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Human cytomegalovirus (HCMV) encodes multiple putative G protein-coupled receptors (GPCRs). US28 functions as a viral chemokine receptor and is expressed during both latent and lytic phases of virus infection. US28 actively promotes cellular migration, transformation, and plays a major role in mediating viral latency and reactivation; however, knowledge about the interaction partners involved in these processes is still incomplete. Herein, we utilized a proximity-dependent biotinylating enzyme (TurboID) to characterize the US28 interactome when expressed in isolation, and during both latent (CD34+ hematopoietic progenitor cells) and lytic (fibroblasts) HCMV infection. Our analyses indicate that the US28 signalosome converges with RhoA and EGFR signal transduction pathways, sharing multiple mediators that are major actors in processes such as cellular proliferation and differentiation. Integral members of the US28 signaling complex were validated in functional assays by immunoblot and small-molecule inhibitors. Importantly, we identified RhoGEFs as key US28 signaling intermediaries. In vitro latency and reactivation assays utilizing primary CD34+ hematopoietic progenitor cells (HPCs) treated with the small-molecule inhibitors Rhosin or Y16 indicated that US28 -RhoGEF interactions are required for efficient viral reactivation. These findings were recapitulated in vivo using a humanized mouse model where inhibition of RhoGEFs resulted in a failure of the virus to reactivate. Together, our data identifies multiple new proteins in the US28 interactome that play major roles in viral latency and reactivation, highlights the utility of proximity-sensor labeling to characterize protein interactomes, and provides insight into targets for the development of novel anti-HCMV therapeutics.

ABSTRACT 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. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns – conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we – a broad group of working virologists – seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.

Rhesus cytomegalovirus–based (RhCMV-based) vaccine vectors induce immune responses that protect ~60% of rhesus macaques (RMs) from SIVmac239 challenge. This efficacy depends on induction of effector memory–based (EM-biased) CD8+ T cells recognizing SIV peptides presented by major histocompatibility complex-E (MHC-E) instead of MHC-Ia. The phenotype, durability, and efficacy of RhCMV/SIV-elicited cellular immune responses were maintained when vector spread was severely reduced by deleting the antihost intrinsic immunity factor phosphoprotein 71 (pp71). Here, we examined the impact of an even more stringent attenuation strategy on vector-induced immune protection against SIV. Fusion of the FK506-binding protein (FKBP) degradation domain to Rh108, the orthologue of the essential human CMV (HCMV) late gene transcription factor UL79, generated RhCMV/SIV vectors that conditionally replicate only when the FK506 analog Shield-1 is present. Despite lacking in vivo dissemination and reduced innate and B cell responses to vaccination, Rh108-deficient 68-1 RhCMV/SIV vectors elicited high-frequency, durable, EM-biased, SIV-specific T cell responses in RhCMV-seropositive RMs at doses of ≥ 1 × 106 PFU. Strikingly, elicited CD8+ T cells exclusively targeted MHC-Ia–restricted epitopes and failed to protect against SIVmac239 challenge. Thus, Rh108-dependent late gene expression is required for both induction of MHC-E–restricted T cells and protection against SIV.

ABSTRACT Human cytomegalovirus (HCMV) is a highly prevalent beta-herpesvirus and a significant cause of morbidity and mortality following hematopoietic and solid organ transplant, as well as the leading viral cause of congenital abnormalities. A key feature of the pathogenesis of HCMV is the ability of the virus to establish a latent infection in hematopoietic progenitor and myeloid lineage cells. The study of HCMV latency has been hampered by difficulties in obtaining and culturing primary cells, as well as an inability to quantitatively measure reactivating virus, but recent advances in both in vitro and in vivo models of HCMV latency and reactivation have led to a greater understanding of the interplay between host and virus. Key differences in established model systems have also led to controversy surrounding the role of viral gene products in latency establishment, maintenance, and reactivation. This review will discuss the details and challenges of various models including hematopoietic progenitor cells, monocytes, cell lines, and humanized mice. We highlight the utility and functional differences between these models and the necessary experimental design required to define latency and reactivation, which will help to generate a more complete picture of HCMV infection of myeloid-lineage cells.

Viruses have evolved diverse strategies to manipulate cellular signaling pathways in order to promote infection and/or persistence. Human cytomegalovirus (HCMV) possesses a number of unique properties that allow the virus to alter cellular events required for infection of a diverse array of host cell types and long-term persistence. Of specific importance is infection of bone marrow derived and myeloid lineage cells, such as peripheral blood monocytes and CD34+ hematopoietic progenitor cells (HPCs) because of their essential role in dissemination of the virus and for the establishment of latency. Viral induced signaling through the Epidermal Growth Factor Receptor (EGFR) and other receptors such as integrins are key control points for viral-induced cellular changes and productive and latent infection in host organ systems. This review will explore the current understanding of HCMV strategies utilized to hijack cellular signaling pathways, such as EGFR, to promote the wide-spread dissemination and the classic life-long herpesvirus persistence.

Human cytomegalovirus (HCMV) causes serious disease in immunocompromised individuals and is a significant problem during transplantation. The virus can establish a latent infection in CD34 +

Viral dissemination is a key mechanism responsible for persistence and disease following human cytomegalovirus (HCMV) infection. Monocytes play a pivotal role in viral dissemination to organ tissue during primary infection and following reactivation from latency. For example, during primary infection, infected monocytes migrate into tissues and differentiate into macrophages, which then become a source of viral replication. In addition, because differentiated macrophages can survive for months to years, they provide a potential persistent infection source in various organ systems. We broadly note that there are three phases to infection and differentiation of HCMV-infected monocytes: (1) Virus enters and traffics to the nucleus through a virus receptor ligand engagement event that activates a unique signalsome that initiates the monocyte-to-macrophage differentiation process. (2) Following initial infection, HCMV undergoes a “quiescence-like state” in monocytes lasting for several weeks and promotes monocyte differentiation into macrophages. While, the initial event is triggered by the receptor-ligand engagement, the long-term cellular activation is maintained by chronic viral-mediated signaling events. (3) Once HCMV infected monocytes differentiate into macrophages, the expression of immediate early viral (IE) genes is detectable, followed by viral replication and long term infectious viral particles release. Herein, we review the detailed mechanisms of each phase during infection and differentiation into macrophages and discuss the biological significance of the differentiation of monocytes in the pathogenesis of HCMV.

Abstract The strict species specificity of Human Cytomegalovirus (HCMV) has impeded our understanding of antiviral adaptive immune responses in the context of a human immune system. We have previously shown that HCMV infection of human hematopoietic progenitor cells engrafted in immune deficient mice (huNSG) results in viral latency that can be reactivated following G-CSF treatment. In this study, we characterized the functional human adaptive immune responses in HCMV latently-infected huBLT (humanized Bone marrow-Liver-Thymus) mice. Following infection, huBLT mice generate human effector and central memory CD4+ and CD8+ T-cell responses reactive to peptides corresponding to both IE and pp65 proteins. Additionally, both HCMV specific IgM and IgG B-cell responses with the ability to neutralize virus were detected. These results indicate that the HCMV huBLT mouse model may provide a valuable tool to study viral latency and reactivation as well as evaluate HCMV vaccines and immune responses in the context of a functional human immune system.

ABSTRACT Human cytomegalovirus (HCMV) infection of CD34+ hematopoietic progenitor cells (CD34+ HPCs) provides a critical reservoir of virus in stem cell transplant patients, and viral reactivation remains a significant cause of morbidity and mortality. The HCMV chemokine receptor US28 is implicated in the regulation of viral latency and reactivation. To explore the role of US28 signaling in latency and reactivation, we analyzed protein tyrosine kinase signaling in CD34+ HPCs expressing US28. US28-ligand signaling in CD34+ HPCs induced changes in key regulators of cellular activation and differentiation. In vitro latency and reactivation assays utilizing CD34+ HPCs indicated that US28 was required for viral reactivation but not latency establishment or maintenance. Similarly, humanized NSG mice (huNSG) infected with TB40E-GFP-US28stop failed to reactivate upon treatment with granulocyte-colony-stimulating factor, but viral genome levels were maintained. Interestingly, HCMV-mediated changes in hematopoiesis during latency in vivo and in vitro was also dependent upon US28, as US28 directly promoted differentiation toward the myeloid lineage. To determine whether US28 constitutive activity and/or ligand-binding activity were required for latency and reactivation, we infected both huNSG mice and CD34+ HPCs in vitro with HCMV TB40E-GFP containing the US28-R129A mutation (no CA) or Y16F mutation (no ligand binding). TB40E-GFP-US28-R129A was maintained during latency and exhibited normal reactivation kinetics. In contrast, TB40E-GFP-US28-Y16F exhibited high levels of viral genome during latency and reactivation, indicating that the virus did not establish latency. These data indicate that US28 is necessary for viral reactivation and ligand binding activity is required for viral latency, highlighting the complex role of US28 during HCMV latency and reactivation. IMPORTANCE Human cytomegalovirus (HCMV) can establish latency following infection of CD34+ hematopoietic progenitor cells (HPCs), and reactivation from latency is a significant cause of viral disease and accelerated graft failure in bone marrow and solid-organ transplant patients. The precise molecular mechanisms of HCMV infection in HPCs are not well defined; however, select viral gene products are known to regulate aspects of latency and reactivation. The HCMV-encoded chemokine receptor US28, which binds multiple CC chemokines as well as CX3CR1, is expressed both during latent and lytic phases of the virus life cycle and plays a role in latency and reactivation. However, the specific timing of US28 expression and the role of ligand binding in these processes are not well defined. In this report, we determined that US28 is required for reactivation but not for maintaining latency. However, when present during latency, US28 ligand binding activity is critical to maintaining the virus in a quiescent state. We attribute the regulation of both latency and reactivation to the role of US28 in promoting myeloid lineage cell differentiation. These data highlight the dynamic and multifunctional nature of US28 during HCMV latency and reactivation.

Human cytomegalovirus (HCMV) infection is a serious complication in hematopoietic stem cell transplant (HSCT) recipients due to virus-induced myelosuppression and impairment of stem cell engraftment. Despite the clear clinical link between myelosuppression and HCMV infection, little is known about the mechanism(s) by which the virus inhibits normal hematopoiesis because of the strict species specificity and the lack of surrogate animal models. In this study, we developed a novel humanized mouse model system that recapitulates the HCMV-mediated engraftment failure after hematopoietic cell transplantation. We observed significant alterations in the hematopoietic populations in peripheral lymphoid tissues following engraftment of a subset of HCMV+ CD34+ hematopoietic progenitor cells (HPCs) within the transplant, suggesting that a small proportion of HCMV-infected CD34+ HPCs can profoundly affect HPC differentiation in the bone marrow microenvironment. This model will be instrumental to gain insight into the fundamental mechanisms of HCMV myelosuppression after HSCT and provides a platform to assess novel treatment strategies.

ABSTRACT The ability of human cytomegalovirus (HCMV) to reactivate from latent infection of hematopoietic progenitor cells (HPCs) is intimately linked to cellular differentiation. HCMV encodes UL7 that our group has shown is secreted from infected cells and induces angiogenesis. In this study, we show that UL7 is a ligand for Fms-like tyrosine kinase 3 receptor (Flt-3R), a well-known critical factor in HPC differentiation. We observed that UL7 directly binds Flt-3R and induces downstream signaling cascades, including phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways. Importantly, we show that UL7 protein induces differentiation of both CD34+ HPCs and CD14+ monocytes. Last, we show that an HCMV mutant lacking UL7 fails to reactivate in CD34+ HPCs in vitro as well as in humanized mice. These observations define the first virally encoded differentiation factor with significant implications not only for HCMV reactivation but also for alteration of the hematopoietic compartment in transplant patients. IMPORTANCE Human cytomegalovirus (HCMV) remains a significant cause of morbidity and mortality in allogeneic hematopoietic stem cell transplant recipients. CD34+ hematopoietic progenitor cells (HPCs) represent a critical reservoir of latent HCMV in the transplant population, thereby providing a source of virus for dissemination to visceral organs. HCMV reactivation has been linked to HPC/myeloid cellular differentiation; however, the mechanisms involved in these events are poorly understood at the molecular level. In this study, we show that a viral protein is a ligand for Fms-like tyrosine kinase 3 receptor (Flt-3R) and that the binding of HCMV UL7 to the Flt-3R triggers HPC and monocyte differentiation. Moreover, the loss of UL7 prevents viral reactivation in HPCs in vitro as well as in humanized mice. These observations define the first virally encoded differentiation factor with significant implications not only for HCMV reactivation but also for alteration of the hematopoietic compartment in transplant patients.

ABSTRACT Genetic engineering of cytomegalovirus (CMV) currently relies on generating a bacterial artificial chromosome (BAC) by introducing a bacterial origin of replication into the viral genome using in vivo recombination in virally infected tissue culture cells. However, this process is inefficient, results in adaptive mutations, and involves deletion of viral genes to avoid oversized genomes when inserting the BAC cassette. Moreover, BAC technology does not permit the simultaneous manipulation of multiple genome loci and cannot be used to construct synthetic genomes. To overcome these limitations, we adapted synthetic biology tools to clone CMV genomes in Saccharomyces cerevisiae. Using an early passage of the human CMV isolate Toledo, we first applied transformation-associated recombination (TAR) to clone 16 overlapping fragments covering the entire Toledo genome in Saccharomyces cerevisiae. Then, we assembled these fragments by TAR in a stepwise process until the entire genome was reconstituted in yeast. Since next-generation sequence analysis revealed that the low-passage-number isolate represented a mixture of parental and fibroblast-adapted genomes, we selectively modified individual DNA fragments of fibroblast-adapted Toledo (Toledo-F) and again used TAR assembly to recreate parental Toledo (Toledo-P). Linear, full-length HCMV genomes were transfected into human fibroblasts to recover virus. Unlike Toledo-F, Toledo-P displayed characteristics of primary isolates, including broad cellular tropism in vitro and the ability to establish latency and reactivation in humanized mice. Our novel strategy thus enables de novo cloning of CMV genomes, more-efficient genome-wide engineering, and the generation of viral genomes that are partially or completely derived from synthetic DNA. IMPORTANCE The genomes of large DNA viruses, such as human cytomegalovirus (HCMV), are difficult to manipulate using current genetic tools, and at this time, it is not possible to obtain, molecular clones of CMV without extensive tissue culture. To overcome these limitations, we used synthetic biology tools to capture genomic fragments from viral DNA and assemble full-length genomes in yeast. Using an early passage of the HCMV isolate Toledo containing a mixture of wild-type and tissue culture-adapted virus. we directly cloned the majority sequence and recreated the minority sequence by simultaneous modification of multiple genomic regions. Thus, our novel approach provides a paradigm to not only efficiently engineer HCMV and other large DNA viruses on a genome-wide scale but also facilitates the cloning and genetic manipulation of primary isolates and provides a pathway to generating entirely synthetic genomes.

ABSTRACT Human cytomegalovirus (HCMV), a betaherpesvirus, persists indefinitely in the human host through poorly understood mechanisms. The UL136 gene is carried within a genetic locus important to HCMV latency termed the UL133/8 locus, which also carries UL133, UL135, and UL138. Previously, we demonstrated that UL136 is expressed as five protein isoforms ranging from 33-kDa to 19-kDa, arising from alternative transcription and, likely, translation initiation mechanisms. We previously showed that the UL136 isoforms are largely dispensable for virus infection in fibroblasts, a model for productive virus replication. In our current work, UL136 has emerged as a complex regulator of HCMV infection in multiple contexts of infection relevant to HCMV persistence: in an endothelial cell (EC) model of chronic infection, in a CD34+ hematopoietic progenitor cell (HPC) model of latency, and in an in vivo NOD-scid IL2Rγcnull humanized (huNSG) mouse model for latency. The 33- and 26-kDa isoforms promote replication, while the 23- and 19-kDa isoforms suppress replication in ECs, in CD34+ HPCs, and in huNSG mice. The role of the 25-kDa isoform is context dependent and influences the activity of the other isoforms. These isoforms localize throughout the secretory pathway, and loss of the 33- and 26-kDa UL136 isoforms results in virus maturation defects in ECs. This work reveals an intriguing functional interplay between protein isoforms that impacts virus replication, latency, and dissemination, contributing to the overall role of the UL133/8 locus in HCMV infection. IMPORTANCE The persistence of DNA viruses, and particularly of herpesviruses, remains an enigma because we have not completely defined the viral and host factors important to persistence. Human cytomegalovirus, a herpesvirus, persists in the absence of disease in immunocompetent individuals but poses a serious disease threat to transplant patients and the developing fetus. There is no vaccine, and current therapies do not target latent reservoirs. In an effort to define the viral factors important to persistence, we have studied viral genes with no known viral replication function in contexts important to HCMV persistence. Using models relevant to viral persistence, we demonstrate opposing roles of protein isoforms encoded by the UL136 gene in regulating latent and replicative states of infection. Our findings reveal an intriguing interplay between UL136 protein isoforms and define UL136 as an important regulator of HCMV persistence.

ABSTRACT Persistent human cytomegalovirus (HCMV) infection has been linked to several diseases, including atherosclerosis, transplant vascular sclerosis (TVS), restenosis, and glioblastoma. We have previously shown that factors secreted from HCMV-infected cells induce angiogenesis and that this process is due, at least in part, to increased secretion of interleukin-6 (IL-6). In order to identify the HCMV gene(s) responsible for angiogenesis promotion, we constructed a large panel of replication-competent HCMV recombinants. One HCMV recombinant deleted for UL1 to UL10 was unable to induce secretion of factors necessary for angiogenesis. Fine mapping using additional HCMV recombinants identified UL7 as a viral gene required for production of angiogenic factors from HCMV-infected cells. Transient expression of pUL7 induced phosphorylation of STAT3 and ERK1/2 MAP kinases and production of proangiogenic factors, including IL-6. Addition of recombinant pUL7 to cells was sufficient for angiogenesis and was again associated with increased IL-6 expression. Analysis of the UL7 structure revealed a conserved domain similar to the immunoglobulin superfamily domain and related to the N-terminal V-like domain of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Our report therefore identifies UL7 as a novel HCMV-encoded molecule that is both structurally and functionally related to cellular CEACAM1, a proangiogenic factor highly expressed during vasculogenesis. IMPORTANCE A hallmark of cytomegalovirus (CMV) infection is its ability to modulate the host cellular machinery, resulting in the secretion of factors associated with long-term diseases such as vascular disorders and cancer. We previously demonstrated that HCMV infection alters the types and quantities of bioactive proteins released from cells (designated the HCMV secretome) that are involved in the promotion of angiogenesis and wound healing. A key proangiogenic and antiapoptotic factor identified from a proteomic-based approach was IL-6. In the present report, we show for the first time that HCMV UL7 encodes a soluble molecule that is a structural and functional homologue of the CEACAM1 proangiogenic cellular factor. This report thereby identifies a critical component of the HCMV secretome that may be responsible, at least in part, for the vascular dysregulation associated with persistent HCMV infection.

Clinical strains of HCMV encode 20 putative ORFs within a region of the genome termed ULb' that are postulated to encode functions related to persistence or immune evasion. We have previously identified ULb'-encoded pUL138 as necessary, but not sufficient, for HCMV latency in CD34+ hematopoietic progenitor cells (HPCs) infected in vitro. pUL138 is encoded on polycistronic transcripts that also encode 3 additional proteins, pUL133, pUL135, and pUL136, collectively comprising the UL133-UL138 locus. This work represents the first characterization of these proteins and identifies a role for this locus in infection. Similar to pUL138, pUL133, pUL135, and pUL136 are integral membrane proteins that partially co-localized with pUL138 in the Golgi during productive infection in fibroblasts. As expected of ULb' sequences, the UL133-UL138 locus was dispensable for replication in cultured fibroblasts. In CD34+ HPCs, this locus suppressed viral replication in HPCs, an activity attributable to both pUL133 and pUL138. Strikingly, the UL133-UL138 locus was required for efficient replication in endothelial cells. The association of this locus with three context-dependent phenotypes suggests an exciting role for the UL133-UL138 locus in modulating the outcome of viral infection in different contexts of infection. Differential profiles of protein expression from the UL133-UL138 locus correlated with the cell-type dependent phenotypes associated with this locus. We extended our in vitro findings to analyze viral replication and dissemination in a NOD-scid IL2Rγ(c) (null)-humanized mouse model. The UL133-UL138(NULL) virus exhibited an increased capacity for replication and/or dissemination following stem cell mobilization relative to the wild-type virus, suggesting an important role in viral persistence and spread in the host. As pUL133, pUL135, pUL136, and pUL138 are conserved in virus strains infecting higher order primates, but not lower order mammals, the functions encoded likely represent host-specific viral adaptations.

Human outbreaks of Ebola virus (EBOV) are a serious human health concern in Central Africa. Great apes (gorillas/chimpanzees) are an important source of EBOV transmission to humans due to increased hunting of wildlife including the 'bush-meat' trade. Cytomegalovirus (CMV) is an highly immunogenic virus that has shown recent utility as a vaccine platform. CMV-based vaccines also have the unique potential to re-infect and disseminate through target populations regardless of prior CMV immunity, which may be ideal for achieving high vaccine coverage in inaccessible populations such as great apes.We hypothesize that a vaccine strategy using CMV-based vectors expressing EBOV antigens may be ideally suited for use in inaccessible wildlife populations. To establish a 'proof-of-concept' for CMV-based vaccines against EBOV, we constructed a mouse CMV (MCMV) vector expressing a CD8+ T cell epitope from the nucleoprotein (NP) of Zaire ebolavirus (ZEBOV) (MCMV/ZEBOV-NP(CTL)). MCMV/ZEBOV-NP(CTL) induced high levels of long-lasting (>8 months) CD8+ T cells against ZEBOV NP in mice. Importantly, all vaccinated animals were protected against lethal ZEBOV challenge. Low levels of anti-ZEBOV antibodies were only sporadically detected in vaccinated animals prior to ZEBOV challenge suggesting a role, at least in part, for T cells in protection.This study demonstrates the ability of a CMV-based vaccine approach to protect against an highly virulent human pathogen, and supports the potential for 'disseminating' CMV-based EBOV vaccines to prevent EBOV transmission in wildlife populations.

The generation of humanized mouse models in which immune deficient mice are engrafted with human tissues allows for the direct in vivo investigation of human-restricted viruses. These humanized mouse models have been developed and improved over the past 30 years. It is now possible to achieve high levels of human cell engraftment producing human myeloid and lymphoid lineage cells. Humanized mouse models have been increasingly utilized in the study of human cytomegalovirus (HCMV), a human-specific beta-herpesvirus that infects myeloprogenitor cells and establishes a life-long latency in the infected host. This review focuses on the strengths and limitations of the current humanized mouse models used to study HCMV replication, pathogenesis and treatment. [ABSTRACT FROM AUTHOR]

Infection with human cytomegalovirus (HCMV) remains a significant cause of morbidity and mortality following hematopoietic stem cell transplant (HSCT) because of various hematologic problems, including myelosuppression. Here, we demonstrate that latently expressed HCMV miR-US5-2 downregulates the transcriptional repressor NGFI-A binding protein (NAB1) to induce myelosuppression of uninfected CD34+ hematopoietic progenitor cells (HPCs) through an increase in TGF-β production. Infection of HPCs with an HCMVΔmiR-US5-2 mutant resulted in decreased TGF-β expression and restoration of myelopoiesis. In contrast, we show that infected HPCs are refractory to TGF-β signaling as another HCMV miRNA, miR-UL22A, downregulates SMAD3, which is required for maintenance of latency. Our data suggest that latently expressed viral miRNAs manipulate stem cell homeostasis by inducing secretion of TGF-β while protecting infected HPCs from TGF-β-mediated effects on viral latency and reactivation. These observations provide a mechanism through which HCMV induces global myelosuppression following HSCT while maintaining lifelong infection in myeloid lineage cells. • Latent HCMV infection of stem cells induces the myelosuppressive cytokine TGF-β • HCMV miR-US5-2 targets the transcriptional repressor NAB1 to mediate TGF-β expression • HCMV miR-UL22A downregulates SMAD3 to block TGF-β signaling in the infected cell • Blocking TGF-β signaling is critical for HCMV latency and genome maintenance HCMV infection causes significant morbidity and mortality in stem cell transplant recipients due to virus-induced myelosuppression. Hancock et al. show that HCMV miRNAs produced during latent infection induce the expression of the myelosuppressive cytokine TGF-β while protecting the infected cell from TGF-β signaling for efficient viral latency. [ABSTRACT FROM AUTHOR]