Christine M. O'COnnor

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.

The innate immune response is the first line of defense against invading pathogens, including the betaherpesvirus human cytomegalovirus (CMV). The host’s innate response acts as the first line of defense, and CMV, like other viruses, has consequently evolved multiple mechanisms to manipulate host interferon (IFN) responses. DEAD-box helicase 41 (DDX41) is an intracellular dsDNA sensor that, upon activation by Bruton’s tyrosine kinase (BTK), triggers type I IFN production through the stimulator of interferon genes (STING) signaling pathway. Here, we show the activation of this signaling pathway during lytic CMV infection, wherein BTK, DDX41, and STING are activated through tyrosine phosphorylation, and both DDX41 and BTK interact with STING. Furthermore, CMV infection re-localizes a fraction of DDX41 from the nucleus to the cytoplasm. Here, DDX41 phosphorylation is attenuated, suggesting that cytoplasmic redistribution leads to a less active or inactive form. Additionally, a pool of DDX41 co-localizes in the virus assembly compartment (vAC) with the CMV tegument proteins, pp65 and pp71, each of which interact with DDX41 in immunoprecipitation assays. We further demonstrate the protective role of this signaling pathway, as treatment with the BTK inhibitor orelabrutinib attenuates DDX41 phosphorylation/activation and supports increased expression of viral proteins and virus replication. In sum, our work highlights the important role of BTK-DDX41-STING signaling in the innate immune response against CMV, which the virus subverts by attenuating its cytoplasmic activity, thereby diverting it from its typically protective function. IMPORTANCE: Human cytomegalovirus (CMV) is an ubiquitous pathogen that poses a significant threat to immunocompromised individuals, highlighting the critical role of innate immunity in controlling this viral infection. Despite extensive research, the complex mechanisms underlying innate immunity against CMV and the virus’s strategies for evading immune detection remain only partially understood. This study identifies the activation of the cellular BTK-DDX41-STING innate signaling axis during lytic CMV infection, which ultimately results in protective interferon responses. Our findings show that CMV infection triggers the cytoplasmic redistribution of the cellular protein, DDX41, leading to reduced phosphorylation and activity, thereby undermining its protective function. Additionally, pharmacological inhibition of BTK enhances viral protein expression and replication, highlighting the importance of this pathway in immune defense. Our work identifies BTK- and DDX41-dependent STING signaling as important for innate immune responses against CMV and further advances understanding of CMV’s manipulation of these responses.

Human cytomegalovirus (CMV) is a ubiquitous pathogen that establishes lifelong, latent infection in hematopoietic cells. Immune-competent individuals are usually asymptomatic for disease. However, immune dysregulation in latently infected individuals can result in viral reactivation, often causing further complications. Viral gene transcription during latency is restricted, although the CMV-encoded G protein-coupled receptor homologs, US28 and UL78, are expressed. We and others find US28 is critical for establishing and maintaining viral latency, in part, through regulating host cell signaling. How US28 switches from pro-latent to pro-lytic during reactivation, however, is unknown, though our findings herein reveal a role for UL78. Myeloid cells infected with a UL78 open reading frame deletion mutant maintain viral latency yet fail to efficiently reactivate. However, the UL78 G protein-coupling domain is not required for reactivation, suggesting UL78-mediated signaling is not critical for reactivation. Prior work revealed that UL78 and US28 interact, resulting in altered US28-mediated signaling. Additionally, we showed that US28 attenuates extracellular signal-regulated kinase (ERK) phosphorylation during latency, while ERK is phosphorylated upon reactivation; however, the mechanism underlying this switch is unknown. Thus, we hypothesized the UL78:US28 interaction is important for altering US28-mediated signaling upon viral reactivation. We find US28 and UL78 interact during lytic infection of fibroblasts and colocalize in myeloid cells upon their differentiation. Furthermore, reactivation in myeloid cells latently infected with wild-type virus results in upregulated ERK phosphorylation, while parallel cultures infected with the UL78-deficient virus fail to do so. Our data reveal the first function for UL78 in myeloid cells, where it influences cellular signaling during reactivation. IMPORTANCE: Cytomegalovirus (CMV) is a ubiquitous human herpesvirus, infecting the majority of the population worldwide. As with all herpesviruses, once an individual is infected with CMV, the virus remains in the person’s blood cells for life in a silenced state called latency, and this infection, for the most part, remains asymptomatic. When an infected individual’s immune system fails to function properly, however, CMV can become active (termed viral reactivation), which allows the virus to replicate and cause downstream disease. Our understanding of the cellular and viral factors that dictate this switch from silenced to activated remains incomplete. Here, we show a viral protein, UL78, is required for this switch. We find that UL78 helps to reshape cellular signaling, changing the cell environment from one that favors latency to one that supports reactivation. This highlights a new avenue for therapeutic intervention to prevent CMV reactivation and downstream disease.

Sex differences in cancer outcome, including that of glioblastoma (GBM), are shaped by biological, hormonal, and immunological factors and impact disease progression, treatment responses, survival, and tumor microenvironment (TME) interactions. Platelets regulate the immune responses and tumor progression in many cancers, but it is not clear how they contribute to these sex-based differences by affecting the dynamics of the TME. Here, we show that GBM patients exhibit heightened platelet reactivity driven by PAR4 signaling. In murine GBM models, both pharmacological inhibition of PAR4 using BMS986120 and genetic deletion of PAR4 significantly prolong survival in females but not males. This survival advantage is estrogen dependent: it is preserved in chromosomal male-hormonal female mice within the four-core genotype model and is rescued in ovariectomized mice treated with estrogen. The survival benefit is TME specific and is mediated by platelet-driven enhancement of CD8⁺ T cell infiltration into the tumor. Inhibition of platelet PAR4 signaling increases calcium signaling through an estrogen-dependent interaction between PAR4 and estrogen receptor β (ERβ)—a receptor interaction not previously described. PAR4-activated platelets within the TME suppress CD8⁺ T cell function, and depletion of CD8⁺ T cells abolishes both the tumor-induced platelet reactivity and the survival benefit conferred by PAR4 inhibition. These findings establish platelet-mediated PAR4 signaling as a critical driver of tumor progression and identify sex-specific immune responses as key to therapeutic efficacy.

Human cytomegalovirus (CMV) is a ubiquitous pathogen that establishes life-long, latent infection in hematopoietic cells. Immune-competent individuals are usually asymptomatic for disease. However, immune dysregulation in latently-infected individuals can result in viral reactivation, often causing further complications. Viral gene transcription during latency is restricted, although the CMV-encoded G-protein coupled receptor homologs, US28 and UL78, are expressed. We and others find US28 is critical for establishing and maintaining viral latency, in part, through regulating host cell signaling. How US28 switches from pro-latent to pro-lytic during reactivation, however, is unknown, though our findings herein reveal a role for UL78. Myeloid cells infected with a UL78 ORF deletion mutant maintain viral latency yet fail to efficiently reactivate. However, the UL78 G protein-coupling domain is not required for reactivation, suggesting UL78-mediated signaling is not critical for reactivation. Prior work revealed UL78 and US28 interact, resulting in altered US28-mediated signaling. Additionally, we showed US28 attenuates ERK phosphorylation during latency, while ERK is phosphorylated upon reactivation; however, the mechanism underlying this switch is unknown. Thus, we hypothesized the UL78:US28 interaction is important for altering US28-mediating signaling upon viral reactivation. We find US28 and UL78 interact during lytic infection of fibroblasts and colocalize in myeloid cells upon their differentiation. Further, reactivation in myeloid cells latently-infected with wild-type virus results in upregulated ERK phosphorylation, while parallel cultures infected with the UL78-deficient virus fail to do so. Our data reveal the first function for UL78 in myeloid cells, where it influences cellular signaling to switch from pro-latent to pro-lytic. Importance Cytomegalovirus (CMV) is a ubiquitous human herpesvirus, infecting the majority of the population worldwide. As with all herpesviruses, once an individual is infected with CMV, the virus remains in a person’s blood cells for their life in a silenced state called latency, and this infection, for the most part, remains asymptomatic. When an infected individual’s immune system fails to function properly, however, CMV can become active (termed viral reactivation), which allows the virus to replicate and cause downstream disease. Our understanding of the cellular and viral factors that dictate this switch from silenced to activated remains incomplete. Here, we show a viral protein, UL78, is required for this switch. We find UL78 helps to reshape cellular signaling, changing the cell environment from one that favors latency to one that instead supports reactivation. This highlights a new avenue for therapeutic intervention to prevent CMV reactivation and downstream disease.

Reactivation of latent human cytomegalovirus (CMV) can lead to severe complications in individuals with dysregulated immune systems. While antiviral therapies for CMV are approved, these compounds are limited by their toxicity and inability to specifically target the latent reservoir or prevent reactivation. Herein we show that CMV reactivation in Kasumi-3 cells, a CD34 ⁺ hematopoietic cell line, requires mitogen-activated protein kinase (MAPK) activation. Importantly, pharmacological inhibition of the MAPK signaling pathway, including MEK and ERK, restricts viral reactivation in Kasumi-3 cells. In sum, our findings show MAPK signaling is critical for CMV reactivation, revealing a potential avenue for therapeutic intervention to prevent viral reactivation and downstream pathogenesis that is often detrimental for immunosuppressed and immunocompromised patients.

Three-dimensional chromatin control of eukaryotic transcription is pivotal for regulating gene expression. This additional layer of epigenetic regulation is also utilized by DNA viruses, including herpesviruses. Dynamic, spatial genomic organization often involves looping of chromatin anchored by host-encoded CCCTC-binding factor (CTCF) and other factors, which control crosstalk between promoters and enhancers. Herein, we review the contribution of CTCF-mediated looping in regulating transcription during herpesvirus infection, with a specific focus on the betaherpesvirus, human cytomegalovirus (HCMV).

The innate immune response is the first line of defense against invading pathogens, including the betaherpesvirus, human cytomegalovirus (CMV). The host’s innate response acts as the first line of defense, and CMV, like other viruses, has consequently evolved multiple mechanisms to manipulate host interferon (IFN) responses. DEAD-box Helicase 41 (DDX41) is an intracellular dsDNA sensor that, upon activation by Bruton’s tyrosine kinase (BTK), triggers type I IFN production through the Stimulator of Interferon Genes (STING) signaling pathway. Here, we show the activation of this signaling pathway during lytic CMV infection, wherein BTK, DDX41, and STING are activated through tyrosine phosphorylation, and both DDX41 and BTK interact with STING. Further, CMV infection re-localizes DDX41 from the nucleus to the cytoplasm, where it localizes to the perinuclear virus assembly compartment (vAC). Here, DDX41 phosphorylation is attenuated, suggesting cytoplasmic redistribution leads to a less active or inactive form. Additionally, DDX41 co-localizes in the vAC with the CMV tegument proteins, pp65 and pp71, each of which interact with DDX41 in immunoprecipitation assays. We further demonstrate the protective role of this signaling pathway, as treatment with the BTK inhibitor, orelabrutinib, attenuates DDX41 phosphorylation/activation and supports increased expression of viral proteins and virus replication. In sum, our work highlights the important role of BTK-DDX41-STING signaling in the innate immune response against CMV, which the virus subverts by attenuating its cytoplasmic activity, thereby diverting it from its typically protective function. Importance: Human cytomegalovirus (CMV) is a ubiquitous pathogen that poses a significant threat to immunocompromised individuals, highlighting the critical role of innate immunity in controlling this viral infection. Despite extensive research, the complex mechanisms underlying innate immunity against CMV and the virus’s strategies for evading immune detection remain only partially understood. This study identifies the activation of the cellular BTK-DDX41-STING innate signaling axis during lytic CMV infection, which ultimately results in protective interferon responses. Our findings show that CMV infection triggers the cytoplasmic redistribution of the cellular protein, DDX41, leading to reduced phosphorylation and activity, thereby undermining its protective function. Additionally, pharmacological inhibition of BTK enhances viral protein expression and replication, highlighting the importance of this pathway in immune defense. Our work identifies BTK- and DDX41-dependent STING signaling as important for innate immune responses against CMV and further advances our understanding of CMV’s manipulation of these responses.