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.

Inhibition of the FACT Complex Reduces Transcription from the Human Cytomegalovirus Major Immediate Early Promoter in Models of Lytic and Latent Replication.

The successful colonization of the majority of the population by human cytomegalovirus is a direct result of the virus's ability to establish and, more specifically, reactivate from latency. The underlying cellular factors involved in viral reactivation remain unknown. Here, we show that the host complexfacilitateschromatintranscription (FACT) binds to the major immediate early promoter (MIEP) and that inhibition of this complex reduces MIEP transactivation, thus inhibiting viral reactivation.

Human cytomegalovirus miR-UL112-1 promotes the down-regulation of viral immediate early-gene expression during latency to prevent T-cell recognition of latently infected cells.

Human cytomegalovirus, a member of the herpesvirus family, can cause significant morbidity and mortality in immune compromised patients resulting from either primary lytic infection or reactivation from latency. Latent infection is associated with a restricted viral transcription programme compared to lytic infection which consists of defined protein coding RNAs but also includes a number of virally encoded microRNAs (miRNAs). One of these, miR-UL112-1, is known to target the major lytic IE72 transcript but, to date, a functional role for miR-UL112-1 during latent infection has not been shown. To address this, we have analysed latent infection in myeloid cells using a virus in which the target site for miR-UL112-1 in the 3' UTR of IE72 was removed such that any IE72 RNA present during latent infection would no longer be subject to regulation by miR-UL112-1 through the RNAi pathway. Our data show that removal of the miR-UL112-1 target site in IE72 results in increased levels of IE72 RNA in experimentally latent primary monocytes. Furthermore, this resulted in induction of immediate early (IE) gene expression that is detectable by IE-specific cytotoxic T-cells (CTLs); no such CTL recognition of monocytes latently infected with wild-type virus was observed. We also recapitulated these findings in the more tractable THP-1 cell line model of latency. These observations argue that an important role for miR-UL112-1 during latency is to ensure tight control of lytic viral immediate early (IE) gene expression thereby preventing recognition of latently infected cells by the host's potent pre-existing anti-viral CTL response.

A human herpesvirus 6A-encoded microRNA: role in viral lytic replication.

UNLABELLED: Human herpesvirus 6A (HHV-6A), a member of the betaherpesvirus family, is associated with several human diseases. Like all herpesviruses, HHV-6A establishes a lifelong, latent infection in its host. Reactivation of HHV-6A is frequent within the immunosuppressed and immunocompromised populations and results in lytic viral replication within multiple organs, often leading to severe disease. MicroRNAs (miRNAs) are key regulators of multiple cellular processes that regulate the translation of specific transcripts. miRNAs carried by herpesviruses play important roles in modulating the host cell, thereby facilitating a suitable environment for productive viral infection and/or latency. Currently, there are approximately 150 known human herpesvirus-encoded miRNAs, although an miRNA(s) encoded by HHV-6A has yet to be reported. We hypothesized that HHV-6A, like other members of the human herpesvirus family, encodes miRNAs, which function to promote viral infection. We utilized deep sequencing of small RNA species isolated from cells harboring HHV-6A to identify five novel small noncoding RNA species that originate from the viral genome, one of which has the characteristics of a viral miRNA. These RNAs are expressed during productive infection by either bacterial artificial chromosome (BAC)-derived virus in Jjhan cells or wild-type HHV-6A strain U1102 virus in HSB2 cells and are associated with the RNA induced silencing complex (RISC) machinery. Growth analyses of mutant viruses that lack each individual miRNA revealed that a viral miRNA candidate (miR-U86) targets the HHV-6A IE gene U86, thereby regulating lytic replication. The identification and biological characterization of this HHV-6A-specific miRNA is the first step to defining how the virus regulates its life cycle. IMPORTANCE: A majority of the human population is infected with human herpesvirus 6A (HHV-6A), a betaherpesvirus family member. Infections usually occur in young children, and upon resolution, the virus remains in a latent state within the host. Importantly, during times of weakened immune responses, the virus can reactivate and is correlated with significant disease states. Viruses encode many different types of factors that both undermine the host antiviral response and regulate viral replication, including small RNA species called microRNAs (miRNAs). Here we report that HHV-6A encodes at least one miRNA, which we named miR-U86. We have characterized the requirement of this viral miRNA and its impact on the viral life cycle and found that it functions to regulate a viral protein important for efficient viral replication. Our data suggest that viral miRNAs are important for HHV-6A and that they may serve as an important therapeutic target to inhibit the virus.

Condensin II subunit dCAP-D3 restricts retrotransposon mobilization in Drosophila somatic cells.

Retrotransposon sequences are positioned throughout the genome of almost every eukaryote that has been sequenced. As mobilization of these elements can have detrimental effects on the transcriptional regulation and stability of an organism's genome, most organisms have evolved mechanisms to repress their movement. Here, we identify a novel role for the Drosophila melanogaster Condensin II subunit, dCAP-D3 in preventing the mobilization of retrotransposons located in somatic cell euchromatin. dCAP-D3 regulates transcription of euchromatic gene clusters which contain or are proximal to retrotransposon sequence. ChIP experiments demonstrate that dCAP-D3 binds to these loci and is important for maintaining a repressed chromatin structure within the boundaries of the retrotransposon and for repressing retrotransposon transcription. We show that dCAP-D3 prevents accumulation of double stranded DNA breaks within retrotransposon sequence, and decreased dCAP-D3 levels leads to a precise loss of retrotransposon sequence at some dCAP-D3 regulated gene clusters and a gain of sequence elsewhere in the genome. Homologous chromosomes exhibit high levels of pairing in Drosophila somatic cells, and our FISH analyses demonstrate that retrotransposon-containing euchromatic loci are regions which are actually less paired than euchromatic regions devoid of retrotransposon sequences. Decreased dCAP-D3 expression increases pairing of homologous retrotransposon-containing loci in tissue culture cells. We propose that the combined effects of dCAP-D3 deficiency on double strand break levels, chromatin structure, transcription and pairing at retrotransposon-containing loci may lead to 1) higher levels of homologous recombination between repeats flanking retrotransposons in dCAP-D3 deficient cells and 2) increased retrotransposition. These findings identify a novel role for the anti-pairing activities of dCAP-D3/Condensin II and uncover a new way in which dCAP-D3/Condensin II influences local chromatin structure to help maintain genome stability.

Host microRNA regulation of human cytomegalovirus immediate early protein translation promotes viral latency.

UNLABELLED: Reactivation of human cytomegalovirus (HCMV) is a significant cause of disease and death in immunocompromised patients, underscoring the need to understand how latency is controlled. Here we demonstrate that HCMV has evolved to utilize cellular microRNAs (miRNAs) in cells that promote latency to regulate expression of a viral protein critical for viral reactivation. Our data reveal that hsa-miR-200 miRNA family members target the UL122 (immediate early protein 2) 3' untranslated region, resulting in repression of this viral protein. Utilizing recombinant viruses that mutate the miRNA-binding site compared to the sequence of the wild-type virus results in lytic rather than latent infections in ex vivo infections of primary CD34+ cells. Cells permissive for lytic replication demonstrate low levels of these miRNAs. We propose that cellular miRNA regulation of HCMV is critical for maintenance of viral latency. IMPORTANCE: Human cytomegalovirus (HCMV) is a herpesvirus that infects a majority of the population. Once acquired, individuals harbor the virus for life, where the virus remains, for the most part, in a quiet or latent state. Under weakened immune conditions, the virus can reactivate, which can cause severe disease and often death. We have found that members of a family of small RNAs, termed microRNAs, encoded by human myeloid progenitor cells are capable of repressing a key viral protein, thus enabling the virus to ensure a quiet/latent state. As these progenitor cells mature further down the myeloid lineage toward cells that support active viral replication, the levels of these microRNAs decrease. Together, our data suggest that host cell microRNA regulation of HCMV is important for the quiet/latent state of this pathogen.

BclAF1 restriction factor is neutralized by proteasomal degradation and microRNA repression during human cytomegalovirus infection.

Cell proteins can restrict the replication of viruses. Here, we identify the cellular BclAF1 protein as a human cytomegalovirus restriction factor and describe two independent mechanisms the virus uses to decrease its steady-state levels. Immediately following infection, the viral pp71 and UL35 proteins, which are delivered to cells within virions, direct the proteasomal degradation of BclAF1. Although BclAF1 reaccumulates through the middle stages of infection, it is subsequently down-regulated at late times by miR-UL112-1, a virus-encoded microRNA. In the absence of BclAF1 neutralization, viral gene expression and replication are inhibited. These data identify two temporally and mechanistically distinct functions used by human cytomegalovirus to down-regulate a cellular antiviral protein.

Human herpesvirus 6A infection and immunopathogenesis in humanized Rag2⁻/⁻ γc⁻/⁻ mice.

Although serious human diseases have been correlated with human herpesvirus 6A (HHV-6A) and HHV-6B, the lack of animal models has prevented studies which would more definitively link these viral infections to disease. HHV-6A and HHV-6B have recently been classified as two distinct viruses, and in this study we focused specifically on developing an in vivo model for HHV-6A. Here we show that Rag2⁻/⁻γc⁻/⁻ mice humanized with cord blood-derived human hematopoietic stem cells produce human T cells that express the major HHV-6A receptor, CD46. Both cell-associated and cell-free viral transmission of HHV-6A into the peritoneal cavity resulted in detectable viral DNA in at least one of the samples (blood, bone marrow, etc.) analyzed from nearly all engrafted mice. Organs and cells positive for HHV-6A DNA were the plasma and cellular blood fractions, bone marrow, lymph node, and thymic samples; control mice had undetectable viral DNA. We also noted viral pathogenic effects on certain T cell populations. Specific thymocyte populations, including CD3⁻ CD4⁺ CD8⁻ and CD3⁺ CD4⁻ cells, were significantly modified in humanized mice infected by cell-associated transmission. In addition, we detected significantly increased proportions of CD4⁺ CD8⁺ cells in the blood of animals infected by cell-free transmission. These findings provide additional evidence that HHV-6A may play a role in human immunodeficiencies. These results indicate that humanized mice can be used to study HHV-6A in vivo infection and replication as well as aspects of viral pathogenesis.

Herpes Simplex Virus-1 targets the 2'-3'cGAMP importer SLC19A1 as an antiviral countermeasure.

To establish a successful infection, herpes simplex virus-1 (HSV-1), a virus with high seropositivity in the human population, must undermine host innate and intrinsic immune defense mechanisms, including the stimulator of interferon genes (STING) pathway. Recently it was discovered that not only de novo produced intracellular 2'-3'cGAMP, but also extracellular 2'-3'cGAMP activates the STING pathway by being transported across the cell membrane via the folate transporter, SLC19A1, the first identified extracellular antiporter of this signaling molecule. We hypothesized that the import of exogenous 2'-3'cGAMP functions to establish an antiviral state like that seen with the paracrine antiviral activities of interferon. Further, to establish a successful infection, HSV-1 must undermine this induction of the STING pathway by inhibiting the biological functions of SLC19A1. Herein, we report that treatment of the monocytic cell line, THP-1 cells, epithelial cells (ARPE-19) and SH-SY5Y neuronal cell line with exogenous 2'-3'cGAMP induces interferon production and establishes an antiviral state. Using either pharmaceutical inhibition or genetic knockout of SLC19A1 blocks the 2'-3'cGAMP-induced antiviral state. Additionally, HSV-1 infection results in the reduction of SLC19A1 transcription, translation, and importantly, the rapid removal of SLC19A1 from the cell surface of infected cells. Our data indicate SLC19A1 functions as a newly identified antiviral mediator for extracellular 2'-3'cGAMP which is undermined by HSV-1. This work presents novel and important findings about how HSV-1 manipulates the host's immune environment for viral replication and discovers details about an antiviral mechanism which information could aid in the development of better antiviral drugs in the future. Importance: HSV-1 has evolved multiple mechanisms to neutralize of the host's innate and intrinsic defense pathways, such as the STING pathway. Here, we identified an antiviral response in which extracellular 2'-3'cGAMP triggers IFN production via its transporter SLC19A1. Moreover, we report that HSV-1 blocks the functions of this transporter thereby impeding the antiviral response, suggesting exogenous 2'-3'cGAMP can act as an immunomodulatory molecule in uninfected cells to activate the STING pathway, and priming an antiviral state, similar to that seen in interferon responses. The details of this mechanism highlight important details about HSV-1 infections. This work presents novel findings about how HSV-1 manipulates the host's immune environment for viral replication and reveals details about a novel antiviral mechanism. These findings expand our understanding of how viral infections undermine host responses and may help in the development of better broad based antiviral drugs in the future.

Import of extracellular 2'-3'cGAMP by the folate transporter, SLC19A1, establishes an antiviral response that limits herpes simplex virus-1.

Recently it was discovered that extracellular 2'-3'cGAMP can activate the STING pathway in a cGAS-independent fashion by being transported across the cell membrane via the folate transporter, SLC19A1, the first identified extracellular antiporter of this critical signaling molecule in cancer cells. We hypothesized that this non-canonical activation of STING pathway would function to establish an antiviral state similar to that seen with the paracrine antiviral activities of interferon. Herein, we report that treatment of the monocytic cell line, THP-1 cells and SH-SY5Y neuronal cell line with exogenous 2'-3'cGAMP induces interferon production and establishes an antiviral state that limits herpes simplex virus-1 (HSV-1), a ubiquitous virus with high seropositivity in the human population. Using either pharmaceutical inhibition or genetic knockout of SLC19A1 blocks the 2'-3'cGAMP-induced inhibition of viral replication. Our data indicate SLC19A1 functions as a newly identified antiviral mediator for extracellular 2'-3'cGAMP. This work presents novel and important findings about an antiviral mechanism which information could aid in the development of better antiviral drugs in the future.

Targeting the host transcription factor HSF1 prevents human cytomegalovirus replication in vitro and in vivo.

FDA-approved antivirals against HCMV have several limitations, including only targeting the later stages of the viral replication cycle, adverse side effects, and the emergence of drug-resistant strains. Antivirals targeting host factors specifically activated within infected cells and necessary for viral replication could address the current drawbacks of anti-HCMV standard-of-care drugs. In this study, we found HCMV infection stimulated the activation of the stress response transcription factor heat shock transcription factor 1 (HSF1). HCMV entry into fibroblasts rapidly increased HSF1 activity and subsequent relocalization from the cytoplasm to the nucleus, which was maintained throughout viral replication and in contrast to the transient burst of activity induced by canonical heat shock. Prophylactic pharmacological inhibition or genetic depletion of HSF1 prior to HCMV infection attenuated the expression of all classes of viral genes, including immediate early (IE) genes, and virus production, suggesting HSF1 promotes the earliest stages of the viral replication cycle. Therapeutic treatment with SISU-102, an HSF1 inhibitor tool compound, after IE expression also reduced the levels of L proteins and progeny production, suggesting HSF1 regulates multiple steps along the HCMV replication cycle. Leveraging a newly developed human skin xenograft transplant murine model, we found prophylactic treatment with SISU-102 significantly attenuated viral replication in transplanted human skin xenografts as well as viral dissemination to distal sites. These data demonstrate HCMV infection rapidly activates and relocalizes HSF1 to the nucleus to promote viral replication, which can be exploited as a host-directed antiviral strategy. One Sentence Summary: Inhibiting of HSF1 as a host-directed antiviral therapy attenuates HCMV replication in vitro and in vivo.

A myeloid progenitor cell line capable of supporting human cytomegalovirus latency and reactivation, resulting in infectious progeny.

Human cytomegalovirus (HCMV) is a herpesvirus that establishes a lifelong, latent infection within a host. At times when the immune system is compromised, the virus undergoes a lytic reactivation producing infectious progeny. The identification and understanding of the biological mechanisms underlying HCMV latency and reactivation are not completely defined. To this end, we have developed a tractable in vitro model system to investigate these phases of viral infection using a clonal population of myeloid progenitor cells (Kasumi-3 cells). Infection of these cells results in maintenance of the viral genome with restricted viral RNA expression that is reversed with the addition of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, also known as PMA). Additionally, a latent viral transcript (LUNA) is expressed at times where viral lytic transcription is suppressed. Infected Kasumi-3 cells initiate production of infectious virus following TPA treatment, which requires cell-to-cell contact for efficient transfer of virus to other cell types. Importantly, lytically infected fibroblast, endothelial, or epithelial cells can transfer virus to Kasumi-3 cells, which fail to initiate lytic replication until stimulated with TPA. Finally, inflammatory cytokines, in addition to the pharmacological agent TPA, are sufficient for transcription of immediate-early (IE) genes following latent infection. Taken together, our findings argue that the Kasumi-3 cell line is a tractable in vitro model system with which to study HCMV latency and reactivation.

The Human Cytomegalovirus Latency-Associated Gene Product Latency Unique Natural Antigen Regulates Latent Gene Expression.

Human cytomegalovirus (HCMV) infection can lead to either lytic or latent infection, which is dependent on the regulation of the viral major immediate early promoter (MIEP). Suppression of the MIEP is a pre-requisite for latency and is driven by repressive epigenetic modifications at the MIEP during latent infection. However, other viral genes are expressed during latency and this is correlated with activatory epigenetic modifications at latent gene promoters. Yet the molecular basis of the differential regulation of latent and lytic gene expression by epigenetics is unclear. LUNA, a latent viral transcript, has been suggested to be important for HCMV latency and has also been shown to be important for efficient reactivation likely through its known deSUMOylase activity. Intriguingly, we and others have also observed that LUNA enhances latency-associated expression of the viral UL138 gene. Here, we show that in the absence of LUNA, the expression of multiple latency-associated transcripts is reduced during latent infection, which is correlated with a lack of activatory marks at their promoters. Interestingly, we also show that LUNA interacts with the hematopoietic transcription factor GATA-2, which has previously been shown to bind to a number of latency-associated gene promoters, and that this interaction is dependent on the deSUMOylase domain of LUNA. Finally, we show that the deSUMOylase activity of LUNA is required for the establishment and/or maintenance of an open chromatin configuration around latency-associated gene promoters. As such, LUNA plays a key role in efficient latency-associated viral gene expression and carriage of viral genome during latent carriage.

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.

Design of a US28 ORF Deletion Virus in a Temperature-Sensitive Cytomegalovirus Strain Fails to Promote Lytic Replication in Hematopoietic Cells.

Human cytomegalovirus (CMV) is a ubiquitous pathogen that latently resides in hematopoietic cells. Latently infected individuals with dysfunctional immune systems often experience CMV reactivation, which can cause devastating disease and mortality. While factors dictating the balance between latency and reactivation are not completely understood, CMV US28 is required for maintaining latent infection, and viral mutants that alter US28 function result in a lytic-like, rather than latent, infection in hematopoietic cells. In turn, viral lytic factors alter the host cell, making it challenging to characterize the US28-specific changes in the cellular milieu. To circumvent this, we generated a temperature-sensitive TB40/E recombinant virus, TB40/EgfpC510G (tsC510G), into which we engineered an amino acid change at position 510 (C510G) of IE2, as previously described in the CMV Towne strain. Using tsC510G, we then deleted the US28 ORF, termed tsC510G-US28Δ. Consistent with previous findings, tsC510G-US28Δ fails to undergo latency in Kasumi-3 cells at the permissive temperature. However, parallel cultures maintained at the non-permissive temperature showed a significant reduction in infectious center frequency, as measured by limiting dilution assay. Thus, we generated a new US28 mutant virus for use as a tool to study US28-specific changes in latently infected hematopoietic cells in the absence of induced lytic replication.

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.

Suppression of immediate-early viral gene expression by herpesvirus-coded microRNAs: implications for latency.

A quantitative algorithm was developed and applied to predict target genes of microRNAs encoded by herpesviruses. Although there is almost no conservation among microRNAs of different herpesvirus subfamilies, a common pattern of regulation emerged. The algorithm predicts that herpes simplex virus 1, human cytomegalovirus, Epstein-Barr virus, and Kaposi's sarcoma-associated herpesvirus all employ microRNAs to suppress expression of their own genes, including their immediate-early genes. In the case of human cytomegalovirus, a virus-coded microRNA, miR-112-1, was predicted to target the viral immediate-early protein 1 mRNA. To test this prediction, mutant viruses were generated that were unable to express the microRNA, or encoded an immediate-early 1 mRNA lacking its target site. Analysis of RNA and protein within infected cells demonstrated that miR-UL112-1 inhibits expression of the major immediate-early protein. We propose that herpesviruses use microRNA-mediated suppression of immediate-early genes as part of their strategy to enter and maintain latency.

Reevaluating the Impact of Epstein-Barr Virus Noncoding RNAs on the Interferon Response.

The necessity of viruses to modulate the innate immune response often dictates the outcome of viral infection. As such, viruses encode many factors that undermine these potent antiviral responses. A recent study by Bouvet et al. (M. Bouvet, S. Voigt, T. Tagawa, M. Albanese, et al., mBio 12:e03440-20, 2021, https://doi.org/10.1128/mBio.03440-20) revisits the impact of virus-encoded noncoding RNAs on key components of the interferon pathway and sheds light on how the extensive biological functions of Epstein-Barr virus (EBV) microRNAs (miRNAs) are on targeting both the induction and signaling cascades of interferon.