Vargab Baruah

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.

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.

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.

Fulminant hepatic failure (FHF) is a lethal manifestation of hepatitis A virus (HAV) infection, whose underlying mechanisms are poorly understood. We aimed to evaluate the importance of the modulation of RANTES‐CCR5 signalling axis and its immunomodulatory effects in directing hepatitis A disease pathogenesis using an in‐silico, in‐vitro and patient cohort based approach.In‐silico interaction studies were performed using computation approaches with suitable software. Differential expression of relevant cytokines and immune cell markers were studied using qRT‐PCR, ELISA and flow‐cytometry‐based methods. In HepG2 cell line, we studied inflammatory responses and susceptibility to HAV infection following RANTES stimulation and antibody blockade of CCR5.The HAV‐VP3 region exhibited high interaction in CCR5: HAV complexes. RANTES levels were significantly increased in FHF cases. A reduced monocyte and T‐cell activation was observed in FHF cases. RANTES expression inversely correlated with viremia but positively correlated with pro‐inflammatory responses. Hyper Th1‐biased immune responses, marked by high IL‐12/IL‐10 ratio were observed in FHF cases, which were also characterized by upregulated TNF‐α expression and reduced IFN‐γ expression. In‐vitro, RANTES was protective against HAV infection, but resulted in upregulated TNF‐α expression. Although viral load increased upon regulation of inflammatory responses by CCR5 blocking, it was still significantly lower compared to control HAV‐infected cells.Our study suggests the importance of RANTES‐CCR5 signalling and linked‐immunomodulation in HAV disease pathogenesis, as well as highlights the utility of CCR5 antagonists as a risk‐reduction strategy in FHF patients. Our findings therefore have important implications for the management of high‐risk HAV infections.

The 2019 novel coronavirus (2019‐nCoV) outbreak has caused a large number of deaths with thousands of confirmed cases worldwide, especially in East Asia. This study took an immunoinformatics approach to identify significant cytotoxic T lymphocyte (CTL) and B cell epitopes in the 2019‐nCoV surface glycoprotein. Also, interactions between identified CTL epitopes and their corresponding major histocompatibility complex (MHC) class I supertype representatives prevalent in China were studied by molecular dynamics simulations. We identified five CTL epitopes, three sequential B cell epitopes and five discontinuous B cell epitopes in the viral surface glycoprotein. Also, during simulations, the CTL epitopes were observed to be binding MHC class I peptide‐binding grooves via multiple contacts, with continuous hydrogen bonds and salt bridge anchors, indicating their potential in generating immune responses. Some of these identified epitopes can be potential candidates for the development of 2019‐nCoV vaccines.

We aimed to evaluate the significance of the RANTES‐CCR5 axis and resulting immunomodulatory status in Dengue pathogenesis involving a Guwahati, India based population where Dengue cases have increased alarmingly. An increased CC‐chemokine receptor type 5 (CCR5) messenger RNA expression and CCR5 positive cell count profile was observed in Dengue cases, the highest being in severe cases. CCR5 ligand RANTES expression was significantly decreased in Dengue cases and inversely correlated with Dengue viremia fold change in severe cases. Monocytes are involved in Dengue virus homing and replication. Its levels and activation profile were higher in Dengue cases. A hyper Th1‐biased immunomodulatory profile with upregulated tumor necrosis factor‐α levels, and downregulated expression of antiviral cytokine interferon‐γ and key regulatory Th2 anti‐inflammatory cytokine interleukin 10 was observed in severe Dengue cases compared with mild Dengue cases and controls. The results, therefore, suggest the significance of RANTES‐CCR5 axis deregulation and resulting altered immunomodulation in Dengue pathogenesis, and holds prognostic and therapeutic significance.