We hypothesized that these proteins would be induced by heat stress in young animals, and that aging would attenuate this response. Young (6 mo) and old (24 mo) male Fischer 344 rats were exposed to a two-heat stress protocol, and livers were harvested at several time points after the second heat stress. Ref-1 and Trx-1 were evaluated by immunoblot and immunohistochemistry. In young rats, Ref-1 was induced by ~50% immediately (0 h) after heat stress, and returned to control levels at 2 h. We observed no change in Ref-1 after hyperthermia in old rats; however, aging was associated with a 2-fold increase in Ref-1 expression. At 2 h after heat stress, Trx-1 was increased in old rats, but there was no change in young rats. In tissue sections, we observed frequent ductular reactions in the old rats that were positive for both Ref-1 and Trx-1. The impairment in the induction of Ref-1 suggests a mechanism for the increased oxidative injury observed in old rats after heat stress. Furthermore, the observation of ductular reactions positive for both Ref-1 and Trx-1 demonstrates a proliferative cellular niche that develops with aging.