Abstract Infection with Human Cytomegalovirus (HCMV) can result in a significant burden of disease in those that are immunocompromised or immunonaïve. HCMV encodes a repertoire of glycoproteins that facilitate its extensive viral tropism, some of which remain to be characterized. Currently, there is no effective vaccine or cure for HCMV, therefore emphasizing the need to identify viral proteins of critical function. UL14 was selected as an open reading frame of interest due to its high scoring on an in-silico prediction algorithm, as well as its conservation amongst CMVs. Our goal was to elucidate the function of this uncharacterized viral open reading frame. We hypothesized that UL14 functions in the establishment of infection in epithelial cells, due to its predicted structural similarity to UL141. This study demonstrates that HCMV UL14 is a glycosylated viral protein packaged with the virion. Importantly, the deletion of UL14 resulted in a significant reduction of viral growth in epithelial cells, whereas no growth defect was observed in fibroblasts. Mechanistically, we found this defect to be a result of post entry, pre-IE transcription in the establishment of infection, consistent with a defect endosomal escape. Taken together, our results suggest that UL14 functions in the establishment of infection in an epithelial cell-specific manner and may be a novel target for future vaccines or antiviral therapies. Author Summary HCMV is found in a wide variety of human cells during the course of viral infection. As such, HCMV encodes several glycoprotein complexes that dictate tropism. In this work we report the identification of a novel glycoprotein, UL14, that is involved in establishing productive infections of epithelial cells, a common site of HCMV induced disease. We report that deletion of UL14 from the viral genome impacts its ability to infect ARPE19 cells at a stage indicative of viral events post viral entry but prior to viral transcriptional activation. Further, trans complementation of UL14 by expansion of mutant virus in cells expressing the viral glycoprotein, restore viral infectivity suggesting that UL14 mediates events early in viral infection. Importantly, the characterization of this viral envelope protein provides key insights into viral tropism and identifies a novel target for vaccine design and antiviral therapies.

ABSTRACT Cytomegalovirus (CMV) has been implicated in glioblastoma (GBM) progression. Ongoing clinical trials are assessing therapeutic approaches targeting CMV in GBM but to date no new therapy has been approved outside the standard of care. Previous preclinical studies have highlighted the potential of the antiviral drug Cidofovir (CDV) in GBM; however, its clinical use is limited by dose-dependent nephrotoxicity and poor cellular uptake, necessitating high intravenous doses to achieve therapeutic activity. Brincidofovir (BCV), a lipid conjugate of CDV has been developed, which does not induce nephrotoxicity and has significantly greater cellular bioavailability. Here we examined the effects of BCV in a newly established CMV-driven GBM model (SB28) and in patient-derived tumor neurospheres. We show that BCV prolongs survival in vivo and exerts both CMV-dependent and independent antitumor effects. Mechanistically, BCV induces DNA damage and cell cycle dysregulation in GBM cells and inhibits proliferation of patient-derived neurospheres in a dose-dependent manner. These data identify BCV as a dual-action therapeutic that suppresses viral oncomodulation while directly targeting tumor cell viability.

Infection with human cytomegalovirus (HCMV) can result in a significant disease burden within the immunosuppressed and immunocompromised patient populations. Current standard of care (SOC) relies on direct-acting antivirals which target a limited group of viral proteins including the viral polymerase (UL54), terminase (UL56), and protein kinase (UL97). Incomplete inhibition of virally encoded proteins result in a selective pressure towards the generation of “breakthrough” drug resistant variants. One limitation in evaluating novel antivirals is the difficulty in profiling their antiviral activity against variants resistant to current SOC interventions, as these resistant variants have arisen in different genetic backgrounds with distinct replication kinetics and yields.To limit strain variation we generated a targeted mutant panel of viruses in a bacterial artificial chromosome (BAC) derived clinically relevant laboratory strain of HCMV, TB40e, that expresses the fluorescent proteins mCherry upon viral entry and eGFP at times after viral DNA replication. This unique construct allows for the monitoring of viral entry and viral DNA replication independently. This panel consists of WT and seven mutant viruses harboring mutations that confer resistance to ganciclovir, maribavir, cidofovir, and letermovir. In addition, we characterized a host-targeted sirtuin 2 deacetylase (Sirt2) inhibitor, FLS-359, against the SOC resistant variants. We observed that mutant viruses demonstrated increased EC50 concentrations for SOC inhibition, and that host directed FLS-359 demonstrated broad-spectrum antiviral activity against known SOC drug-resistant mutants. This panel represents a much-needed comparatively innovative platform for screening the efficacy of new direct-acting antivirals and host-directed antivirals against HCMV variants refractive to therapeutic interventions.

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.

ABSTRACT Post-translational modifications (PTMs) are key regulators of various processes important for cell survival. These modifications are critical for dealing with stress conditions, such as those observed in disease states, and during infections with various pathogens. We previously reported that during infection of primary dermal fibroblasts, multiple human cytomegalovirus (HCMV)-encoded proteins were post-translationally modified by the addition of a nitric oxide group to cysteine residues, a modification called protein-S-nitrosylation. For example, tegument protein pp71 is nitrosylated, diminishing its ability to inhibit STING, a protein necessary for DNA virus immune response. Herein, we report that an additional HCMV tegument protein, pp65, responsible for the inhibition of cGAS is also modified by protein-S-nitrosylation on two cysteine residues. Utilizing site-directed mutagenesis to generate recombinant viruses that encode a pp65 that cannot be protein-S-nitrosylated, we evaluated the impact of this PTM on viral replication and how the virus impacts the cGAS/STING pathway. We report that the nitrosylation of pp65 negatively impacts its ability to block cGAS enzymatic functions. pp65 protein-S-nitrosylation mutants demonstrated a decrease in cGAS/STING-induced IRF3 and TBK1 phosphorylation. Additionally, we observed a reduction in IFN-β1 secretion in NuFF-1 cells expressing a nitrosylation-resistant pp65. We report that HCMV expressing a protein-S-nitrosylation-deficient pp65 is resistant to the activation of cGAS in the infection of primary dermal fibroblasts. Our work suggests that nitrosylation of viral proteins may serve as a broadly neutralizing mechanism in HCMV infection. IMPORTANCE Post-translational modifications (PTM) are utilized by host cells to limit an invading pathogen's ability to establish a productive infection. A potent PTM, called protein-S-nitrosylation, has anti-bacterial and anti-viral properties. Increasing protein-S-nitrosylation with the addition of nitric oxide donor compounds reduced HCMV replication in fibroblasts and epithelial cells. We previously reported that protein-S-nitrosylation of HCMV pp71 limits its ability to inhibit STING. Herein, we report that the protein-S-nitrosylation of HCMV pp65 impacts its ability to limit cGAS activity, an additional protein important in regulating interferon response. Therapeutically, patients provided nitric oxide by inhalation reduced viral replication in coronavirus disease 2019, influenza, and even impacted bacterial growth within patients' lungs. It is thought that an increase in free nitric oxide increases the frequency of nitrosylated proteins. Understanding how protein-S-nitrosylation regulates a common DNA virus like HCMV will provide insights into the development of broadly neutralizing therapeutics in drug-resistant viral infections.

Cytomegalovirus (CMV) infection has been implicated in glioblastoma (GBM) progression, with effects on stemness, immune evasion, and angiogenesis. Our work investigates the impact of CMV infection on spheroid formation, oncogenic pathways including SOX2, retinoblastoma protein (pRB), phospho-AKT (pAKT), and STAT3/IL-6 signaling, as well as cytokine modulation. Additionally, we evaluate the therapeutic potential of ganciclovir in reversing CMV-induced effects. GBM cell lines LN229, U251, U87, U138, and T98G were infected with the GFP-expressing CMV TB40 strain at a multiplicity of infection (MOI) of 0.5. Infection was confirmed via fluorescence microscopy and Western blotting for CMV pp65 protein. The stable cell line with infection was confirmed after multiple passage and presence of CMV protein. Spheroid formation assays assessed morphological changes and aggressiveness in vitro. Western blotting analyzed the expression of SOX2, pRB, pAKT, and phospho-STAT3 (pSTAT3), while cytokine profiles, including PD-L1, VEGF, angiopoietin-2, and IL-6, were measured in non-infected, infected, and ganciclovir-treated cells using Luminex panel. CMV infection was confirmed in all GBM cell lines, with infection rates ranging from 60% to 90%. CMV-infected cells exhibited accelerated and more aggressive spheroid formation compared to non-infected cells. Infection led to upregulation of SOX2, pRB, pAKT, and pSTAT3, accompanied by elevated IL-6, indicating enhanced stemness, proliferation, and pro-inflammatory signaling. Cytokine profiling revealed significant increases in PD-L1and angiopoietin-2 with decrease in soluble VEGF levels following infection and treatment with Gancylovir, reflecting immune suppression and angiogenesis. Ganciclovir treatment modulate pro-tumorigenic cytokines, creating a more favorable tumor microenvironment. CMV infection accelerates spheroid formation and enhances the aggressive phenotype in GBM via upregulation of SOX2, pRB, pAKT, and STAT3/IL-6 signaling, alongside immune evasion and angiogenic cytokine shifts. Our data suggest the therapeutic potential of targeting CMV and its downstream pathways in GBM. These findings highlight the critical role of CMV in GBM pathogenesis and its promise as a therapeutic target. Vida Tajiknia, Noe Mercado, Jacquelin Real, Eain Murphy, Sean Lawler. Cytomegalovirus infection enhances spheroid formation and drives aggressive phenotype in glioblastoma via upregulation of SOX2 pRB pAKT and STAT3/IL-6 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3070.

The extracellular addition of the STING agonist, 2-3cGAMP, induces an antiviral state that inhibits HSV-1 replication in a cell type dependent manner via the transportation of the cyclic-dinucleotide through the folate antiporter SLC19A1. To establish a successful infection, herpes simplex virus-1 (HSV-1), a ubiquitous virus with high seropositivity in the human population, must undermine a multitude of host innate and intrinsic immune defense mechanisms, including key players of the STimulator of INterferon Genes (STING) pathway. Herein, we report that 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 protein ICP27. 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 important antiviral mechanism.

All viruses that establish successful infections express proteins that inhibit innate anti-viral pathways such as the stimulator of interferon genes (STING) pathway. In response, cells have evolved mechanisms to limit viruses by modifying these viral proteins via post-translational modifications (PTMs). One potent PTM, protein-S-nitrosylation, inhibits the ability of human cytomegalovirus (HCMV) to undermine the establishment of an anti-viral state. The direct nitrosylation of HCMV tegument protein pp71 at a central cysteine within its pRB binding domain reduces pp71's ability to limit the activity of STING. Two different proteins encoded by unrelated DNA viruses, adenovirus (AdV) E1A and human papillomavirus (HPV) E7, also contain a pRB binding domain and inhibit STING like pp71. Herein, we report that E1A and E7 are both protein-S-nitrosylated like pp71. Stable cell lines expressing a WT, or mutants in which the predicted modified cysteine was changed to the closely related serine amino acid, thus blocking protein-S-nitrosylation, revealed that E1A and E7 are both protein-S-nitrosylated. Furthermore, induction of the STING pathway promoted IFN-β1 transcript production and the phosphorylation of IRF3, which was limited in E1A and E7 stable cell lines. Mutant stable cell lines exhibited a stronger inhibition of IFN-β1 transcription and reduced IRF3 phosphorylation, suggesting that the PTM limits WT viral protein inhibition of STING. Furthermore, both E1a and E7 can complement the replication of a HCMV that lacks pp71 during times of STING activation. These observations support a model in which protein-S-nitrosylation of viral virulence factors may function as an anti-viral mechanism in DNA virus infections. IMPORTANCE DNA viruses, such as HCMV, AdV, and HPV, have the capacity to cause significant disease. Infection with AdV can cause severe lower respiratory and liver disease in children, and HPV infection is persistent and is a causative agent of cancer. Thus, these infections can be a severe health risk. Host cells have adapted innate responses like protein S-nitrosylation to limit viral replication. Our previous work reported that direct nitrosylation of two HCMV viral proteins, pp65 and pp71, limits their ability to undermine host anti-viral responses. Herein, we investigated whether protein-S-nitrosylation of AdV and HPV proteins inhibits their functions, suggesting that this PTM is an anti-viral mechanism. This may provide insight into the development of broad anti-viral therapeutics for persistent viral infections.

ABSTRACT Human cytomegalovirus (HCMV) is a betaherpesvirus capable of infecting numerous cell types and persisting throughout an infected individual’s life. Disease usually occurs in individuals with compromised or underdeveloped immune systems. Several antivirals exist but have limitations relating to toxicity and resistance. HCMV replication involves upregulation of host proteasomal activities, which play important roles in the temporal stages of replication. Here, we defined the impact on replication kinetics of the proteasome inhibitor, bortezomib. We demonstrate that bortezomib significantly reduces levels of viral genomes and infectious virions produced from a population of cells. Inhibition reduced expression of viral proteins that are influenced by genome synthesis. When added prior to 24 hpi, we observe decreases in PCNA and Cdk1 while increases in p21 whose regulations contribute to efficient replication. This response synergized with an antiviral, maribavir. Since some replication occurred, we tested the hypothesis that a subset of infected cells might break through inhibition. Initially, we simulated bortezomib activities using a mechanistic computational model of late-lytic replication. Upon reducing multiplicity of infection (MOI) in silico , we observed near-identical simulated results compared to experimental data. Next, we analyzed replication using live-cell imaging. This revealed treated cultures do contain a population of cells with fully developed late-stage cytoplasmic assembly compartments but at significantly lower numbers. We refer to this as the effective MOI. Overall, our studies support a hypothesis in which 20S proteasome inhibition disrupts HCMV replication by reducing the MOI to an effective MOI, defined by a fraction of infected cells capable of progressing to fulminant infection. IMPORTANCE Human cytomegalovirus (HCMV) infection and reactivation continues to contribute to morbidity and mortality around the world. Antiviral compounds are available but have limitations. Here, we have defined the impact of the proteasome inhibitor bortezomib on HCMV replication. Proteasomal activities play a critical role in temporal changes required for replication. We demonstrate that disrupting these activities inhibits viral replication while likely supporting increased antiviral activity of the anti-HCMV agent, maribavir. Using a combination of live-cell imaging and computational tools, we discover that a subset of infected cells progresses to fulminant infection, which we define as the effective multiplicity of infection, and this subset would otherwise be missed when analyzing the average of the population.

In the United States (US), biosafety and biosecurity oversight of research on viruses is being reappraised. Safety in virology research is paramount and oversight frameworks should be reviewed periodically. Changes should be made with care, however, to avoid impeding science that is essential for rapidly reducing and responding to pandemic threats as well as addressing more common challenges caused by infectious diseases. Decades of research uniquely positioned the US to be able to respond to the COVID-19 crisis with astounding speed, delivering life-saving vaccines within a year of identifying the virus. We should embolden and empower this strength, which is a vital part of protecting the health, economy, and security of US citizens. Herein, we offer our perspectives on priorities for revised rules governing virology research in the US.

INTRODUCTION:Herpes simplex virus 1 (HSV-1) infection alters critical markers of Alzheimer's Disease (AD) in neurons. One key marker of AD is the hyperphosphorylation of Tau, accompanied by altered levels of Tau isoforms. However, an imbalance in these Tau splice variants, specifically resulting from altered 3R to 4R MAPT splicing of exon 10, has yet to be directly associated with HSV-1 infection. METHODS:To this end, we infected 2D and 3D human neural models with HSV-1 and monitored MAPT splicing and Tau phosphorylation. Further, we transduced SH-SY5Y-neurons with HSV-1 ICP27 which alters RNA splicing to analyze if ICP27 alone is sufficient to induce altered MAPT exon 10 splicing. RESULTS:We show that HSV-1 infection induces altered splicing of MAPT exon 10, increasing 4R-Tau protein levels, Tau hyperphosphorylation, and Tau oligomerization. DISCUSSION:Our experiments reveal a novel link between HSV-1 infection and the development of cytopathic phenotypes linked with AD progression. HIGHLIGHTS:HSV-1 infection in forebrain organoids reduces the neurite length of MAP2-positive neurons.HSV-1 infection increases Tau hyperphosphorylation in both two-month-old and four-month-old forebrain organoids. HSV-1 infection increases Exon 10 containing (4R) MAPT mRNA and 4R-Tau protein expression in both forebrain organoids and human SH-SY5Y-neurons. HSV-1 ICP27 is both necessary and sufficient to induce increased 4R MAPT mRNA and 4R-Tau protein expression in SH-SY5Y-neurons. HSV-1 infection increases Tau oligomerization in both forebrain organoids and SH-SY5Y-neurons.

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.

Abstract BACKGROUND Cytomegalovirus (CMV) has been implicated in glioblastoma (GBM) pathogenesis by affecting stemness, angiogenesis and immune pathways. Clinical trials targeting CMV in GBM patients have shown some early promise, and CMV seropositivity has been associated with poorer outcomes. However, the underlying mechanisms remain unclear. Here we investigated the effects of CMV infection on critical signaling pathways in GBM (IL6/STAT3, and Akt signaling) based on the hypothesis that CMV may contribute to poorer outcomes in GBM by increasing oncogenic signaling. MATERIALS AND METHODS Human U251 and LN229 GBM cell lines were infected with mCherry expressing human CMV TB40 strain at a multiplicity of infection (MOI) of 0.5. We performed basic phenotypic studies in vitro and investigated the status of IL6/STAT3, and Akt signaling by Western blotting. RESULTS Both LN229 and U251 cell lines were readily infectable by CMV. Infection of U251 cells was over 90% and in LN229 cells was approximately 60%. Interestingly expression of CMV was sustained over many passages in these cell lines. Infection was also verified by the presence of the CMV pp65 protein assessed by Western blotting. Measurement of cell proliferation indicated faster growth after CMV infection. Interrogation of key pro-oncogenic signaling pathways in GBM revealed a robust upregulation of IL6, as well as phospho(Tyr705)-STAT3 and phospho(Thr308)-Akt. This elevation of pro-oncogenic signaling pathways was observed in both cell lines compared with controls. CONCLUSIONS Here we show for the first time that infection of human GBM cell lines with CMV results in a more aggressive phenotype associated with upregulation of IL6/STAT3 signaling as well as a significant increase in phospho-Akt levels. We are currently studying sensitivity to standard therapies and additional molecular changes in these cells. These data support the hypothesis that CMV causes more aggressive disease and that CMV is a relevant therapeutic target in GBM.

Introduction: The pathogenicity of cytomegalovirus (CMV) has predominantly been recognized in immunosuppressed populations & newborns. However, a recent study in persons with CF observed that CMV seropositive individuals died from lung disease 8 years sooner than those who were seronegative. Further, CMV DNA was identified in those individuals' sputum (Parkins 2019, ERJ). This observation aligns with increasing evidence associating CMV infection with worse outcomes in many inflammatory conditions, and a higher overall mortality. To date, models of CMV infection of human bronchial epithelial (HBE) cells have not been established, limiting our ability to study its impact in CF airways. Methods: This pilot project aimed to develop a model to evaluate airways responses to CMV infection. Primary HBEs were infected with TB40/E CMV (expressing mCherry & eGFP fluorophores at cell binding and replication, respectively) in submersion. Cells were harvested for DNA isolation & RNA sequencing at 1–7 days post-infection (DPI). Supernatants were collected for detection of inflammatory/remodelling proteins via ELISA. Results: CMV was found to bind to HBEs within 15 minutes, with peak binding observed and maintained at 3 hours PI, as seen by fluorescence. Viral replication was first noticed at 3 DPI (confirmed with qPCR). Supernatants showed increased levels of IL8, CXCL10, & amphiregulin at 1 & 2 DPI, though these did not achieve statistical significance. Upon CMV infection, HBEs showed a significant transcriptional up-regulation in 16 genes at 1 DPI (Table 1) – many of which have previously been identified as modifiers of CF disease severity (Kormann 2017, PLOS One) Conclusions: This study is the first to infect HBEs with CMV, showing increases in several inflammatory and antiviral pathways known to be activated in CF. This model is suitable for investigation of CMV-mediated inflammation using CF-derived HBEs with & without concomitant bacterial infection.

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.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals.

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.