The DEAD-box helicase DHX29 plays a critical role in the translation of mRNAs containing complex RNA secondary structure in their 5' untranslated regions (UTRs). The human cytomegalovirus (HCMV) genome has a high GC content, suggesting that the 5' UTRs of viral mRNAs may contain significant secondary structure and require DHX29 for efficient translation. We found that depleting DHX29 from primary human fibroblasts prior to infection reduced viral mRNA and protein levels and decreased HCMV replication. The defect in HCMV replication correlated with decreased expression of the HCMV immediate-early proteins IE1 and IE2, which are necessary for the establishment of lytic infection. Analysis of polysome-associated mRNAs revealed that the defect in IE1 and IE2 expression is due to decreased mRNA translation efficiency. DHX29 depletion led to reduced levels of the eIF4F translation initiation complex, resulting from decreased translation of the eIF4G1 mRNA. However, in line with our previous results showing a minimal role for the eIF4F complex in viral mRNA translation, we found that depleting eIF4G prior to infection did not impact IE1 and IE2 translation. Together, our results define a new role for DHX29 in regulating eIF4F-dependent translation and identify a critical role for DHX29 in the translation of HCMV mRNAs. IMPORTANCE:Expression of the human cytomegalovirus (HCMV) immediate-early proteins IE1 and IE2 is critical for the establishment of lytic replication and the reactivation of latent HCMV infections. Defining the mechanisms controlling HCMV IE1 and IE2 protein expression has the potential to identify new strategies for therapeutic interventions that can limit HCMV disease in immune-naïve and immune-compromised individuals. Our finding that the cellular DHX29 helicase is necessary for the efficient translation of mRNAs encoding IE1 and IE2 suggests that therapies that inhibit DHX29 could potentially be useful in treating HCMV disease and adds to the growing body of literature suggesting that DHX29 activity is a disease driver in multiple indications, including viral disease, inflammation, and cancer.

The siderophore yersiniabactin (Ybt) produced by a subset of intestinal adherent-invasive Escherichia coli (AIEC) drive intestinal fibrosis in murine model of Crohn's disease (CD). This is linked to the Ybt-induced disruption of host metal homeostasis and activation of the hypoxia-inducible factor 1-alpha (HIF-1α) in macrophages. Elevated glycolytic activity has been documented in both intestinal tissues and macrophages from patients with CD, indicating that metabolic reprogramming is a characteristic feature of the disease. Here, we show that HIF-1α stabilization by Ybt+ AIEC requires active host glycolysis. This effect is independent of Hif1a transcription and lipopolysaccharide stimulation and is not solely explained by intracellular bacterial load but instead relies on host metabolic activity. Mechanistically, Ybt+ AIEC activated the Akt-mTOR pathway to support HIF-1α translation. Inhibition of glycolysis suppressed this signaling axis, reducing HIF-1α translation and nuclear localization. Given the association between Ybt+ AIEC and fibrosis in CD, these findings suggest that targeting host glycolysis may limit AIEC-driven macrophage HIF-1α activation and fibrotic progression in CD patients.

The Orthoflavivirus genus (flaviviruses) includes globally significant arboviruses, which cycle between arthropod vectors (mosquitoes and ticks) and vertebrate hosts. In contrast, no-known-vector flaviviruses (NKVFVs) have been isolated from rodents and bats, but not arthropods, so are thought to spread by vector-independent routes. However, little is known about the host range and pathogenic mechanisms of these viruses. To evaluate NKVFV pathogenesis, we infected wild-type, Ifnar1 -/- , and Ifnar1 -/- Ifngr1 -/- mice by footpad inoculation with 12 NKVFVs: Entebbe bat virus (ENTV), Sokuluk virus (SOKV), Yokose virus (YOKV), Modoc virus (MODV), Apoi virus (APOIV), Jutiapa virus (JUTV), Sal Vieja virus (SVV), Dakar bat virus (DBV), Rio Bravo virus (RBV), Montana myotis leukoencephalitis virus (MMLV), Phnom Penh bat virus (PPBV), and Tamana bat virus (TABV). We compared these NKVFVs to the mosquito-borne Zika virus and Kedougou virus and to the tick-borne Langat virus and Kadam virus. We monitored disease signs and measured viremia. 7 NKVFVs (ENTV, SOKV, YOKV, MODV, APOIV, DBV, RBV) were virulent in Ifnar1 -/- mice, causing 100% lethality within 9 dpi, accompanied by viremia. All viruses tested were virulent in Ifnar1 -/- Ifngr1 -/- mice and produced greater viremia compared to Ifnar1 -/- mice. No viremia or disease signs were detected in wild-type mice. We further evaluated RBV, MMLV, and PPBV replication in mouse primary fibroblasts and bone marrow-derived macrophages, as well as in cell lines from three bat species. Altogether, our results provide new information about the virulence and replication phenotypes of NKVFVs, supporting future studies investigating NKVFV-specific and pan-flavivirus pathogenic mechanisms. IMPORTANCE:Flaviviruses that cause human disease are transmitted by mosquitoes and ticks (e.g. West Nile virus, yellow fever virus, tick-borne encephalitis virus). But there are related flaviviruses that are not known to infect arthropods, so are thought to spread by vector-independent routes (no-known-vector flaviviruses, NKVFVs). Not much is known about NKVFVs, but they provide an opportunity to understand flavivirus replication, tropism, and pathogenesis more broadly. We evaluated a panel of 12 NKVFVs for their ability to cause disease in mice with and without antiviral interferon responses as well as their replication in mouse cells. Our findings provide new information about these under-studied viruses and demonstrate which mouse models may be appropriate to use for further studies with NKVFVs.

The SARS-CoV-2 pandemic has underscored the urgent need for broad-spectrum antivirals in pandemic preparedness efforts. Nucleoside analogs targeting viral polymerases are often considered in this context. Here, we employ ensemble biochemical assays and single-molecule magnetic tweezers to characterize the detailed mechanism of action of the adenosine analog CMX521 (developed through Phase 1 clinical studies), a broad-spectrum antiviral against caliciviruses and coronaviruses, against SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). The triphosphate form of CMX521 is efficiently incorporated by RdRp, even against saturating ATP concentrations. Analog incorporation induces only a brief pause in nascent RNA synthesis. When embedded in the template strand, CMX521 causes the polymerase to stall ~9 s on average due to impaired uridine opposite incorporation. Multiple CMX521 residues in the template strand completely inhibit polymerase elongation. When the coronavirus polymerase is associated with the viral helicase, CMX521 strongly promotes copy-back RNA synthesis suggesting a second inhibitory mechanism for CMX521. Collectively, our findings establish a two-pronged mode of coronavirus polymerase inhibition by CMX521.

The papain-like protease (PLpro), one of two essential cysteine proteases in human coronaviruses, is indispensable for viral polyprotein processing and immune evasion through deubiquitination and deISGylation of host proteins. This dual role makes PLpro an attractive antiviral target, either as a monotherapy or in combination with other direct-acting antivirals. Guided by structural insights, we designed and synthesized covalent inhibitors that exploit two key binding elements: Glu167 in the α-cleft (Site I), which mediates recognition of ubiquitin and interferon-stimulated gene (ISG15), and the blocking loop 2 (BL2) hydrophobic groove, which stabilizes inhibitor binding and shapes selectivity. The optimized inhibitors exhibited potent enzymatic inhibition (IC50 = 25-100 nM), strong protein stabilization (ΔTm > 20 °C), and submicromolar antiviral activity in SARS-CoV-2-infected A549-ACE2 cells (EC50 = 130-350 nM), with no measurable cytotoxicity (CC50 > 10 μM). Lead compounds 8 (GL-612), 9 (GL-508), and 10 (GL-637) efficiently inactivated PLpro, with GL-637 achieving a kinact/KI > 18,000 M-1 s-1, and demonstrated selectivity by sparing host deubiquitinases USP7 and USP14 at concentrations up to 30 μM. Docking and mechanistic analyses revealed that cyclic amine-substituted benzamides reinforced electrostatic interactions with Glu167, while the (S)-3-fluoropyrrolidin-1-methyl-phenylthiophene scaffold anchored the BL2 groove, disrupting recognition of ubiquitin and ISG15 substrates. This dual engagement strategy improved covalent efficiency, strengthened binding affinity, and translated into superior antiviral potency compared with benchmark compd. 7 (4). Collectively, these findings establish GL-612, GL-508, and GL-637 as valuable structural templates for developing novel PLpro inhibitors that uniquely target Glu167 and the BL2 groove, providing a strong foundation for broad-spectrum coronavirus therapeutics against current and emerging coronaviruses.

Human cytomegalovirus (HCMV) is a β-herpesvirus that is ubiquitous in the human population. HCMV has the largest genome of the human herpesviruses and encodes an array of genes that affect pathogenesis in different cell types. Given the ability of HCMV to replicate in a range of cell types, investigators have begun to identify viral proteins required for cell type-specific replication. There are four proteins encoded by HCMV that are homologous to G protein-coupled receptors (GPCRs); these viral GPCRs (vGPCRs) are UL33, UL78, US27, and US28. In this study, we find that deletion of all four vGPCR genes severely attenuates HCMV replication in primary human salivary gland epithelial cells and ARPE-19 retinal epithelial cells, as evidenced by decreases in viral gene expression and virus production. Deletion of UL33 from the HCMV genome also results in a failure to efficiently replicate in epithelial cells, and this defect is manifested by decreased levels of viral gene expression and virus production. We find that, similar to US28, UL33 constitutively activates Gαq signaling to high levels in epithelial cells. We also find that UL33 transcription is more complicated than originally believed, and there is the potential for the virus to utilize various 5' UTRs to create novel UL33 proteins that are all capable of constitutive Gαq signaling. Taken together, these studies provide novel molecular and biochemical data regarding UL33 expression, subcellular localization, and signaling, and indicate that UL33 activity is essential for efficient HCMV replication in cells of epithelial origin. IMPORTANCE:Human cytomegalovirus (HCMV) replicates in a number of cell types and tissues in vivo, and the viral genes involved in cell type-specific replication are just beginning to be elucidated. The HCMV-encoded viral G protein-coupled receptors (vGPCRs) UL33, UL78, US27, and US28 are proving to play important roles in multiple aspects of HCMV replication, including the establishment and maintenance of latency. Here, we demonstrate that the HCMV vGPCRs and UL33, in particular, play an important role in driving lytic replication in cells of epithelial origin, including those derived from the salivary gland. This work expands on potential functions of the vGPCRs, will drive future studies to understand mechanistically how they affect tropism, and provides a new target for future therapeutics.

Broad-spectrum antiviral agent (BSAA) drugs are essential in the fight against viral, especially emerging, diseases. However, their development faces several challenges including insufficiently focused funding, fragmented research efforts and limited systematic data on BSAAs. In this review, we highlight these challenges, summarize accessible collections of data on antiviral compounds and viral targets, and explore possible directions toward the development of BSAAs. Whereas most existing BSAAs have been discovered serendipitously, we posit that rational, and feasible, design of direct-acting BSAAs should be focused on homologous targets found in viruses within a single viral family.

The human cytomegalovirus (HCMV) UL135 and UL138 genes play opposing roles regulating latency and reactivation in CD34+ human progenitor cells. We designed an RNA sequencing study to compare the transcriptional profile of HCMV infection in the presence and absence of these genes using the Tohoku Hospital Pediatrics-1 (THP-1) monocytic cell line model for latency. Relative to primary cell models, THP-1 cells offer the strength of a homogenous population that uniformly silences gene expression and will synchronously reexpress viral genes following stimulation to differentiate, which models early phases of viral reactivation. The loss of UL138 resulted in elevated levels of viral gene expression and in spontaneous adhesion of distinct cell populations that support HCMV gene expression and genome synthesis. The loss of UL135 resulted in diminished viral gene expression during an initial burst that occurs as latency is established and in no expression of eleven viral genes from the ULb' region even following differentiation and reexpression of viral genes. Transcriptional network analysis revealed host transcription factors (TFs) with potential to regulate the ULb' genes in coordination with pUL135. We show that the cellular TF peroxisome proliferator-activated receptor gamma binds to the viral genome and influences the expression of UL133-UL138 locus genes. Our results define roles for UL135 and UL138 in regulation of patterns of viral gene expression for the establishment of latency and reexpression of viral genes for reactivation and reveal insights into differentiation-linked mechanisms of transcriptional control of the HCMV genome.

Chikungunya is a mosquito-borne viral disease that causes fever and severe joint pain for which there is no direct acting drug treatments. Vinyl sulfone SGC-NSP2PRO-1 (3) was identified as a potent inhibitor of the nsP2 cysteine protease (nsP2pro) that reduced viral titer against infectious isolates of Chikungunya and other alphaviruses. The covalent warhead in 3 captured the active site C478 and inactivated nsP2pro with a kinact/Ki ratio of 5950 M–1 s–1. The vinyl sulfone 3 was inactive across a panel of 23 other cysteine proteases and demonstrated remarkable proteome-wide selectivity by two chemoproteomic methods. A negative control analog SGC-NSP2PRO-1N (4) retained the isoxazole core and covalent warhead but demonstrated > 100-fold decrease in enzyme inhibition. Both 3 and 4 were stable across a wide range of pH in solution and upon prolonged storage as solids. Vinyl sulfone 3 and its negative control 4 will find utility as high-quality chemical probes to study the role of the nsP2pro in cellular studies of alphaviral replication and virulence.

In mammalian cells, the addition of ADP-ribose to proteins and DNA plays well established roles in regulating cell function. Recently, RNA ribosylation was also found in mammalian cells under conditions of cell stress, though the functional consequences remain unclear. Here we find that infection with chikungunya virus, a positive strand RNA virus that causes frequent widespread epidemics, increases overall levels of RNA ribosylation in human fibroblasts. During infection, viral RNA is ribosylated by the PARP12 ribosyltransferase, which is counteracted by a virally-encoded. Increased viral RNA ribosylation resulted in decreased translation in cell-free systems and infected fibroblasts, and more rapid viral RNA decay. Further, ribosylated RNA potently induced the expression of antiviral host response genes. Together these data show the first functional consequences of RNA ribosylation in mammalian cells by showing that RNA ribosylation inhibits translation, decreases RNA stability, and creates a novel pathogen-associated molecular pattern (PAMP) that activates the host innate immune response. As macrodomains are present in multiple unrelated viruses, our data suggest RNA ribosylation is a novel component of cellular antiviral sensing pathway. These results also provide a starting point for defining functional roles for RNA ribosylation in other mammalian cell stress conditions beyond viral infection. ### Competing Interest Statement The authors have declared no competing interest. National Institute of Allergy and Infectious Diseases, U19 AI17129201, T32 AI007419 University of North Carolina at Chapel Hill, https://ror.org/0130frc33, RNA Discover Center (RDC) Collaborative Team Science Fellowship

Alphaviruses are mosquito-borne viruses that have caused significant outbreaks in the 21st century. Despite multiple recent outbreaks, there are no approved antiviral drugs to treat any alphavirus infection. Therefore, developing broadly acting antiviral drugs effective against multiple alphaviruses is necessary and could provide protection from both current and emerging alphavirus threats. A critical component of the alphavirus replication complex is non-structural protein 2 (nsP2), which is a multifunctional enzyme containing a helicase domain connected to a protease domain by a flexible linker. nsP2 functions as an ATP-dependent helicase, is conserved across the alphavirus genus, and is essential for virus replication, making it a promising target for development of alphavirus broad-acting antiviral drugs. Previous studies identified an enantioselective compound RA-0025298 that inhibited nsP2 ATPase activity and chikungunya virus CHIKV replication. Antiviral testing of RA-25298. against a diverse group of alphaviruses found broad activity except for Sindbis-like viruses. Using this information along with mutational profiling of virus passaged with RA-0025298 we identified the site of RA-0025298 action and confirmed the binding site via biophysical analyses. Finally, we found that the active enantiomer of RA-0025298 (SGC-NSP2hel-1) reduced viral loads in vivo and protected mice from tissue damage and disease caused by CHIKV infection. These findings further describe the mechanism of action of a first-in-class nsP2 helicase inhibitor with the potential for development as a broad spectrum drug for treating or preventing disease caused by current and emerging alphaviruses. One Sentence Summary This study describes the mechanism of action and in vivo efficacy of a first in class broadly acting inhibitor of alphavirus nsP2 helicase activity. ### Competing Interest Statement The authors have declared no competing interest.

Emerging viruses pose an ongoing threat to human health. While certain viral families are common sources of outbreaks, predicting the specific virus within a family that will cause the next outbreak or pandemic is not possible, creating an urgent need for broad spectrum antiviral drugs that are effective against a wide array of related viral pathogens. However, broad spectrum drug development is hindered by the lack of detailed knowledge of compound binding sites that are structurally and functionally conserved between viral family members and are essential for virus replication. To overcome this limitation, we developed an in silico approach that combines AI-driven protein structure prediction, computational fragment soaking, multiple sequence alignment, and protein stability calculations to identify highly conserved target sites that are both solvent-accessible and conserved. We applied this approach to the Togaviridae family, which includes emerging pandemic disease threats such as chikungunya and Venezuelan equine encephalitis virus for which there are currently no approved antiviral therapies. Our analysis identified multiple solvent accessible and structurally conserved pockets in the alphavirus non-structural protein 2 (nsP2) protease domain, which is essential for processing the viral replicase proteins. Mutagenesis of key solvent accessible and conserved residues identified novel pockets that are essential for the replication of multiple alphaviruses, validating these pockets as potential antiviral target sites for nsP2 inhibitors. These findings highlight the potential of artificial intelligence-informed modeling for revealing functionally conserved, accessible pockets as a means of identifying potential target binding sites for broadly active direct acting antivirals.

RA-0002034 (1) is a potent covalent inhibitor targeting the nsP2 cysteine protease. The species-dependent pharmacokinetics and metabolism of 1 were investigated to evaluate its therapeutic potential. Pharmacokinetic profiling revealed rapid clearance in mice, predominantly mediated by glutathione S-transferase (GST)-catalyzed conjugation. This metabolic liability contrasted with slower clearance observed in human hepatocytes and preclinical species, such as rats, dogs, and monkeys. Cross-species studies confirmed the dominance of GST-driven metabolism in mice, whereas oxidative pathways were more pronounced in dogs. Despite rapid systemic clearance, 1 achieved antiviral efficacy in mice, reducing chikungunya (CHIKV) viral loads in multiple tissues. These cross-species pharmacokinetic and metabolism studies support the continued evaluation of 1 as a potential antialphaviral therapeutic to further define the contribution of hepatic and non-hepatic GST metabolism to its clearance in humans.

Alphaviruses are mosquito-borne RNA viruses that pose a significant public health threat, with no FDA-approved antiviral therapeutics available. The nonstructural protein 2 helicase (nsP2hel) is an enzyme involved in unwinding dsRNA essential for alphavirus replication. This study reports the discovery and optimization of first-in-class oxaspiropiperidine inhibitors targeting nsP2hel. Structure-activity relationship (SAR) studies identified potent cyclic sulfonamide analogs with nanomolar antiviral activity against chikungunya virus (CHIKV). Biochemical analyses of nsP2hel ATPase and RNA unwindase activities showed these compounds act in a noncompetitive mode, suggesting that they are allosteric inhibitors. Viral resistance mutations mapped to nsP2hel and a fluorine-labeled analog exhibited direct binding to the protein by 19F NMR. The lead inhibitor, 2o, demonstrated broad-spectrum antialphaviral activity, reducing titers of CHIKV, Mayaro virus (MAYV), and Venezuelan equine encephalitis virus (VEEV). These findings support nsP2hel as a viable target for the development of broad-spectrum, direct-acting antialphaviral drugs.

Viruses have evolved unique strategies to circumvent host control of protein synthesis and enable viral protein synthesis in the face of the host response. Defining the factors that regulate viral messenger RNA (mRNA) translation is thus critical to understand how viruses replicate and cause disease. To identify factors that might regulate viral mRNA translation, we developed a technique for identifying proteins associated with a native RNA expressed from its endogenous promoter and genomic locus. This approach uses a guide RNA to target dCas13b fused to a biotin ligase domain to a specific RNA, where it covalently labels proteins in close proximity. Using this approach, we identified multiple proteins associated with transcripts encoding the human cytomegalovirus (HCMV) IE1 and IE2 proteins and found that several associated proteins positively or negatively regulate HCMV replication. We confirmed that one such protein, the cellular Y-box binding protein 1 (YBX1), binds to HCMV immediate early mRNAs and is required for efficient viral protein expression and virus replication. Ablating YBX1 expression reduced the association of HCMV immediate early mRNAs with polysomes, demonstrating a role for YBX1 as a positive regulator of viral RNA translation. These results provide a powerful tool for unraveling RNA-protein interactions that can be used in a wide range of biological processes and reveal a role for YBX1 as a critical regulator of HCMV immediate early gene expression.

Chikungunya virus (CHIKV) replication relies on the multifunctional nsP2 protein, making it an attractive target for antiviral drug discovery. Here, we report the resolution of oxaspiropiperidine 1, a first-in-class inhibitor of the CHIKV nsP2 RNA helicase (nsP2hel), into its constitutive enantiomers and characterization of their antiviral activity. The enantiomer (R)-1 exhibited potent inhibition of viral replication, nsP2hel ATPase activity, and dsRNA unwinding, while the (S)-1 enantiomer was >100-fold less active. The (R)-1 enantiomer also demonstrated a high selectivity for CHIKV over other RNA viruses and for nsP2hel over other RNA helicases. Direct binding of (R)-1 to the nsP2hel protein was confirmed by 19F NMR. Biophysical and structural studies revealed conformational polymorphism in the spirocyclic scaffold of (R)-1, suggesting a potential role of thermal mobility of the ligand in allosteric inhibition of nsP2hel. Collectively, these findings designate (R)-1 (RA-NSP2-1) as a high-quality chemical probe and (S)-1 (RA-NSP2-1N) as a negative control for probing the biology of alphavirus RNA helicases.

We describe the design, synthesis, and antialphaviral activity of spirodioxolane inhibitors targeting the alphavirus nsP2 helicase (nsP2hel). The spirodioxolanes are a new series of direct-acting antivirals that retain key molecular features required for inhibition of nsP2hel, including a highly substituted piperidine acetamide with its associated conformational isomerism and thermal mobility. Unlike the related oxaspiropiperidine nsP2hel inhibitors, the spirodioxolanes showed no enantioselectivity in their antiviral activity. The spirodioxolanes demonstrated antialphaviral activity against the Old World alphavirus Chikungunya virus, with some analogs also showing activity against the New World alphavirus Venezuelan equine encephalitis virus. Importantly, certain spirodioxolane analogs, such as 6b, maintained activity against viral mutants that displayed resistance to first-generation oxaspiropiperidine inhibitors, indicating their potential for optimization as a new class of broad-spectrum antialphaviral drugs.

The emergence of mosquito-borne alphaviruses that cause chronic arthritis or encephalitis underscores the urgent need for broad-spectrum antiviral therapeutics. The viral nsP2 cysteine protease, which is essential for alphavirus replication, is a promising antiviral target. Vinyl sulfone covalent inhibitors potently inhibit nsP2 protease but suffer from glutathione reactivity and species-dependent systemic clearance catalyzed by glutathione S-transferase. To address these liabilities, we explored alternative electrophilic warheads and identified acetamide inhibitors bearing N-alkyl sulfamate warheads with improved biochemical and antiviral profiles. 2-((5-(2-Ethoxyphenyl)-1H-pyrazol-3-yl)amino)-2-oxoethyl methylsulfamate emerged as a lead compound with potency against New and Old World alphaviruses, low GSH reactivity, and proteome-wide selectivity. Despite its promising antialphaviral activity, 2-((5-(2-ethoxyphenyl)-1H-pyrazol-3-yl)amino)-2-oxoethyl methylsulfamate exhibited rapid clearance due to hepatic glucuronidation. Structure-activity studies revealed modifications that improve metabolic stability while retaining antiviral activity. These findings introduce sulfamate acetamides as a new class of covalent nsP2 protease inhibitors and direct-acting pan-alphavirus drugs.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that infects the majority of the world's population. Lytic HCMV replication in immunocompromised individuals or neonates can lead to severe disease in multiple organ systems and even death. The establishment of lytic replication is driven by the first viral proteins expressed upon infection, the immediate early proteins, which play a key role in creating an intracellular environment conducive to virus replication. Two immediate early proteins, the functional orthologs pTRS1 and pIRS1, stimulate immediate early gene expression by suppressing antiviral PKR/eIF2α signaling and enhance the translation of viral mRNAs independent of PKR antagonism. To better understand the molecular functions of pTRS1, we used proximity labeling proteomics to identify proteins that interact with pTRS1 in infected cells. Multiple novel host and viral interactors were identified, including the catalytic subunits of the protein phosphatase 1 (PP1) holoenzyme. Mutations to a PP1 catalytic subunit known to disrupt binding to PP1 regulatory subunits decreased binding to pTRS1. pTRS1 immune complexes contained phosphatase activity, and inhibition of phosphatase activity in transfected or infected cells reversed the ability of pTRS1 to inhibit the antiviral kinase PKR. Depletion of individual PP1 catalytic subunits decreased virus replication and increased the phosphorylation of the PKR substrate eIF2α. Taken together, our data suggest potential novel functions for pTRS1 and define a novel role for PP1 as an antagonist of the antiviral PKR/eIF2α signaling axis during HCMV infection.IMPORTANCEThe human cytomegalovirus (HCMV) pTRS1 and pIRS1 proteins are critical regulators of HCMV replication, both during primary infection and during reactivation from viral latency. Thus, defining the molecular functions of pTRS1/pIRS1 is important for understanding the molecular events controlling HCMV replication and viral disease. These data provide new insights into potential pTRS1 functional roles, providing a starting point for others to understand new features of infected cell biology. Another important result of this study is the finding that specific protein phosphatase 1 (PP1) regulatory subunits are required to suppress PKR/eIF2α signaling, a critical cellular innate immune defense to viral infection. These data lay the groundwork for future efforts to discover therapeutics that disrupt pTRS1 interaction with PP1 allowing cellular defenses to limit HCMV replication and disease.

HCMV genes UL135 and UL138 play opposing roles regulating latency and reactivation in CD34+ human progenitor cells (HPCs). Using the THP-1 cell line model for latency and reactivation, we designed an RNA sequencing study to compare the transcriptional profile of HCMV infection in the presence and absence of these genes. The loss of UL138 results in elevated levels of viral gene expression and increased differentiation of cell populations that support HCMV gene expression and genome synthesis. The loss of UL135 results in diminished viral gene expression during an initial burst that occurs as latency is established and no expression of eleven viral genes from the ULb' region even following stimulation for differentiation and reactivation. Transcriptional network analysis revealed host transcription factors with potential to regulate the ULb' genes in coordination with pUL135. These results reveal roles for UL135 and UL138 in regulation of viral gene expression and potentially hematopoietic differentiation.