Lower Humoral and Cellular Immunity Following Asymptomatic SARS-CoV-2 Infection Compared to Symptomatic Infection in Education (The ACE Cohort).

PURPOSE: Asymptomatic SARS-CoV-2 infections were widely reported during the COVID-19 pandemic, acting as a hidden source of infection. Many existing studies investigating asymptomatic immunity failed to recruit true asymptomatic individuals. Thus, we conducted a longitudinal cohort study to evaluate humoral- and cell-mediated responses to infection and vaccination in well-defined asymptomatic young adults (the Asymptomatic COVID-19 in Education [ACE] cohort). METHODS: Asymptomatic testing services located at three UK universities identified asymptomatic young adults who were subsequently recruited with age- and sex-matched symptomatic and uninfected controls. Blood and saliva samples were collected after SARS-CoV-2 Wuhan infection, and again after vaccination. 51 participant's anti-spike antibody titres, neutralizing antibodies, and spike-specific T-cell responses were measured, against both Wuhan and Omicron B.1.1.529.1. RESULTS: Asymptomatic participants exhibited reduced Wuhan-specific neutralization antibodies pre- and post-vaccination, as well as fewer Omicron-specific neutralization antibodies post-vaccination, compared to symptomatic participants. Lower Wuhan and Omicron-specific IgG titres in asymptomatic individuals were also observed pre- and post-vaccination, compared to symptomatic participants. There were no differences in salivary IgA levels. Conventional flow cytometry analysis and multi-dimensional clustering analysis indicated unvaccinated asymptomatic participants had significantly fewer Wuhan-specific IL-2 secreting CD4+ CD45RA+ T cells and activated CD8+ T cells than symptomatic participants, though these differences dissipated after vaccination. CONCLUSIONS: Asymptomatic infection results in decreased antibody and T cell responses to further exposure to SARS-CoV-2 variants, compared to symptomatic infection. Post-vaccination, antibody responses are still inferior, but T cell immunity increases to match symptomatic subjects, emphasising the importance of vaccination to help protect asymptomatic individuals against future variants.

Spontaneous, persistent, T cell-dependent IFN-γ release in patients who progress to Long Covid.

After acute infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), a proportion of patients experience persistent symptoms beyond 12 weeks, termed Long Covid. Understanding the mechanisms that cause this debilitating disease and identifying biomarkers for diagnostic, therapeutic, and monitoring purposes are urgently required. We detected persistently high levels of interferon-γ (IFN-γ) from peripheral blood mononuclear cells of patients with Long Covid using highly sensitive FluoroSpot assays. This IFN-γ release was seen in the absence of ex vivo peptide stimulation and remains persistently elevated in patients with Long Covid, unlike the resolution seen in patients recovering from acute SARS-CoV-2 infection. The IFN-γ release was CD8+ T cell-mediated and dependent on antigen presentation by CD14+ cells. Longitudinal follow-up of our study cohort showed that symptom improvement and resolution correlated with a decrease in IFN-γ production to baseline levels. Our study highlights a potential mechanism underlying Long Covid, enabling the search for biomarkers and therapeutics in patients with Long Covid.

Atypical B cells and impaired SARS-CoV-2 neutralization following heterologous vaccination in the elderly.

Suboptimal responses to a primary vaccination course have been reported in the elderly, but there is little information regarding the impact of age on responses to booster third doses. Here, we show that individuals 70 years or older (median age 73, range 70-75) who received a primary two-dose schedule with AZD1222 and booster third dose with mRNA vaccine achieve significantly lower neutralizing antibody responses against SARS-CoV-2 spike pseudotyped virus compared with those younger than 70 (median age 66, range 54-69) at 1 month post booster. Impaired neutralization potency and breadth post third dose in the elderly is associated with circulating "atypical" spike-specific B cells expressing CD11c and FCRL5. However, when considering individuals who received three doses of mRNA vaccine, we did not observe differences in neutralization or enrichment in atypical B cells. This work highlights the finding that AdV and mRNA COVID-19 vaccine formats differentially instruct the memory B cell response.

Long COVID: what is known and what gaps need to be addressed.

INTRODUCTION: Long COVID is a chronic condition that follows after acute COVID-19 and is characterized by a wide range of persistent, cyclic symptoms. SOURCES OF DATA: PubMed search for publications featuring 'Long COVID' or 'post-acute sequelae of COVID-19'. AREAS OF AGREEMENT: Long COVID occurs frequently post-acute COVID-19, with a majority of people experiencing at least one symptom (such as cough, fatigue, myalgia, anosmia and dyspnoea) 4 weeks after infection. AREAS OF CONTROVERSY: The specific symptoms and the minimum duration of symptoms required to be defined as Long COVID. GROWING POINTS: There is a consistent reduction in Long COVID incidence amongst vaccinated individuals, although the extent of this effect remains unclear. AREAS TIMELY FOR DEVELOPING RESEARCH: There is an urgent need to understand the causes of Long COVID, especially extreme fatigue more than 6 months after infection. We must understand who is at risk and whether reinfections similarly risk Long COVID.

Reduced Incidence of Long Coronavirus Disease Referrals to the Cambridge University Teaching Hospital Long Coronavirus Disease Clinic.

Long coronavirus disease (COVID [LC]) constitutes a potential health emergency as millions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections lead to chronic symptoms. We must understand whether vaccines reduce LC because this has major implications for health policy. We report a 79% reduction in LC referrals correlating with vaccination in the United Kingdom.

Cytomegalovirus US28 regulates cellular EphA2 to maintain viral latency.

Cytomegalovirus (CMV) reactivation from latency following immune dysregulation remains a serious risk for patients, often causing substantial morbidity and mortality. Here, we demonstrate the CMV-encoded G protein-coupled receptor, US28, in coordination with cellular Ephrin receptor A2, attenuates mitogen-activated protein kinase signaling, thereby limiting viral replication in latently infected primary monocytes. Furthermore, treatment of latently infected primary monocytes with dasatinib, a Food and Drug Association-approved kinase inhibitor used to treat a subset of leukemias, results in CMV reactivation. These ex vivo data correlate with our retrospective analyses of the Explorys electronic health record database, where we find dasatinib treatment is associated with a significant risk of CMV-associated disease (odds ratio 1.58, P = 0.0004). Collectively, our findings elucidate a signaling pathway that plays a central role in the balance between CMV latency and reactivation and identifies a common therapeutic cancer treatment that elevates the risk of CMV-associated disease.

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.

Evidence of previous SARS-CoV-2 infection in seronegative patients with long COVID.

BACKGROUND: There is currently no consensus on the diagnosis, definition, symptoms, or duration of COVID-19 illness. The diagnostic complexity of Long COVID is compounded in many patients who were or might have been infected with SARS-CoV-2 but not tested during the acute illness and/or are SARS-CoV-2 antibody negative. METHODS: Given the diagnostic conundrum of Long COVID, we set out to investigate SARS-CoV-2-specific T cell responses in patients with confirmed SARS-CoV-2 infection and/or Long COVID from a cohort of mostly non-hospitalised patients. FINDINGS: We discovered that IL-2 release (but not IFN-γ release) from T cells in response to SARS-CoV-2 peptides is both sensitive (75% +/-13%) and specific (88%+/-7%) for previous SARS-CoV-2 infection >6 months after a positive PCR test. We identified that 42-53% of patients with Long COVID, but without detectable SARS-CoV-2 antibodies, nonetheless have detectable SARS-CoV-2 specific T cell responses. INTERPRETATION: Our study reveals evidence (detectable T cell mediated IL-2 release) of previous SARS-CoV-2 infection in seronegative patients with Long COVID. FUNDING: This work was funded by the Addenbrooke's Charitable Trust (900276 to NS), NIHR award (G112259 to NS) and supported by the NIHR Cambridge Biomedical Research Centre. NJM is supported by the MRC (TSF MR/T032413/1) and NHSBT (WPA15-02). PJL is supported by the Wellcome Trust (PRF 210688/Z/18/Z, 084957/Z/08/Z), a Medical Research Council research grant MR/V011561/1 and the United Kingdom Research and a Innovation COVID Immunology Consortium grant (MR/V028448/1).

Regulation of host and viral promoters during human cytomegalovirus latency via US28 and CTCF.

Viral latency is an active process during which the host cell environment is optimized for latent carriage and reactivation. This requires control of both viral and host gene promoters and enhancers often at the level of chromatin, and several viruses co-opt the chromatin organiser CTCF to control gene expression during latency. While CTCF has a role in the latencies of alpha- and gamma-herpesviruses, it was not known whether CTCF played a role in the latency of the beta-herpesvirus human cytomegalovirus (HCMV). Here, we show that HCMV latency is associated with increased CTCF expression and CTCF binding to the viral major lytic promoter, the major immediate early promoter (MIEP). This increase in CTCF binding is dependent on the virally encoded G protein coupled receptor, US28, and contributes to suppression of MIEP-driven transcription, a hallmark of latency. Furthermore, we show that latency-associated upregulation of CTCF represses expression of the neutrophil chemoattractants S100A8 and S100A9 which we have previously shown are downregulated during HCMV latency. As with downregulation of the MIEP, CTCF binding to the enhancer region of S100A8/A9 drives their suppression, again in a US28-dependent manner. Taken together, we identify CTCF upregulation as an important mechanism for optimizing latent carriage of HCMV at both the levels of viral and cellular gene expression.

CMV-encoded GPCR pUL33 activates CREB and facilitates its recruitment to the MIE locus for efficient viral reactivation.

Human cytomegalovirus (HCMV) establishes life-long latent infection in hematopoietic progenitor cells and circulating monocytes in infected individuals. Myeloid differentiation coupled with immune dysregulation leads to viral reactivation, which can cause severe disease and mortality. Reactivation of latent virus requires chromatin reorganization and the removal of transcriptional repressors in exchange for transcriptional activators. While some factors involved in these processes are identified, a complete characterization of the viral and cellular factors involved in their upstream regulation remains elusive. Herein, we show the HCMV-encoded G protein-coupled receptor (GPCR), UL33, is expressed during latency. Although this viral GPCR is not required to maintain latent infection, our data reveal UL33-mediated signaling is important for efficient viral reactivation. Additionally, UL33 signaling induces cellular cyclic AMP response element binding protein (CREB1, referred to here as CREB) phosphorylation, a transcription factor that promotes reactivation when recruited to the major immediate early (MIE) enhancer/promoter. Finally, targeted pharmacological inhibition of CREB activity reverses the reactivation phenotype of the UL33 signaling-deficient mutant. In sum, our data reveal UL33-mediated signaling functions to activate CREB, resulting in successful viral reactivation.

Activator protein-1 transactivation of the major immediate early locus is a determinant of cytomegalovirus reactivation from latency.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that latently infects hematopoietic cells and has the ability to reactivate when triggered by immunological stress. This reactivation causes significant morbidity and mortality in immune-deficient patients, who are unable to control viral dissemination. While a competent immune system helps prevent clinically detectable viremia, a portrait of the factors that induce reactivation following the proper cues remains incomplete. Our understanding of the complex molecular mechanisms underlying latency and reactivation continues to evolve. We previously showed the HCMV-encoded G protein-coupled receptor US28 is expressed during latency and facilitates latent infection by attenuating the activator protein-1 (AP-1) transcription factor subunit, c-fos, expression and activity. We now show AP-1 is a critical component for HCMV reactivation. Pharmacological inhibition of c-fos significantly attenuates viral reactivation. In agreement, infection with a virus in which we disrupted the proximal AP-1 binding site in the major immediate early (MIE) enhancer results in inefficient reactivation compared to WT. Concomitantly, AP-1 recruitment to the MIE enhancer is significantly decreased following reactivation of the mutant virus. Furthermore, AP-1 is critical for derepression of MIE-driven transcripts and downstream early and late genes, while immediate early genes from other loci remain unaffected. Our data also reveal MIE transcripts driven from the MIE promoter, the distal promoter, and the internal promoter, iP2, are dependent upon AP-1 recruitment, while iP1-driven transcripts are AP-1-independent. Collectively, our data demonstrate AP-1 binding to and activation of the MIE enhancer is a key molecular process controlling reactivation from latency.

The Requirement for US28 During Cytomegalovirus Latency Is Independent of US27 and US29 Gene Expression.

The ability to establish a latent infection with periodic reactivation events ensures herpesviruses, like human cytomegalovirus (HCMV), lifelong infection, and serial passage. The host-pathogen relationship throughout HCMV latency is complex, though both cellular and viral factors influence the equilibrium between latent and lytic infection. We and others have shown one of the viral-encoded G protein-coupled receptors, US28, is required for HCMV latency. US28 potentiates signals both constitutively and in response to ligand binding, and we previously showed deletion of the ligand binding domain or mutation of the G protein-coupling domain results in the failure to maintain latency similar to deletion of the entire US28 open reading frame (ORF). Interestingly, a recent publication detailed an altered phenotype from that previously reported, showing US28 is required for viral reactivation rather than latency, suggesting the US28 ORF deletion impacts transcription of the surrounding genes. Here, we show an independently generated US28-stop mutant, like the US28 ORF deletion mutant, fails to maintain latency in hematopoietic cells. Further, we found US27 and US29 transcription in each of these mutants was comparable to their expression during wild type infection, suggesting neither US28 mutant alters mRNA levels of the surrounding genes. Finally, infection with a US28 ORF deletion virus expressed US27 protein comparable to its expression following wild type infection. In sum, our new data strongly support previous findings from our lab and others, detailing a requirement for US28 during HCMV latent infection.

Interferon-Responsive Genes Are Targeted during the Establishment of Human Cytomegalovirus Latency.

Human cytomegalovirus (HCMV) latency is an active process which remodels the latently infected cell to optimize latent carriage and reactivation. This is achieved, in part, through the expression of viral genes, including the G-protein-coupled receptor US28. Here, we use an unbiased proteomic screen to assess changes in host proteins induced by US28, revealing that interferon-inducible genes are downregulated by US28. We validate that major histocompatibility complex (MHC) class II and two pyrin and HIN domain (PYHIN) proteins, myeloid cell nuclear differentiation antigen (MNDA) and IFI16, are downregulated during experimental latency in primary human CD14+ monocytes. We find that IFI16 is targeted rapidly during the establishment of latency in a US28-dependent manner but only in undifferentiated myeloid cells, a natural site of latent carriage. Finally, by overexpressing IFI16, we show that IFI16 can activate the viral major immediate early promoter and immediate early gene expression during latency via NF-κB, a function which explains why downregulation of IFI16 during latency is advantageous for the virus.IMPORTANCE Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus which infects 50 to 100% of humans worldwide. HCMV causes a lifelong subclinical infection in immunocompetent individuals but is a serious cause of mortality and morbidity in the immunocompromised and neonates. In particular, reactivation of HCMV in the transplant setting is a major cause of transplant failure and related disease. Therefore, a molecular understanding of HCMV latency and reactivation could provide insights into potential ways to target the latent viral reservoir in at-risk patient populations.

Human Cytomegalovirus Upregulates Expression of HCLS1 Resulting in Increased Cell Motility and Transendothelial Migration during Latency.

Human cytomegalovirus establishes a lifelong, latent infection in the human host and can cause significant morbidity and mortality, particularly, in immunocompromised individuals. One established site of HCMV latency and reactivation is in cells of the myeloid lineage. In undifferentiated myeloid cells, such as CD14+ monocytes, virus is maintained latently. We have recently reported an analysis of the total proteome of latently infected CD14+ monocytes, which identified an increase in hematopoietic lineage cell-specific protein (HCLS1). Here we show that this latency-associated upregulation of HCLS1 occurs in a US28-dependent manner and stabilizes actin structure in latently infected cells. This results in their increased motility and ability to transit endothelial cell layers. Thus, latency-associated increases in monocyte motility could aid dissemination of the latently infected reservoir, and targeting this increased motility could have an impact on the ability of latently infected monocytes to distribute to tissue sites of reactivation.

Monocytes Latently Infected with Human Cytomegalovirus Evade Neutrophil Killing.

One site of latency of human cytomegalovirus (HCMV) in vivo is in undifferentiated cells of the myeloid lineage. Although latently infected cells are known to evade host T cell responses by suppression of T cell effector functions, it is not known if they must also evade surveillance by other host immune cells. Here we show that cells latently infected with HCMV can, indeed, be killed by host neutrophils but only in a serum-dependent manner. Specifically, antibodies to the viral latency-associated US28 protein mediate neutrophil killing of latently infected cells. To address this mechanistically, a full proteomic screen was carried out on latently infected monocytes. This showed that latent infection downregulates the neutrophil chemoattractants S100A8/A9, thus suppressing neutrophil recruitment to latently infected cells. The ability of latently infected cells to inhibit neutrophil recruitment represents an immune evasion strategy of this persistent human pathogen, helping to prevent clearance of the latent viral reservoir.

Human cytomegalovirus G protein-coupled receptor US28 promotes latency by attenuating c-fos.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that undergoes latency in cells of the hematopoietic compartment, although the mechanisms underlying establishment and maintenance of latency remain elusive. We previously reported that the HCMV-encoded G protein-coupled receptor (GPCR) homolog US28 is required for successful latent infection. We now show that US28 protein (pUS28) provided in trans complements the US28Δ lytic phenotype in myeloid cells, suggesting that sustained US28 expression is necessary for long-term latency. Furthermore, expression of pUS28 at the time of infection represses transcription from the major immediate early promoter (MIEP) within 24 h. However, this repression is only maintained in the presence of continual pUS28 expression provided in trans Our data also reveal that pUS28-mediated signaling attenuates both expression and phosphorylation of cellular fos (c-fos), an AP-1 transcription factor subunit, to repress MIEP-driven transcription. AP-1 binds to the MIEP and promotes lytic replication, and in line with this we find that US28Δ infection results in an increase in AP-1 binding to the MIEP, compared with WT latent infection. Pharmacological inhibition of c-fos represses the MIEP during US28Δ infection to levels similar to those we observe during WT latent infection. Together, our data reveal that US28 is required for both establishment and long-term maintenance of HCMV latency, which is modulated, at least in part, by repressing functional AP-1 binding to the MIEP.

US28: HCMV's Swiss Army Knife.

US28 is one of four G protein coupled receptors (GPCRs) encoded by human cytomegalovirus (HCMV). The US28 protein (pUS28) is a potent signaling molecule that alters a variety of cellular pathways that ultimately alter the host cell environment. This viral GPCR is expressed not only in the context of lytic replication but also during viral latency, highlighting its multifunctional properties. pUS28 is a functional GPCR, and its manipulation of multiple signaling pathways likely impacts HCMV pathogenesis. Herein, we will discuss the impact of pUS28 on both lytic and latent infection, pUS28-mediated signaling and its downstream consequences, and the influence this viral GPCR may have on disease states, including cardiovascular disease and cancer. We will also discuss the potential for and progress towards exploiting pUS28 as a novel therapeutic to combat HCMV.

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.

Defining the Transcriptional Landscape during Cytomegalovirus Latency with Single-Cell RNA Sequencing.

Primary infection with human cytomegalovirus (HCMV) results in a lifelong infection due to its ability to establish latent infection, with one characterized viral reservoir being hematopoietic cells. Although reactivation from latency causes serious disease in immunocompromised individuals, our molecular understanding of latency is limited. Here, we delineate viral gene expression during natural HCMV persistent infection by analyzing the massive transcriptome RNA sequencing (RNA-seq) atlas generated by the Genotype-Tissue Expression (GTEx) project. This systematic analysis reveals that HCMV persistence in vivo is prevalent in diverse tissues. Notably, we find only viral transcripts that resemble gene expression during various stages of lytic infection with no evidence of any highly restricted latency-associated viral gene expression program. To further define the transcriptional landscape during HCMV latent infection, we also used single-cell RNA-seq and a tractable experimental latency model. In contrast to some current views on latency, we also find no evidence for any highly restricted latency-associated viral gene expression program. Instead, we reveal that latency-associated gene expression largely mirrors a late lytic viral program, albeit at much lower levels of expression. Overall, our work has the potential to revolutionize our understanding of HCMV persistence and suggests that latency is governed mainly by quantitative changes, with a limited number of qualitative changes, in viral gene expression.IMPORTANCE Human cytomegalovirus is a prevalent pathogen, infecting most of the population worldwide and establishing lifelong latency in its hosts. Although reactivation from latency causes significant morbidity and mortality in immunocompromised hosts, our molecular understanding of the latent state remains limited. Here, we examine the viral gene expression during natural and experimental latent HCMV infection on a transcriptome-wide level. In contrast to the classical views on herpesvirus latency, we find no evidence for a restricted latency-associated viral gene expression program. Instead, we reveal that latency gene expression largely resembles a late lytic viral profile, albeit at much lower levels of expression. Taken together, our data transform the current view of HCMV persistence and suggest that latency is mainly governed by quantitative rather than qualitative changes in viral gene expression.

Latency-Associated Expression of Human Cytomegalovirus US28 Attenuates Cell Signaling Pathways To Maintain Latent Infection.

Reactivation of human cytomegalovirus (HCMV) latent infection from early myeloid lineage cells constitutes a threat to immunocompromised or immune-suppressed individuals. Consequently, understanding the control of latency and reactivation to allow targeting and killing of latently infected cells could have far-reaching clinical benefits. US28 is one of the few viral genes that is expressed during latency and encodes a cell surface G protein-coupled receptor (GPCR), which, during lytic infection, is a constitutive cell-signaling activator. Here we now show that in monocytes, which are recognized sites of HCMV latency in vivo, US28 attenuates multiple cell signaling pathways, including mitogen-activated protein (MAP) kinase and NF-κB, and that this is required to establish a latent infection; viruses deleted for US28 initiate a lytic infection in infected monocytes. We also show that these monocytes then become potent targets for the HCMV-specific host immune response and that latently infected cells treated with an inverse agonist of US28 also reactivate lytic infection and similarly become immune targets. Consequently, we suggest that the use of inhibitors of US28 could be a novel immunotherapeutic strategy to reactivate the latent viral reservoir, allowing it to be targeted by preexisting HCMV-specific T cells.IMPORTANCE Human cytomegalovirus (HCMV) is a betaherpesvirus and a leading cause of morbidity and mortality among immunosuppressed individuals. HCMV can establish latent infection, where the viral genome is maintained in an infected cell, without production of infectious virus. A number of genes, including US28, are expressed by HCMV during latent infection. US28 has been shown to activate many cellular signaling pathways during lytic infection, promoting lytic gene expression and virus production. As such, the role of US28 remains unclear and seems at odds with latency. Here, we show that US28 has the opposite phenotype in cells that support latent infection-it attenuates cellular signaling, thereby maintaining latency. Inhibition of US28 with a small-molecule inhibitor causes HCMV latent infection to reactivate, allowing latently infected cells to be detected and killed by the immune system. This approach could be used to treat latent HCMV to clear it from human transplants.