Human Cytomegalovirus Hijacks WD Repeat Domain 11 for Virion Assembly Compartment Formation and Virion Morphogenesis

Human cytomegalovirus (HCMV) has a large (∼235-kb) genome with over 200 predicted open reading frames and exploits numerous cellular factors to facilitate its replication. A key feature of HCMV-infected cells is the emergence of a distinctive membranous cytoplasmic compartment termed the virion assembly compartment (vAC). Here we report that host protein WD repeat domain 11 (WDR11) plays a key role in vAC formation and virion morphogenesis. We found that WDR11 was up-regulated at both mRNA and protein levels during HCMV infection. At the late stage of HCMV replication, WDR11 relocated to the vAC and co-localized with markers of the trans-Golgi network (TGN) and vAC. Depletion of WDR11 hindered HCMV-induced membrane reorganization of the Golgi and TGN, altered vAC formation, and impaired HCMV secondary envelopment and virion morphogenesis. Further, motifs critical for the localization of WDR11 in TGN were identified by alanine-scanning mutagenesis. Mutation of these motifs led to WDR11 mislocation outside of the TGN and loss of vAC formation. Taken together, these data indicate that host protein WDR11 is required for efficient viral replication at the stage of virion assembly, possibly by facilitating the remodeling of the endomembrane system for vAC formation and virion morphogenesis. Importance During the late phase of human cytomegalovirus (HCMV) infection, the endomembrane system is dramatically reorganized, resulting in the formation of a unique structure termed the virion assembly compartment (vAC), which is critical for the assembly of infectious virions. The mechanism of HCMV-induced vAC formation is still not fully understood. In this report, we identified a host factor, WDR11, that plays an important role in vAC formation. Our findings argue that WDR11 contributes to the relocation of the Golgi and trans-Golgi network to the vAC, a membrane reorganization process that appears to be required for efficient virion maturation. The present work provides new insights into the vAC formation and HCMV virion morphogenesis and a potential novel target for anti-viral treatment.

PPAR Ligands Induce Antiviral Effects Targeting Perturbed Lipid Metabolism during SARS-CoV-2, HCV, and HCMV Infection

The manipulation of host metabolisms by viral infections has been demonstrated by several studies, with a marked influence on the synthesis and utilization of glucose, nucleotides, fatty acids, and amino acids. The ability of virus to perturb the metabolic status of the infected organism is directly linked to the outcome of the viral infection. A great deal of research in recent years has been focusing on these metabolic aspects, pointing at modifications induced by virus, and suggesting novel strategies to counteract the perturbed host metabolism. In this review, our attention is turned on PPARs, nuclear receptors controlling multiple metabolic actions, and on the effects played by PPAR ligands during viral infections. The role of PPAR agonists and antagonists during SARS-CoV-2, HCV, and HCMV infections will be analyzed.

Metabolic Reprogramming of Glioblastoma Cells during HCMV Infection Induces Secretome-Mediated Paracrine Effects in the Microenvironment

Glioblastoma (GBM) is an aggressive primary central nervous system neoplasia with limited therapeutic options and poor prognosis. Following reports of cytomegalovirus (HCMV) in GBM tumors, the anti-viral drug Valganciclovir was administered and found to significantly increase the longevity of GBM patients. While these findings suggest a role for HCMV in GBM, the relationship between them is not clear and remains controversial. Treatment with anti-viral drugs may prove clinically useful; however, their results do not explain the underlying mechanism between HCMV infection and GBM progression. We hypothesized that HCMV infection would metabolically reprogram GBM cells and that these changes would allow for increased tumor progression. We infected LN-18 GBM cells and employed a Seahorse Bioanalyzer to characterize cellular metabolism. Increased mitochondrial respiration and glycolytic rates were observed following infection. These changes were accompanied by elevated production of reactive oxygen species and lactate. Due to lactate’s numerous tumor-promoting effects, we examined the impact of paracrine signaling of HCMV-infected GBM cells on uninfected stromal cells. Our results indicated that, independent of viral transmission, the secretome of HCMV-infected GBM cells was able to alter the expression of key metabolic proteins and epigenetic markers. This suggests a mechanism of action where reprogramming of GBM cells alters the surrounding tumor microenvironment to be permissive to tumor progression in a manner akin to the Reverse-Warburg Effect. Overall, this suggests a potential oncomodulatory role for HCMV in the context of GBM.

Mapping and Characterization of HCMV-Specific Unconventional HLA-E-Restricted CD8 T Cell Populations and Associated NK and T Cell Responses Using HLA/Peptide Tetramers and Spectral Flow Cytometry

HCMV drives complex and multiple cellular immune responses, which causes a persistent immune imprint in hosts. This study aimed to achieve both a quantitative determination of the frequency for various anti-HCMV immune cell subsets, including CD8 T, γδT, NK cells, and a qualitative analysis of their phenotype. To map the various anti-HCMV cellular responses, we used a combination of three HLApeptide tetramer complexes (HLA-EVMAPRTLIL, HLA-EVMAPRSLLL, and HLA-A2NLVPMVATV) and antibodies for 18 surface markers (CD3, CD4, CD8, CD16, CD19, CD45RA, CD56, CD57, CD158, NKG2A, NKG2C, CCR7, TCRγδ, TCRγδ2, CX3CR1, KLRG1, 2B4, and PD-1) in a 20-color spectral flow cytometry analysis. This immunostaining protocol was applied to PBMCs isolated from HCMV- and HCMV+ individuals. Our workflow allows the efficient determination of events featuring HCMV infection such as CD4/CD8 ratio, CD8 inflation and differentiation, HCMV peptide-specific HLA-EUL40 and HLA-A2pp65CD8 T cells, and expansion of γδT and NK subsets including δ2-γT and memory-like NKG2C+CD57+ NK cells. Each subset can be further characterized by the expression of 2B4, PD-1, KLRG1, CD45RA, CCR7, CD158, and NKG2A to achieve a fine-tuned mapping of HCMV immune responses. This assay should be useful for the analysis and monitoring of T-and NK cell responses to HCMV infection or vaccines.

Maternal Fc-mediated non-neutralizing antibody responses correlate with protection against congenital human cytomegalovirus infection

Human cytomegalovirus (HCMV) is the most common congenital infection and a leading cause of stillbirth, neurodevelopmental impairment, and pediatric hearing loss worldwide. Development of a maternal vaccine or therapeutic to prevent congenital infection has been hindered by limited knowledge of the immune responses that protect against placental HCMV transmission in maternal primary and nonprimary infection. To identify protective antibody responses, we measured anti-HCMV IgG binding and anti-viral functions in maternal and cord blood sera from HCMV transmitting (n=41) and non- transmitting (n=40) mother-infant dyads identified via a large U.S.-based public cord blood bank. In a predefined immune correlate analysis, maternal monocyte-mediated antibody-dependent cellular phagocytosis (ADCP) and high avidity IgG binding to HCMV envelope glycoproteins were associated with decreased risk of congenital HCMV infection. Moreover, HCMV-specific IgG engagement of FcγRI and FcγRIIA, which mediate non-neutralizing antibody responses, was enhanced in non-transmitting mother-infant dyads and strongly correlated with ADCP. These findings suggest that Fc effector functions including ADCP protect against placental HCMV transmission. Taken together, our data indicate that future active and passive immunization strategies to prevent congenital HCMV infection should target Fc-mediated non-neutralizing antibody responses.

The impact of murine cytomegalovirus (mCMV) route and age at infection upon virus spread, immune responses and the establishment of latency

Cytomegalovirus (CMV) is a ubiquitous human virus, which establishes a characteristic lifetime infection in its host. Murine CMV (mCMV) is a widely-used infection model that has been employed to investigate the nature and extent of CMV's contribution to inflammatory, immunological, and health disturbances in humans. In an effort to assess the role of route and age in modeling hCMV infection in mice, we have performed a comparative analysis of two common experimental modes of infection (intraperitoneal and intranasal) at two different clinically relevant ages (4 weeks, or prepubescent childhood equivalent, and 12 weeks, or young postpubescent adult). We found that while both routes of infection led to similar early viral loads, differential activation of several parameters of innate immune function were observed. In particular, younger, prepubescent mice exhibited the strongest NK activation in the blood in response to i.p. infection, with this trend holding true in NK cells expressing the mCMV-specific receptor Ly49H. Moreover, i.p. infected animals accumulated a larger amount of anti-mCMV IgG and experienced a greater expansion of both acute and latent phase CD8+ T cells. This was especially true for young postpubescent mice, further illustrating a distinction in the bloodborne immune response across not only infection routes, but also ages. These results may be important in the understanding of how a more physiologically applicable model of CMV influences immunity, inflammation, and health over the lifespan.

Impact of new cyclooxygenase 2 inhibitors on human cytomegalovirus replication in vitro

Background Human cytomegalovirus (HCMV) is involved in complications on immunocompromised patients. Current therapeutics are associated with several drawbacks, such as nephrotoxicity. Purpose: As HCMV infection affects inflammation pathways, especially prostaglandin E2 (PGE2) production via cyclooxygenase 2 enzyme (COX-2), we designed 2'-hydroxychalcone compounds to inhibit human cytomegalovirus. Study design We first selected the most efficient new synthetic chalcones for their effect against COX-2-catalyzed PGE2. Study sample Among the selected compounds, we assessed the antiviral efficacy against different HCMV strains, such as the laboratory strain AD169 and clinical strains (naïve or multi-resistant to conventional drugs) and toxicity on human cells. Results The most efficient and less toxic compound (chalcone 7) was tested against HCMV in combination with other antiviral molecules: artesunate (ART), baicalein (BAI), maribavir (MBV), ganciclovir (GCV), and quercetin (QUER) using Compusyn software. Association of chalcone 7 with MBV and BAI is synergistic, antagonistic with QUER, and additive with GCV and ART. Conclusion These results provide a promising search path for potential bitherapies against HCMV.

Human Cytomegalovirus modifies placental small extracellular vesicle secretion and composition towards a proviral phenotype to enhance infection of fetal recipient cells

Although placental small extracellular vesicles (sEVs) are extensively studied in the context of pregnancy, little is known about their role during human cytomegalovirus (hCMV) congenital infection, especially at the beginning of pregnancy. In this study, we examined the consequences of hCMV infection on sEVs production and composition using an immortalized human cytotrophoblast cell line derived from first trimester placenta. By combining complementary approaches of biochemistry, imaging techniques and quantitative proteomic analysis, we showed that hCMV infection increased the yield of sEVs produced by cytotrophoblasts and modified their protein composition towards a proviral phenotype. We further demonstrated that sEVs secreted by hCMV-infected cytotrophoblasts potentiated infection in naive recipient cells of fetal origin, including neural stem cells. Importantly, the enhancement of hCMV infection was also observed with sEVs prepared from either an ex vivo model of infected histocultures from early placenta or from the amniotic fluid of patients naturally infected by hCMV at the beginning of pregnancy. Based on these findings, we propose that placental sEVs could be key actors favoring viral dissemination to the fetal brain during hCMV congenital infection.

Challenging the Conventional Interpretation of HCMV Seronegativity

The majority of adults in the world (around 83%) carry antibodies reactive with HCMV and are thought to retain inactive or latent infections lifelong. The virus is transmitted via saliva, so infection events are likely to be common. Indeed, it is hard to imagine a life without exposure to HCMV. From 45 seronegative individuals (13 renal transplant recipients, 32 healthy adults), we present seven cases who had detectable HCMV DNA in their blood and/or saliva, or a CMV-encoded homologue of IL-10 (vIL-10) in their plasma. One case displayed NK cells characteristic of CMV infection before her HCMV DNA became undetectable. In other cases, the infection may persist with seroconversion blocked by vIL-10. Future research should seek mechanisms that can prevent an individual from seroconverting despite a persistent HCMV infection, as HCMV vaccines may not work well in such people.

The Endothelin Receptor Antagonist Macitentan Inhibits Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) infection is an important cause of morbidity and mortality in immunocompromised patients and a major etiological factor for congenital birth defects in newborns. Ganciclovir and its pro-drug valganciclovir are the preferred drugs in use today for prophylaxis and treatment of viremic patients. Due to long treatment times, patients are at risk for developing viral resistance to ganciclovir and to other drugs with a similar mechanism of action. We earlier found that the endothelin receptor B (ETBR) is upregulated during HCMV infection and that it plays an important role in the life cycle of this virus. Here, we tested the hypothesis that ETBR blockade could be used in the treatment of HCMV infection. As HCMV infection is specific to humans, we tested our hypothesis in human cell types that are relevant for HCMV pathogenesis; i.e., endothelial cells, epithelial cells and fibroblasts. We infected these cells with HCMV and treated them with the ETBR specific antagonist BQ788 or ETR antagonists that are approved by the FDA for treatment of pulmonary hypertension; macitentan, its metabolite ACT-132577, bosentan and ambrisentan, and as an anti-viral control, we used ganciclovir or letermovir. At concentrations expected to be relevant in vivo, macitentan, ACT-132577 and BQ788 effectively inhibited productive infection of HCMV. Of importance, macitentan also inhibited productive infection of a ganciclovir-resistant HCMV isolate. Our results suggest that binding or signaling through ETBR is crucial for viral replication, and that selected ETBR blockers inhibit HCMV infection.