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PurposeMethodsResultsConclusionTo explore the potential risk factors for the occurrence of human cytomegalovirus (HCMV) retinitis (CMVR) in allogeneic hematopoietic stem cell transplantation (allo-HSCT) patients.This is a retrospective, nested case-control study conducted in hematological patients with CMVR who underwent allo-HSCT. Patients diagnosed with CMVR after allo-HSCT were included as the case group, and those without CMVR were matched by a ratio of 1:2 and were recruited as controls. We selected 19 pre- and post-transplant indicators for univariate analysis between the cases and controls, and then Logistic regression analysis was used to calculate the odds ratio (OR) and 95% confidence intervals (CI) for exploration of risk factors of the CMVR occurrence.A total of 1308 allo-HSCT patients from January 1, 2020 to July 31, 2023 were analyzed, and 27 patients were diagnosed CMVR with a median onset time of 222 days after transplantation. In univariate analysis, donors of stem cells source, HLA-match types (including matched sibling donor, haploidentical donor, and unrelated donor), post-transplant Epstein-Barr virus (EBV) viremia, platelet implantation time, and serostatus of CMV-IgG were more easily to develop CMVR than controls (p < 0.001, p = 0.003, p < 0.001, p = 0.032, p = 0.038, respectively). Multivariate logistic regression analysis showed that stem cells source (OR 7.823, 95% CI 1.759-34.800), HLA-match types (OR 7.452, 95% CI 1.099-50.542), and post-transplant EBV infection (OR 7.510, 95% CI 1.903-29.640) were positively associated with the onset of CMVR.Stem cells derived from bone marrow and peripheral blood, HLA-match types, and post-transplant EBV viremia are important risk predictors of CMVR in allo-HSCT patients. These results suggest that clinicians should pay more attention to these indicators when formulating preventive measures pre- and post-transplant. [ABSTRACT FROM AUTHOR]

Cytomegalovirus (CMV)-seropositive adults have large T cell responses to a wide range of CMV proteins; these responses have been associated with chronic inflammation and frailty in people with or without HIV infection. We analyzed the relationships between chronic HIV infection, frailty, and the breadth and polyfunctionality of CD4 and CD8 T cell responses to CMV. Peripheral blood mononuclear cells from 42 men (20 without HIV and 22 with virologically suppressed HIV) in the Multicenter AIDS Cohort Study (MACS) were stimulated with peptide pools spanning 19 CMV open reading frames (ORFs). As measured by flow cytometry and intracellular cytokine staining for IFN-γ, TNF-α, and IL-2, CD8 T cells from men with HIV responded to significant ly more CMV ORFs than those from men without HIV. This was primarily due to a broader response to ORFs that are expressed during the late phase of CMV replication. The number of ORFs to which a participant's T cells responded was positively correlated with the sum of all that individual's T cell responses; these correlations were weaker in men with than without HIV. Polyfunctional CMV-specific CD4 responses (production of more than one cytokine) were significantly lower in men with than without HIV. Frailty status did not substantially affect the breadth or magnitude of the CMV-specific T cell responses. These results suggest that immune control of CMV infection is affected more by chronic HIV infection than by frailty. The differences between men with and without HIV were similar to those reported between young and older adults without HIV. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

After primary infection, human cytomegalovirus (HCMV) establishes lifelong persistence, underpinned by latent carriage of the virus with spontaneous reactivation events. In the immune-competent, primary infection or reactivation from latency rarely causes disease. However, HCMV can cause significant disease in immune-compromised individuals such as immune-suppressed transplant patients. Latency, where the viral genome is carried in the absence of the production of infectious virions, can be established in undifferentiated cells of the myeloid lineage. A number of stimuli can cause virus reactivation from latency to occur, beginning with the induction of viral immediate-early (IE) lytic gene expression. The suppression of viral IE gene expression to establish and maintain latent infection is known to result from a balance of viral and cellular factors. One key viral factor involved in this is the G protein-coupled receptor US28. Recently, we have shown that US28 is targeted for degradation by a modified nanobody (PCTD-Vun100bv) based on the novel PACTAC (PCSK9-antibody clearance-targeting chimeras) approach for targeted protein degradation. Furthermore, we have shown that this PCTD-Vun100bv-induced degradation of US28 results in IE gene expression in experimentally latently infected CD14+ monocytes. However, HCMV also establishes latency in CD34+ bone marrow cells, the progenitors of CD14+ cells. Here, we show that PCTD-Vun100bv also causes US28 degradation in these CD34+ primary cells, again resulting in the induction of viral IE gene expression. Additionally, we show that PCTD-Vun100bv can target US28 in naturally latently infected CD14+ monocytes from an HCMV-seropositive donor, allowing these latently infected cells to be killed by HCMV-specific cytotoxic T cells from that same donor. These observations support the view that targeting US28 for degradation during natural latency could be a tractable 'shock-and-kill' strategy to target the latent HCMV reservoir in myeloid cells. [ABSTRACT FROM AUTHOR]

Introduction: Poor graft function (PGF), characterized by myelosuppression, represents a significant challenge following allogeneic hematopoietic stem cell transplantation (allo-HSCT) with human cytomegalovirus (HCMV) being established as a risk factor for PGF. However, the underlying mechanism remains unclear. Bone marrow endothelial progenitor cells (BM-EPCs) play an important role in supporting hematopoiesis and their dysfunction contributes to PGF development. We aim to explore the effects of CMV on BM-EPCs and its underlying mechanism. Methods: We investigated the compromised functionality of EPCs derived from individuals diagnosed with HCMV viremia accompanied by PGF, as well as after infected by HCMV AD 169 strain in vitro, characterized by decreased cell proliferation, tube formation, migration and hematopoietic support, and increased apoptosis and secretion of TGF-β1. Results: We demonstrated that HCMV-induced TGF-β1 secretion by BM-EPCs played a dominant role in hematopoiesis suppression in vitro experiment. Moreover, HCMV down-regulates Vitamin D receptor (VDR) and subsequently activates p38 MAPK pathway to promote TGF-β1 secretion by BM-EPCs. Discussion: HCMV could infect BM-EPCs and lead to their dysfunction. The secretion of TGF-β1 by BM-EPCs is enhanced by CMV through the activation of p38 MAPK via a VDR-dependent mechanism, ultimately leading to compromised support for hematopoietic progenitors by BM EPCs, which May significantly contribute to the pathogenesis of PGF following allo-HSCT and provide innovative therapeutic strategies targeting PGF. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus is a medically important pathogen. Previously, using murine CMV (MCMV), we provided evidence that both neutralizing and nonneutralizing antibodies can confer protection from viral infection in vivo. In this study, we report that serum derived from infected animals had a greater protective capacity in MCMV‐infected RAG−/− mice than serum from animals immunized with purified virus. The protective activity of immune serum was strictly dependent on functional Fcγ receptors (FcγR). Deletion of individual FcγRs or combined deletion of FcγRI and FcγRIV had little impact on the protection afforded by serum. Adoptive transfer of CD115‐positive cells from noninfected donors demonstrated that monocytes represent important cellular mediators of the protective activity provided by immune serum. Our studies suggest that Fc–FcγR interactions and monocytic cells are critical for antibody‐mediated protection against MCMV infection in vivo. These findings may provide new avenues for the development of novel strategies for more effective CMV vaccines or antiviral immunotherapies. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus (hCMV) is a ubiquitous latent persistent herpesvirus infecting 60–90% of the population worldwide. hCMV carriage in immunocompetent people is asymptomatic; thus, hCMV can be considered a component of normative aging. However, hCMV powerfully modulates many features of the immune, and likely other, systems and organs. Questions remain as to how hCMV carriage affects the human host. We used anti-CMV antibody titers as a stratifying criterion to examine the impact of "intensity" of hCMV infection as a potential biomarker of aging, inflammation, and immune homeostasis in a cohort of 247 participants stratified into younger (21–40 years) and older (> 65 years of age) groups. We showed that anti-CMV antibody titers increased with age and directly correlated to increased levels of soluble tumor necrosis factor (sTNFR) I in younger but not older participants. CD8 + cell numbers were reduced in the older group due to the loss in CD8 + T naïve (Tn) cells. In CMV carriers and, in particular, in anti-CMV Ab-high participants, this loss was mitigated or reversed by an increase in the numbers of CD8 + T effector memory (Tem) and T effector memory reexpressing CD45RA (Temra) cells. Analysis of CD38, HLA-DR, and CD57 expression revealed subset (CD4 or CD8)-specific changes that correlated with anti-CMV Ab levels. In addition, anti-CMV Ab levels predicted anti-CMV CD8 T cell responsiveness to different CMV open reading frames (ORFs) selectively in older participants, which correlated to the transcriptional order of expression of specific CMV ORFs. Implications of these results for the potential predictive value of anti-CMV Ab titers during aging are discussed. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is one of the deadliest brain cancers with a median survival of only 15 months. This poor prognosis has prompted exploration of novel therapeutic targets for GBM patients. Human cytomegalovirus (HCMV) has been implicated in GBM; however, its impact remains poorly defined, and there is conflicting data over the presence of HCMV in tumors. Nonetheless, clinical trials targeting HCMV have shown promising initial data, and evidence suggests that HCMV may negatively impact GBM patient survival by multiple mechanisms including changes in GBM cell behavior and the tumor microenvironment (TME) that potentiate tumor progression as well as therapy-induced virus reactivation. Moreover, HCMV has many effects on host immunity that could impact tumor behavior by altering the TME, which are largely unexplored. The goal of this review is to describe these potential interactions between HCMV and GBM. Better understanding of these processes may allow the development of new therapeutic modalities to improve GBM patient outcomes. [ABSTRACT FROM AUTHOR]

The Pentamer complex of Human Cytomegalovirus (HCMV) consists of the viral glycoproteins gH, gL, UL128, UL130, and UL131 and is incorporated into infectious virions. HCMV strains propagated extensively in vitro in fibroblasts carry UL128, UL130, or UL131 alleles that do not make a functional complex and thus lack Pentamer function. Adding functional Pentamer to such strains decreases virus growth in fibroblasts. Here, we show that the Pentamer inhibits productive HCMV replication in fibroblasts by repressing viral Immediate Early (IE) transcription. We show that ectopic expression of the viral IE1 protein, a target of Pentamer-mediated transcriptional repression, complements the growth defect of a Pentamer-positive virus. Furthermore, we show that the Pentamer also represses viral IE transcription in cell types where HCMV in vitro latency is studied. Finally, we identify UL130 as a functional subunit of the Pentamer for IE transcriptional repression and demonstrate that cyclic AMP Response Element (CRE) and NFkB sites within the Major Immediate Early Promoter that drives IE1 transcription contribute to this repression. We conclude that the HCMV Pentamer represses viral IE transcription. [ABSTRACT FROM AUTHOR]

Background/Objectives: Human cytomegalovirus (HCMV) is the most frequent cause of congenital infections. The HCMV-specific T-cell response in primary infection may help define reliable correlates of immune protection in pregnancy. In this study, the antigen-specific T-cell response against different HCMV proteins (IE-1, pp65, gB, gHgLpUL128L) was investigated in pregnant women with primary infection and in control subjects with remote infection to identify possible components of a vaccine. Methods: Blood samples from 35 pregnant women with HCMV primary infection and 30 HCMV-seropositive healthy adult subjects with remote infection were tested. The antigen-specific T-cell response was measured using cytokine intracellular staining after stimulation with IE-1, pp65, gB and gHgLpUL128L peptides pool. Results: The pp65-specific CD4+ T-cell response was higher in pregnant women with HCMV primary infection at the late time point and in control subjects with remote infection, while the pregnant women at the early time point showed a higher gB-specific CD8+ T-cell response. Regarding the CD4+ and CD8+ T-cell phenotypes, we observed that HCMV-specific CD4+ and CD8+ T cells expressing CD45RA+ remained constant in pregnant women with primary infection at the early and late time points and in subjects with remote infection, while HCMV-specific CD4+ and CD8+ T cells expressing IL-7R+ or producing IL-2 were higher in control subjects with remote infection than in pregnant women with HCMV primary infection. Conclusions: The T-cell response was higher against gB in the early phase of infection and against pp65 in the late phase. Therefore, these proteins should be taken into consideration as candidates for a vaccine. [ABSTRACT FROM AUTHOR]

Background Emerging evidence suggests that viral infections may contribute to Alzheimer's disease (AD) onset and/or progression. However, the extent of their involvement and the mechanisms through which specific viruses increase AD susceptibility risk remain elusive. Methods We used an integrative systems bioinformatics approach to identify viral-mediated pathogenic mechanisms, by which Herpes Simplex Virus 1 (HSV-1), Human Cytomegalovirus (HCMV), Epstein-Barr virus (EBV), Kaposi Sarcoma-associated Herpesvirus (KSHV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Influenza A Virus (IAV) and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) could facilitate AD pathogenesis via virus-host protein-protein interactions (PPIs). We also explored potential synergistic pathogenic effects resulting from herpesvirus reactivation (HSV-1, HCMV, and EBV) during acute SARS-CoV-2 infection, potentially increasing AD susceptibility. Results Herpesviridae members (HSV-1, EBV, KSHV, HCMV) impact AD-related processes like amyloid-β (Aβ) formation, neuronal death, and autophagy. Hepatitis viruses (HBV, HCV) influence processes crucial for cellular homeostasis and dysfunction, they also affect microglia activation via virus-host PPIs. Reactivation of HCMV during SARS-CoV-2 infection could potentially foster a lethal interplay of neurodegeneration, via synergistic pathogenic effects on AD-related processes like response to unfolded protein, regulation of autophagy, response to oxidative stress, and Aβ formation. Conclusions These findings underscore the complex link between viral infections and AD development. Viruses impact AD-related processes through shared and distinct mechanisms, potentially influencing variations in AD susceptibility. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus (HCMV) glycoprotein B (gB) is a class III membrane fusion protein required for viral entry. HCMV vaccine candidates containing gB have demonstrated moderate clinical efficacy, but no HCMV vaccine has been approved. Here, we used structure-based design to identify and characterize amino acid substitutions that stabilize gB in its metastable prefusion conformation. One variant containing two engineered interprotomer disulfide bonds and two cavity-filling substitutions (gB-C7), displayed increased expression and thermostability. A 2.8 Å resolution cryoelectron microscopy structure shows that gB-C7 adopts a prefusion-like conformation, revealing additional structural elements at the membrane-distal apex. Unlike previous observations for several class I viral fusion proteins, mice immunized with postfusion or prefusion-stabilized forms of soluble gB protein displayed similar neutralizing antibody titers, here specifically against an HCMV laboratory strain on fibroblasts. Collectively, these results identify initial strategies to stabilize class III viral fusion proteins and provide tools to probe gB-directed antibody responses. [ABSTRACT FROM AUTHOR]

Genetic parasites, including viruses and transposons, exploit components from the host for their own replication. However, little is known about virus-transposon interactions within host cells. Here, we discover a strategy where human cytomegalovirus (HCMV) hijacks L1 retrotransposon encoded protein during its replication cycle. HCMV infection upregulates L1 expression by enhancing both the expression of L1-activating transcription factors, YY1 and RUNX3, and the chromatin accessibility of L1 promoter regions. Increased L1 expression, in turn, promotes HCMV replicative fitness. Affinity proteomics reveals UL44, HCMV DNA polymerase subunit, as the most abundant viral binding protein of the L1 ribonucleoprotein (RNP) complex. UL44 directly interacts with L1 ORF2p, inducing DNA damage responses in replicating HCMV compartments. While increased L1-induced mutagenesis is not observed in HCMV for genetic adaptation, the interplay between UL44 and ORF2p accelerates viral DNA replication by alleviating replication stress. Our findings shed light on how HCMV exploits host retrotransposons for enhanced viral fitness. Human cytomegalovirus (HCMV) hijacks host L1 retrotransposon to supercharge its replication. This novel virus-transposon interaction reveals a strategy for viruses to exploit host machinery, accelerating viral DNA replication and enhancing fitness. [ABSTRACT FROM AUTHOR]

The human cytomegalovirus (HCMV) glycoprotein B (gB) is the viral fusogen required for entry into cells and for direct cell-to-cell spread of the virus. We have previously demonstrated that the exchange of the carboxy-terminal domain (CTD) of gB for the CTD of the structurally related fusion protein G of the vesicular stomatitis virus (VSV-G) resulted in an intrinsically fusion-active gB variant (gB/VSV-G). In this present study, we employed a dual split protein (DSP)-based cell fusion assay to further characterize the determinants of fusion activity in the CTD of gB. We generated a comprehensive library of gB CTD truncation mutants and identified two mutants, gB-787 and gB-807, which were fusion-competent and induced the formation of multinucleated cell syncytia in the absence of other HCMV proteins. Structural modeling coupled with site-directed mutagenesis revealed that gB fusion activity is primarily mediated by the CTD helix 2, and secondarily by the recruitment of cellular SH2/WW-domain-containing proteins. The fusion activity of gB-807 was inhibited by gB-specific monoclonal antibodies (MAbs) targeting the antigenic domains AD-1 to AD-5 within the ectodomain and not restricted to MAbs directed against AD-4 and AD-5 as observed for gB/VSV-G. This finding suggested a differential regulation of the fusion-active conformational state of both gB variants. Collectively, our findings underscore a pivotal role of the CTD in regulating the fusogenicity of HCMV gB, with important implications for understanding the conformations of gB that facilitate membrane fusion, including antigenic structures that could be targeted by antibodies to block this essential step in HCMV infection. [ABSTRACT FROM AUTHOR]

(1) Background: Intrinsic defense mechanisms are pivotal host strategies to restrict viruses already at early stages of their infection. Here, we addressed the question of how the autophagy receptor sequestome 1 (SQSTM1/p62, hereafter referred to as p62) interferes with human cytomegalovirus (HCMV) infection. (2) Methods: CRISPR/Cas9-mediated genome editing, mass spectrometry and the expression of p62 phosphovariants from recombinant HCMVs were used to address the role of p62 during infection. (3) Results: The knockout of p62 resulted in an increased release of HCMV progeny. Mass spectrometry revealed an interaction of p62 with cellular proteins required for nucleocytoplasmic transport. Phosphoproteomics further revealed that p62 is hyperphosphorylated at position S272 in HCMV-infected cells. Phosphorylated p62 showed enhanced nuclear retention, which is concordant with enhanced interaction with viral proteins relevant for genome replication and nuclear capsid egress. This modification led to reduced HCMV progeny release compared to a non-phosphorylated version of p62. (4) Conclusions: p62 is a restriction factor for HCMV replication. The activity of the receptor appears to be regulated by phosphorylation at position S272, leading to enhanced nuclear localization, viral protein degradation and impaired progeny production. [ABSTRACT FROM AUTHOR]

The signal sequences of the human cytomegalovirus (CMV) UL40 protein and its rhesus CMV (RhCMV) counterpart, Rh67, contain a peptide (VMAPRT[L/V][F/I/L/ V]L, VL9) that is presented by major histocompatibility complex (MHC) antigen E (MHC-E). The CMV VL9 peptides replace VL9 peptides derived from classical MHC (Ia) signal sequences, which are lost when CMV disrupts antigen processing and presentation and MHC Ia expression. This allows infected cells to maintain MHC-E surface expression and escape killing by Natural Killer cells. We demonstrate that processing of the Rh67 VL9 peptide mirrors that of UL40, despite the lack of sequence conservation between the two proteins. Processing of both VL9 peptides is dependent on cleavage of their signal sequences by the host protease signal peptide peptidase. As previously shown for UL40, up-regulation of MHC-E expression by Rh67 requires only its signal sequence, with sequences upstream of VL9 critical for conferring independence from TAP, the transporter associated with antigen processing. Our results also suggest that the mature UL40 and Rh67 proteins contribute to CMV immune evasion by decreasing surface expression of MHC Ia. Unexpectedly, while the Rh67 VL9 peptide is resistant to the effects of Rh67, UL40 can partially counteract the up-regulation of MHC-E expression mediated by its own VL9 peptide. This suggests differences in the mechanisms by which the two VL9 peptides up-regulate MHC-E, and further work will be required to determine if any such differences have implications for translating a RhCMV-vectored simian immunodeficiency virus (SIV) vaccine to HIV-1 using human CMV as a vector. IMPORTANCE The protective immune response induced by a rhesus cytomegalovirus (RhCMV)-vectored simian immunodeficiency virus (SIV) vaccine in rhesus macaques depends on the presence of the viral Rh67 gene in the vaccine. The Rh67 protein contains a peptide that allows the RhCMV-infected cells to maintain expression of major histocompatibility complex (MHC) antigen E at the cell surface. We show that production of this peptide, referred to as “VL9,” mirrors that of the equivalent peptide present in the human cytomegalovirus (CMV) protein UL40, despite the little sequence similarity between the two CMV proteins. We also show that the mature UL40 and Rh67 proteins, which have no previously described function, also contribute to CMV immune evasion by reducing cell surface expression of MHC proteins important for the immune system to detect infected cells. Despite these similarities, our work also reveals possible differences between Rh67 and UL40, and these may have implications for the use of human CMV as the vector for a potential HIV-1 vaccine. [ABSTRACT FROM AUTHOR]

Congenital cytomegalovirus (cCMV) infection is the most common congenital infection in developed countries and the most common infectious cause of sensorineural hearing loss and neurodevelopmental impairment. Only a small propor-tion of children with cCMV have a symptomatic disease at birth, while a larger proportion of them are asymptomatic. Nevertheless, all children with cCMV are at risk of developing long-term neurodevelopmental impairment, especially sensorineural hearing loss. According to the incidence, we would currently expect 24 children with cCMV annually in Slovenia, four to five of whom would have a neurodevelopmental impairment due to cCMV. Diagnosis of cCMV is based on evidence of viral DNA in a saliva, urine or blood sample taken within the first three weeks after birth. The gold standard for diagnosis of cCMV is evidence of viral DNA in urine, while confirmation of viral DNA in dried blood spots collected within the first days after birth represents the gold standard for retrospective diagnosis of cCMV in children older than three weeks. After establishing a molecular diagnosis of cCMV, it is necessary to assess the organ impairment with laboratory, neuro-radiological, audiological and ophthalmological tests, on which depends the treatment decision. This review article describes the past and modern diagnostics of cCMV, which allows an individualized management approach and predicts the long-term neurodevelopmental outcome of the disease.

Establishing a nonproductive, quiescent infection within monocytes is essential for the spread of human cytomegalovirus (HCMV). We investigated the mechanisms through which HCMV establishes a quiescent infection in monocytes. US28 is a virally encoded G protein–coupled receptor (GPCR) that is essential for silent infections within cells of the myeloid lineage. We found that preformed US28 was rapidly delivered to monocytes by HCMV viral particles, whereas the de novo synthesis of US28 was delayed for several days. A recombinant mutant virus lacking US28 (US28Δ) was unable to establish a quiescent infection, resulting in a fully productive lytic infection able to produce progeny virus. Infection with US28Δ HCMV resulted in the phosphorylation of the serine and threonine kinase Akt at Ser473 and Thr308, in contrast with the phosphorylation of Akt only at Ser473 after WT viral infection. Inhibiting the dual phosphorylation of Akt prevented the lytic replication of US28Δ, and ectopic expression of a constitutively phosphorylated Akt variant triggered lytic replication of wild-type HCMV. Mechanistically, we found that US28 was necessary and sufficient to attenuate epidermal growth factor receptor (EGFR) signaling induced during the entry of WT virus, which led to the site-specific phosphorylation of Akt at Ser473. Thus, particle-delivered US28 fine-tunes Akt activity by limiting HCMV-induced EGFR activation during viral entry, enabling quiescent infection in monocytes. Editor's summary: Human cytomegalovirus (HCMV) infection is highly prevalent and rapidly spreads throughout an infected host, in part because HCMV can infect blood monocytes without initiating lytic replication. Mahmud et al. investigated how HCMV subverts host signaling pathways to set up a nonlytic, quiescent infection. The authors found that during the early stages of infection, delivery of a protein called US28 by viral particles was critical for limiting the activation of the receptor EGFR. Consequently, the downstream activation of the kinase Akt was restricted in infected monocytes, which enabled a quiescent infection to be established. Thus, limiting the induction of Akt activity by US28 during early infection ensures the survival of HCMV-infected monocytes and prevents the initiation of lytic replication. —Wei Wong [ABSTRACT FROM AUTHOR]

The dynamic regulation of mitochondria shape via fission and fusion is critical for cellular responses to stimuli. In homeostatic cells, two modes of mitochondrial fission, midzone and peripheral, provide a decision fork between either proliferation or clearance of mitochondria. However, the relationship between specific mitochondria shapes and functions remains unclear in many biological contexts. While commonly associated with decreased bioenergetics, fragmented mitochondria paradoxically exhibit elevated respiration in several disease states, including infection with the prevalent pathogen human cytomegalovirus (HCMV) and metastatic melanoma. Here, incorporating super-resolution microscopy with mass spectrometry and metabolic assays, we use HCMV infection to establish a molecular mechanism for maintaining respiration within a fragmented mitochondria population. We establish that HCMV induces fragmentation through peripheral mitochondrial fission coupled with suppression of mitochondria fusion. Unlike uninfected cells, the progeny of peripheral fission enter mitochondria-ER encapsulations (MENCs) where they are protected from degradation and bioenergetically stabilized during infection. MENCs also stabilize pro-viral inter-mitochondria contacts (IMCs), which electrochemically link mitochondria and promote respiration. Demonstrating a broader relevance, we show that the fragmented mitochondria within metastatic melanoma cells also form MENCs. Our findings establish a mechanism where mitochondria fragmentation can promote increased respiration, a feature relevant in the context of human diseases. During HCMV infection, mitochondrial progenies derived from peripheral fragmentation can be protected from mitophagy and result in elevated respiration through a mechanism leveraging ER-mitochondria and inter-mitochondria membrane contacts. [ABSTRACT FROM AUTHOR]

Protein expression is regulated through multiple mechanisms, including post-translational modifications (PTMs), which can alter protein structure, stability, localization, and function. Among these, citrullination stands out due to its ability to convert arginine residues into citrulline, altering protein charge and mass. This modification is catalyzed by calcium-dependent protein arginine deiminases (PADs), enzymes implicated in various inflammatory diseases. We have recently shown that human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1) exploit these enzymes to enhance their replication capabilities. Although the role of PADs in HCMV and HSV-1 infections is well documented, their involvement in HSV-2 infection has not yet been thoroughly investigated. Here, we demonstrate that HSV-2 manipulates the overall protein citrullination profile by activating three PAD isoforms: PAD2, PAD3, and PAD4. However, as previously observed during HSV-1 infection, PAD3 is the most significantly upregulated isoform, both at the mRNA and protein levels. Consistently, we demonstrate that inhibiting PAD3, either through the specific inhibitor CAY10727 or via CRISPR/Cas9-mediated gene silencing, markedly reduces HSV-2 replication and viral protein expression. Lastly, we show that CAY10727 displays an IC50 value of 0.3 μM, which is extremely close to what was previously observed for HSV-1. Overall, our findings highlight the crucial role of PAD3 in the life cycle of HSV-2 and suggest that the targeted inhibition of PAD3 may represent a promising approach for treating HSV-2 infections, especially in cases resistant to existing antiviral therapies. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus (HCMV) is the most common congenital infection. Several HCMV vaccines are in development, but none have yet been approved. An understanding of the kinetics of CMV replication and transmission may inform the rational design of vaccines to prevent this infection. The salivary glands (SG) are an important site of sustained CMV replication following primary infection and during viral reactivation from latency. As such, the strength of the immune response in the SG likely influences viral dissemination within and between hosts. To study the relationship between the immune response and viral replication in the SG, and viral dissemination from the SG to other tissues, mice were infected with low doses of murine CMV (MCMV). Following intra-SG inoculation, we characterized the viral and immunological dynamics in the SG, blood, and spleen, and identified organ-specific immune correlates of protection. Using these data, we constructed compartmental mathematical models of MCMV infection. Model fitting to data and analysis indicate the importance of cellular immune responses in different organs and point to a threshold of infection within the SG necessary for the establishment and spread of infection. Author summary: Cytomegalovirus (CMV) is the most common congenital infection and causes an enormous burden of childhood disease. To gain insight into the immune requirements for controlling infection, we used a mouse model to reproduce characteristics of natural CMV infection, employing a low viral inoculum, and delivering the virus to the salivary glands (SG), a key site of CMV replication. Our results provide detailed data on the spatial and temporal spread of infection throughout the body and identify key immune correlates of the control of viral replication. By translating these findings into mechanistic mathematical models, we revealed the importance of organ-specific immune responses, particularly the requirement of TNF-α and IFN-γ to control infection within the salivary glands. Furthermore, our mathematical modeling allowed us to compare known characteristics of human CMV infection related to infection establishment and spread to those predicted in mice, underscoring the suitability of the MCMV model to study its human homologue. These insights provide guidance for developing targeted vaccines to prevent CMV infection and disease. [ABSTRACT FROM AUTHOR]

Herpesviral protein kinases, such as the therapy-relevant pUL97 of human cytomegalovirus (HCMV), are important for viral replication efficiency as well as pathogenesis, and represent key antiviral drug targets. HCMV pUL97 is a viral cyclin-dependent kinase (CDK) ortholog, as it shares functional and structural properties with human CDKs. Recently, the formation of vCDK/pUL97–cyclin complexes and the phosphorylation of a variety of viral and cellular substrate proteins has been demonstrated. Genetic mapping and structural modeling approaches helped to define two pUL97 interfaces, IF1 and IF2, responsible for cyclin binding. In particular, the regulatory importance of interactions between vCDK/pUL97 and host cyclins as well as CDKs has been highlighted, both as determinants of virus replication and as a novel drug-targeting option. This aspect was substantiated by the finding that virus replication was impaired upon cyclin type H knock-down, and that such host-directed interference also affected viruses resistant to existing therapies. Beyond the formation of binary interactive complexes, a ternary pUL97–cyclin H–CDK7 complex has also been described, and in light of this, an experimental trans-stimulation of CDK7 activity by pUL97 appeared crucial for virus–host coregulation. In accordance with this understanding, several novel antiviral targeting options have emerged. These include kinase inhibitors directed to pUL97, to host CDKs, and to the pUL97–cyclin H interactive complexes. Importantly, a statistically significant drug synergy has recently been reported for antiviral treatment schemes using combinations of pharmacologically relevant CDK7 and vCDK/pUL97 inhibitors, including maribavir. Combined, such findings provide increased options for anti-HCMV control. This review focuses on regulatory interactions of vCDK/pUL97 with the host cyclin–CDK apparatus, and it addresses the functional relevance of these key effector complexes for viral replication and pathogenesis. On this basis, novel strategies of antiviral drug targeting are defined. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus (HCMV) infection remains a major complication for solid organ transplant recipients (SOTRs). The aim of this study was to evaluate the role of HCMV-specific T cell immunity measured at the time of the HCMV-DNA peak in predicting the spontaneous clearance of infection. The performance of cytokine flow cytometry using infected dendritic cells (CFC-iDC), infected cell lysate (CFC-iCL) and pp65 peptide pool (CFC-pp65 pool) as stimuli, as well as ELISPOT assays using infected cell lysate (ELISPOT-iCL) and the pp65 peptide pool (ELISPOT-pp65 pool), was analysed. Among the 40 SOTRs enrolled, 16 patients (40%) required antiviral treatment for an HCMV infection (Non-Controllers), while the others spontaneously cleared the infection (Controllers). At the HCMV-DNA peak, the number of HCMV-specific CD4+ T cells detected by the CFC-iDC, CFC-iCL and CFC-pp65 pool assays in Controllers was higher than that detected in Non-Controllers, while no difference was observed in terms of HCMV-specific CD8+ T cell response. The same trend was observed when the HCMV-specific T cell response was measured by ELISPOT-iCL and ELISPOT-pp65 pool. We observed that the CD4+ CFC-pp65 pool assay was the best predictor of self-resolving HCMV infection at the time of the HCVM-DNA peak. The CFC-pp65 pool assay is able to discriminate between CD4+ and CD8+ T cell responses and could be used in daily clinical practice. [ABSTRACT FROM AUTHOR]

Background MF59-adjuvanted gB subunit (gB/MF59) vaccine demonstrated approximately 50% efficacy against human cytomegalovirus (HCMV) acquisition in multiple clinical trials, suggesting that efforts to improve this vaccine design might yield a vaccine suitable for licensure. Methods A messenger RNA (mRNA)–based vaccine candidate encoding HCMV gB and pentameric complex (PC), mRNA-1647, is currently in late-stage efficacy trials. However, its immunogenicity has not been compared to the partially effective gB/MF59 vaccine. We assessed neutralizing and Fc-mediated immunoglobulin G (IgG) effector antibody responses induced by mRNA-1647 in both HCMV-seropositive and -seronegative vaccinees from a first-in-human clinical trial through 1 year following third vaccination using a systems serology approach. Furthermore, we compared peak anti-gB antibody responses in seronegative mRNA-1647 vaccinees to that of seronegative gB/MF59 vaccine recipients. Results mRNA-1647 vaccination elicited and boosted HCMV-specific IgG responses in seronegative and seropositive vaccinees, respectively, including neutralizing and Fc-mediated effector antibody responses. gB-specific IgG responses were lower than PC-specific IgG responses. gB-specific IgG and antibody-dependent cellular phagocytosis responses were lower than those elicited by gB/MF59. However, mRNA-1647 elicited higher neutralization and antibody-dependent cellular cytotoxicity (ADCC) responses. Conclusions Overall, mRNA-1647 vaccination induced polyfunctional and durable HCMV-specific antibody responses, with lower gB-specific IgG responses but higher neutralization and ADCC responses compared to the gB/MF59 vaccine. Clinical Trials Registration NCT03382405 (mRNA-1647) and NCT00133497 (gB/MF59). [ABSTRACT FROM AUTHOR]

Objective To investigate the intrauterine infection rate of human cytomegalovirus (HCMV) in fetuses and analyze the ultrasound findings and pregnancy outcomes of fetuses with intrauterine HCMV infection from 2016 to 2021 at Shenzhen Baoan Woman′s and Children′s Hospital. Methods Amniotic fluid samples from 4 685 pregnant women who underwent amniocentesis at our hospital were tested for HCMV-DNA. A retrospective analysis was conducted on HCMV-IgG and IgM test results in a subset of cases. Chromosomal karyotyping, ultrasound examinations, and pregnancy outcomes were tracked for 11 cases with positive HCMV-DNA. Results Among the 4 685 amniotic fluid samples, 4 674 were HCMV-DNA negative. Of 2 076 pregnant women, 26 had positive serum HCMV-IgM, and among 1 482 tested, 1 454 were positive for HCMV-IgG. For the 11 pregnant women with positive amniotic fluid HCMV-DNA, the following results were observed: serum HCMV-IgM positive in 3 cases and negative in 8 cases; HCMV-IgG positive in 8 cases, with 3 cases not tested; HCMV-DNA in urine was positive in 2 cases, negative in 7 cases, and not tested in 2 cases; serum HCMV-DNA was negative in 9 cases, with 2 cases not tested. Of the 11 cases with intrauterine HCMV infection, 2 had positive HCMV-IgM before amniocentesis. The remaining 9 underwent amniocentesis due to abnormal fetal ultrasound findings or high-risk Down syndrome screening. Ultrasound results related to HCMV infection were observed in 7 cases: 6 resulted in termination, and 1 newborn was without abnormalities. Four cases showed no infection-related findings on ultrasound, and all had normal deliveries with no abnormalities detected postpartum. Conclusion Amniotic fluid HCMV-DNA testing provides accurate diagnosis of congenital CMV infection. Serum Ig and DNA tests in pregnant women have limited diagnostic value. Some fetuses with intrauterine HCMV infection may present ultrasound abnormalities, while others may be asymptomatic at birth. [ABSTRACT FROM AUTHOR]

Viruses use various strategies and mechanisms to deal with cells and proteins of the immune system that form a barrier against infection. One of these mechanisms is the encoding and production of viral microRNAs (miRNAs), whose function is to regulate the gene expression of the host cell and the virus, thus creating a suitable environment for survival and spreading viral infection. miRNAs are short, single-stranded, non-coding RNA molecules that can regulate the expression of host and viral proteins, and due to their non-immunogenic nature, they are not eliminated by the cells of the immune system. More than half of the viral miRNAs are encoded and produced by Orthoherpesviridae family members. Human cytomegalovirus (HCMV) produces miRNAs that mediate various processes in infected cells to contribute to HCMV pathogenicity, including immune escape, viral latency, and cell apoptosis. Here, we discuss which cellular and viral proteins or cellular pathways and processes these mysterious molecules target to evade immunity and support viral latency in infected cells. We also discuss current evidence that their function of bypassing the host's innate and adaptive immune system is essential for the survival and multiplication of the virus and the spread of HCMV infection. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus (HCMV) infection is common in tumor tissues across different types of cancer. While HCMV has not been recognized as a cancer-causing virus, numerous studies hint at its potential role in cancer development where its presence in various cancers corresponds with the hallmarks of cancer. Herein, we discuss and demonstrate that high-risk HCMV-DB and BL strains have the potential to trigger transformation in epithelial cells, including human mammary epithelial cells (HMECs), ovarian epithelial cells (OECs), and prostate epithelial cells (PECs), through the generation of polyploid giant cancer cells (PGCCs). A discussion is provided on how HCMV infection creates a cellular environment that promotes oncogenesis, supporting the continuous growth of CMV-transformed cells. The aforementioned transformed cells, named CTH, CTO, and CTP cells, underwent giant cell cycling with PGCC generation parallel to dedifferentiation, displaying stem-like characteristics and an epithelial–mesenchymal transition (EMT) phenotype. Furthermore, we propose that giant cell cycling through PGCCs, increased EZH2 expression, EMT, and the acquisition of malignant traits represent a deleterious response to the cellular stress induced by high-risk oncogenic HCMV strains, the latter being the origin of the transformation process in epithelial cells upon HCMV infection and leading to adenocarcinoma of poor prognosis. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus has a linear DNA genome with a total length of approximately 235 kb. This large genome is divided into two domains, "Long" and "Short". There are four isomers of the cytomegalovirus genome with different orientations of each domain. To confirm the presence of four types of isomers, it is necessary to identify the sequence of the junction between the domains. However, due to the presence of repeat sequences, it is difficult to determine the junction sequences by next-generation sequencing analysis. To solve this problem, long-read sequencing was performed using the Oxford Nanopore sequencer and the junctions were successfully identified in four isomers in strain Merin and ATCC-2011-3. Nanopore sequencing also revealed the presence of multiple copies of the "a" sequence (a-seq) in the junctions, indicating the diversity of the junction sequences. These results strongly suggest that long-read sequencing using the nanopore sequencer would be beneficial for identifying the complex structure of the cytomegalovirus genome. [ABSTRACT FROM AUTHOR]

Introduction: Human cytomegalovirus (HCMV) is the most common viral infection seen in newborns. The major route of transmission for acquired human cytomegalovirus infection is breast milk from mothers who are HCMV seropositive to the infants. Thus, a rapid, economical, and simple method to perform HCMV test in breast milk is crucial and necessary for preventing acquired HCMV infection, especially in underdeveloped regions with limited laboratory resources. Methods: In this study, an effective technique for the detection of HCMV was constructed by combining multienzyme isothermal rapid amplification (MIRA) and lateral flow chromatography strip (LFD). Primers for the conserved HCMV sequence UL83 were utilized for MIRA-LFD testing. Results: Our results showed that the entire MIRA reaction could be completed in 12 minutes at 37°C, and LFD outcomes could be observed visibly after 10 minutes. The detection sensitivity of this method reached 50 copy/ml. Samples of breast milk were examined to compare MIRA-LFD and conventional qPCR. The accuracy of MIRA-LFD was 100%. Discussion: The straightforward, rapid, economic features of the test can provide the significant advantages for the prevention of breast milk-acquired cytomegalovirus infection, particularly in resource-limited locations with high seroprevalence of cytomegalovirus. [ABSTRACT FROM AUTHOR]

Congenital human cytomegalovirus (HCMV) infection is a major cause of abnormalities and disorders in the central nervous system (CNS) and/or the peripheral nervous system (PNS). However, the complete pathogenesis of neural differentiation disorders caused by HCMV infection remains to be fully elucidated. Stem cells from human exfoliated deciduous teeth (SHEDs) are mesenchymal stem cells (MSCs) with a high proliferation and neurogenic differentiation capacity. Since SHEDs originate from the neural crest of the early embryonic ectoderm, SHEDs were hypothesized to serve as a promising cell line for investigating the pathogenesis of neural differentiation disorders in the PNS caused by congenital HCMV infection. In this work, SHEDs were demonstrated to be fully permissive to HCMV infection and the virus was able to complete its life cycle in SHEDs. Under neurogenic inductive conditions, HCMV infection of SHEDs caused an abnormal neural morphology. The expression of stem/neural cell markers was also disturbed by HCMV infection. The impairment of neural differentiation was mainly due to a reduction of intracellular cholesterol levels caused by HCMV infection. Sterol regulatory element binding protein-2 (SREBP2) is a critical transcription regulator that guides cholesterol synthesis. HCMV infection was shown to hinder the migration of SREBP2 into nucleus and resulted in perinuclear aggregations of SREBP2 during neural differentiation. Our findings provide new insights into the prevention and treatment of nervous system diseases caused by congenital HCMV infection. [ABSTRACT FROM AUTHOR]

Oral rotavirus vaccines demonstrate diminished immunogenicity in low-income settings where human cytomegalovirus infection is acquired early in childhood and modulates immunity. We hypothesized that human cytomegalovirus infection around the time of vaccination may influence immunogenicity. We measured plasma human cytomegalovirus-specific immunoglobulin M antibodies in rotavirus vaccinated infants from 6 weeks to 12 months old and compared rotavirus immunoglobulin A antibody titers between human cytomegalovirus seropositive and seronegative infants. There was no evidence of an association between human cytomegalovirus serostatus at 9 months and rotavirus-specific antibody titers at 12 months (geometric mean ratio 1.01, 95% CI: 0.70, 1.45; P = 0.976) or fold-increase in RV-IgA titer between 9 and 12 months (risk ratio 0.999, 95%CI: 0.66, 1.52; P = 0.995) overall. However, HIV-exposed-uninfected infants who were seropositive for human cytomegalovirus at 9 months old had a 63% reduction in rotavirus antibody geometric mean titers at 12 months compared to HIV-exposed-uninfected infants who were seronegative for human cytomegalovirus (geometric mean ratio 0.37, 95% CI: 0.17, 0.77; P = 0.008). While the broader implications of human cytomegalovirus infections on oral rotavirus vaccine response might be limited in the general infant population, the potential impact in the HIV-exposed-uninfected infants cannot be overlooked. This study highlights the complexity of immunological responses and the need for targeted interventions to ensure oral rotavirus vaccine efficacy, especially in vulnerable subpopulations. Among rotavirus vaccinated infants, there was no evidence of an association between HCMV-IgM serostatus at 9 months with RV-IgA titer at 12 months (GMR 1.01, 95%CI: 0.70, 1.45; P  = 0.976). However, HIV-exposed-uninfected infants who were HCMV-IgM seropositive at 9 months old had a 63% reduction in RV-IgA geometric mean titers at 12 months compared to HIV-exposed-uninfected infants who were HCMV-IgM seronegative (geometric mean ratio 0.37, 95%CI: 0.17, 0.77; P  = 0.008). Created with BioRender.com Graphical Abstract [ABSTRACT FROM AUTHOR]

The complement system is an evolutionarily ancient component of innate immunity that serves as an important first line of defense against pathogens, including viruses. In response to infection, the complement system can be activated by three distinct yet converging pathways (classical, lectin, and alternative) capable of engaging multiple antiviral host responses to confront acute, chronic, and recurrent viral infections. Complement can exert profound antiviral effects via multiple mechanisms including the induction of inflammation and chemotaxis to sites of infection, neutralization/opsonization of viruses and virally infected cells, and it can even shape adaptive immune responses. With millions of years of co-evolution and the ability to establish life-long infections, herpesviruses have evolved unique mechanisms to counter complement-mediated antiviral defenses, thus enabling their survival and replication within humans. This review aims to comprehensively summarize how human herpesviruses engage with the complement system and highlight our understanding of the role of complement in human cytomegalovirus (HCMV) infection, immunity, and viral replication. Herein we describe the novel and unorthodox roles of complement proteins beyond their roles in innate immunity and discuss gaps in knowledge and future directions of complement and HCMV research. [ABSTRACT FROM AUTHOR]

Since the significance of viral infections in children and adolescents with nephrotic syndrome (NS) is yet to be defined, this study intended to estimate the occurrence, pattern, and outcomes of some DNA viral infections in children with NS. Methods: A prospective study was conducted to determine the genome identification of the viruses Epstein-Barr (EBV), human cytomegalovirus (HCMV), human herpesvirus 6 (HHV-6 type A and type B) and 7 (HHV-7), polyomavirus (BKV), and human adenovirus (HAdV) in plasma and urine samples of pediatric patients with NS. Results: A total of 35 patients aged 1 to 18 years with NS and under immunosuppressant drugs participated in the study. Plasma and urine samples were collected at regular intervals during a median follow-up of 266 days (range 133–595), and DNA was analyzed to detect the selected DNA viruses. Eleven patients (31.4%) had active virus infections, and patterns were classified as coinfection, recurrent, and consecutive. Of these, six patients (54.5%) presented viral coinfection, six (54.5%) viral recurrence, and seven patients (63.3%) had viral consecutive infection. Ten of the eleven patients with active infection had a proteinuria relapse (91%) and eight (72.7%) were hospitalized (p = 0.0022). Active HCMV infection was the most frequent infection and was observed in six patients (54.5%), three of the eleven patients (27.2%) had suspected HCMV disease in the gastrointestinal tract, and one had HHV-7 coinfection. The frequency of other infections was: 9% for HHV-6, 45.5% for BKV, 27.3% for HHV-7, 18.2% for EBV, and 18.2% for HAdV. Conclusion: viral infections, especially HCMV, can be an important cause of morbidity and nephrotic syndrome relapse in children. [ABSTRACT FROM AUTHOR]

Objective This study aimed to investigate the relationship between clinical characteristics and human cytomegalovirus (HCMV) glycoprotein N (gN) genotypes in children. Methods HCMV gN gene polymorphisms in 544 patients were analyzed using semi-nested polymerase chain reaction and restriction fragment length polymorphism. Results The highest proportion was observed for the gN3a genotype (126/544, 23.2%). The proportion of children with mixed infections presenting with the hepatitis phenotype (65/69, 94.2%) was significantly higher than that of each gN genotype, except for gN3b (34/43, 79.1%, all p < 0.0083). Patients infected with 4b genotype (56/56, 100%) had a significantly higher proportion of anemia symptoms than those infected with all other gN genotypes (all p < 0.0083). There were also significant differences in the proportion of patients infected with different gN genotypes who presented with clinical features, such as jaundice, pneumonia, and thrombocytopenic purpura. Patients with the gN2 genotype had significantly higher albumin levels than those with the gN3a genotype (p = 0.042). Conclusion The clinical phenotypes and laboratory indicators of HCMV infection in children with different gN genotypes are somewhat different, suggesting that precise typing of gN genes has clinical value. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus (HCMV) is a representative β-herpesvirus that establishes persistent infections in humans, and exhibits high seropositivity rates in adults. It has co-evolved with its human host and employs various strategies to evade antiviral mechanisms by utilizing a significant portion of its genome. HCMV-encoded proteins and miRNAs have been implicated in regulating these mechanisms, enabling viral survival within the human body. During viral infections, autophagy, a conserved catabolic process essential for cellular homeostasis, acts as an antiviral defense mechanism. Multiple studies have reported that HCMV can modulate autophagy through its proteins and miRNAs, thereby influencing its survival within the host. In this study, we showed the potential involvement of HCMV miRNAs in cellular autophagy. We employed various bioinformatic tools to predict putative HCMV miRNAs that target autophagy-related genes and their corresponding cellular autophagy genes. Our results show that the 3'UTR of autophagy-related genes, including ATG9A, ATG9B, ATG16L2, SQSTM1, and EIF2AK2, harbors potential binding sites for hcmv-miR-UL70-3p. Experimental manipulation involving ectopic expression of hcmv-miR-UL70-3p demonstrated a significant reduction in rapamycin-induced autophagy, with ATG9A as its functional target. These findings establish that hcmv-miR-UL70-3p acts as an autophagy inhibitor by suppressing the expression of ATG9A. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus (HCMV) displays a broad cell tropism, and the infection of biologically relevant cells such as epithelial, endothelial, and hematopoietic cells supports viral transmission, systemic spread, and pathogenesis in the human host. HCMV strains differ in their ability to infect and replicate in these cell types, but the genetic basis of these differences has remained incompletely understood. In this study, we investigated HCMV strain VR1814, which is highly infectious for epithelial cells and macrophages and induces cell-cell fusion in both cell types. A VR1814-derived bacterial artificial chromosome (BAC) clone, FIX-BAC, was generated many years ago but has fallen out of favor because of its modest infectivity. By sequence comparison and genetic engineering of FIX, we demonstrate that the high infectivity of VR1814 and its ability to induce syncytium formation in epithelial cells and macrophages depends on VR1814-specific variants of the envelope glycoproteins gB, UL128, and UL130. We also show that UL130-neutralizing antibodies inhibit syncytium formation, and a FIX-specific mutation in UL130 is responsible for its low infectivity by reducing the amount of the pentameric glycoprotein complex in viral particles. Moreover, we found that a VR1814- specific mutation in US28 further increases viral infectivity in macrophages, possibly by promoting lytic rather than latent infection of these cells. Our findings show that variants of gB and the pentameric complex are major determinants of infectivity and syncytium formation in epithelial cells and macrophages. Furthermore, the VR1814-adjusted FIX strains can serve as valuable tools to study HCMV infection of myeloid cells. IMPORTANCE Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and the leading cause of congenital infections. HCMV infects various cell types, including epithelial cells and macrophages, and some strains induce the fusion of neighboring cells, leading to the formation of large multinucleated cells called syncytia. This process may limit the exposure of the virus to host immune factors and affect pathogenicity. However, the reason why some HCMV strains exhibit a broader cell tropism and why some induce cell fusion more than others is not well understood. We compared two closely related HCMV strains and provided evidence that small differences in viral envelope glycoproteins can massively increase or decrease the virus infectivity and its ability to induce syncytium formation. The results of the study suggest that natural strain variations may influence HCMV infection and pathogenesis in humans. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus (HCMV) is a β-herpesvirus that poses severe disease risk for immunocompromised patients who experience primary infection or reactivation. Development and optimization of safe and effective anti-HCMV therapeutics is of urgent necessity for the prevention and treatment of HCMV-associated diseases in diverse populations. The use of neutralizing monoclonal antibodies (mAbs) to limit HCMV infection poses a promising therapeutic strategy, as anti-HCMV mAbs largely inhibit infection by targeting virion glycoprotein complexes. In contrast, the small-molecule compounds currently approved for patients (e.g., ganciclovir, letermovir, and maribavir) target later stages of the HCMV life cycle. Here, we present a broadly neutralizing human mAb, designated 1C10, elicited from a VelocImmune mouse immunized with infectious HCMV particles. Clone 1C10 neutralizes infection after virion binding to cells by targeting gH/gL envelope complexes and potently reduces infection of diverse HCMV strains in fibroblast, trophoblast, and epithelial cells. Antibody competition assays found that 1C10 recognizes a region of gH associated with broad neutralization and binds to soluble pentamer in the low nanomolar range. Importantly, 1C10 treatment significantly reduced virus proliferation in both fibroblast and epithelial cells. Further, the combination treatment of mAb 1C10 with ganciclovir reduced HCMV infection and proliferation in a synergistic manner. This work characterizes a neutralizing human mAb for potential use as a HCMV treatment, as well as a possible therapeutic strategy utilizing combination-based treatments targeting disparate steps of the viral life cycle. Collectively, the findings support an antibody-based therapy to effectively treat patients at risk for HCMV-associated diseases. IMPORTANCE Human cytomegalovirus is a herpesvirus that infects a large proportion of the population and can cause significant disease in diverse patient populations whose immune systems are suppressed or compromised. The development and optimization of safe anti-HCMV therapeutics, especially those that have viral targets and inhibition mechanisms different from current HCMV treatments, are of urgent necessity to better public health. Human monoclonal antibodies (mAbs) that prevent HCMV entry of cells were identified by immunizing transgenic mice and screened for broad and effective neutralization capability. Here, we describe one such mAb, which was found to target gH/gL envelope complexes and effectively limit HCMV infection and dissemination. Further, administration of the antibody in combination with the antiviral drug ganciclovir inhibited HCMV in a synergistic manner, highlighting this approach and the use of anti-HCMV mAbs more broadly, as a potential therapeutic strategy for the treatment of diverse patient populations. [ABSTRACT FROM AUTHOR]

Donor and recipient human cytomegalovirus (HCMV) seropositive (D+R+) lung transplant recipients (LTRs) often harbor multiple strains of HCMV, likely due to transmitted donor (D) strains and reactivated recipient (R) strains. To date, the extent and timely occurrence of each likely source in shaping the post‐transplantation (post‐Tx) strain population is unknown. Here, we deciphered the D and R origin of the post‐Tx HCMV strain composition in blood, bronchoalveolar lavage (BAL), and CD45+ BAL cell subsets. We investigated either D and/or R formalin‐fixed paraffin‐embedded blocks or fresh D lung tissue from four D+R+ LTRs obtained before transplantation. HCMV strains were characterized by short amplicon deep sequencing. In two LTRs, we show that the transplanted lung is reseeded by R strains within the first 6 months after transplantation, likely by infiltrating CD14+ CD163+/− alveolar macrophages. In three LTRs, we demonstrate both rapid D‐strain dissemination and persistence in the transplanted lung for >1 year post‐Tx. Broad inter‐host diversity contrasts with intra‐host genotype sequence stability upon transmission, during follow‐up and across compartments. In D+R+ LTRs, HCMV strains of both, D and R origin can emerge first and dominate long‐term in subsequent episodes of infection, indicating replication of both sources despite pre‐existing immunity. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus (HCMV) primary infection, re-infection, and reactivation from latency cause morbidity in immune-compromised patients. Consequently, potential therapeutic strategies remain of interest for the treatment of infection. Naturally occurring triterpenoids derived from plants have been demonstrated to have anti-viral activity, although their precise mechanisms of action are not always fully understood. Here, we investigate the activity of Mormordin Ic (Mc) and demonstrate that it is potently anti-viral against HCMV. Through investigation of the mechanistic basis of this anti-viral activity, we identify that it is inhibitory to both viral and host gene expression, and to highly induced genes in particular. We go on to observe that Mc impacts on RNA Pol II activity and, specifically, reduces the occupancy of elongating RNA Pol II at a viral promoter. Next, we demonstrate that Mc is inhibitory to HCMV reactivation, and in doing so identify that it has greater activity against the canonical major immediate early promoter compared to the alternative ip2 promoter located downstream. Finally, we see evidence of RNA Pol II occupancy at the ip2 promoter in undifferentiated myeloid cells. Thus, Mc is potently anti-viral and a potential tool to probe the activity of multiple promoters considered important for controlling HCMV reactivation. [ABSTRACT FROM AUTHOR]

The AAA-type ATPase VPS4 is recruited by proteins of the endosomal sorting complex required for transport III (ESCRT-III) to catalyse membrane constriction and membrane fission. VPS4A accumulates at the cytoplasmic viral assembly complex (cVAC) of cells infected with human cytomegalovirus (HCMV), the site where nascent virus particles obtain their membrane envelope. Here we show that VPS4A is recruited to the cVAC via interaction with pUL71. Sequence analysis, deep-learning structure prediction, molecular dynamics and mutagenic analysis identify a short peptide motif in the C-terminal region of pUL71 that is necessary and sufficient for the interaction with VPS4A. This motif is predicted to bind the same groove of the N-terminal VPS4A Microtubule-Interacting and Trafficking (MIT) domain as the Type 2 MIT-Interacting Motif (MIM2) of cellular ESCRT-III components, and this viral MIM2-like motif (vMIM2) is conserved across β-herpesvirus pUL71 homologues. However, recruitment of VPS4A by pUL71 is dispensable for HCMV morphogenesis or replication and the function of the conserved vMIM2 during infection remains enigmatic. VPS4-recruitment via a vMIM2 represents a previously unknown mechanism of molecular mimicry in viruses, extending previous observations that herpesviruses encode proteins with structural and functional homology to cellular ESCRT-III components. Author summary: Enveloped viruses appropriate cellular membranes from infected cells to generate their membrane coats. Many enveloped viruses accomplish the membrane wrapping of new virus particles by recruiting the cellular membrane-remodelling ESCRT machinery to sites of virus assembly. Herpesviruses have a complex assembly pathway and molecular roles of the ESCRT machinery in the production of new herpesvirus particles remains unclear, but previous studies have shown that the ESCRT-associated ATPase VPS4A is recruited to the cytoplasmic assembly site of human cytomegalovirus (HCMV). Here we show that the HCMV protein pUL71 is necessary for this VPS4A recruitment. Using a combination of biochemical, computational and cell-based techniques we demonstrate that a C-terminal segment of pUL71 binds VPS4A via molecular mimicry of cellular 'MIM2' VPS4-binding motifs. Direct binding to VPS4A via a viral MIM2 (vMIM2) represents a previously unknown mechanism of molecular mimicry in viruses, extending previous observations that herpesviruses encode proteins with structural and functional homology to cellular ESCRT-III components. However, mutational analysis reveals that VPS4A recruitment by pUL71 is not required for HCMV particle assembly, indicating that this novel and conserved HCMV vMIM2 has an unknown function of during infection. [ABSTRACT FROM AUTHOR]

Human leucocyte antigen class I (HLA-I) molecules play a central role for both NK and T-cell responses that prevent serious human cytomegalovirus (HCMV) disease. To create opportunities for viral spread, several HCMV-encoded immunoevasins employ diverse strategies to target HLA-I. Among these, the glycoprotein US10 is so far insufficiently studied. While it was reported that US10 interferes with HLA-G expression, its ability to manipulate classical HLA-I antigen presentation remains unknown. In this study, we demonstrate that US10 recognizes and binds to all HLA-I (HLA-A, -B, -C, -E, -G) heavy chains. Additionally, impaired recruitment of HLA-I to the peptide loading complex was observed. Notably, the associated effects varied significantly dependending on HLA-I genotype and allotype: (i) HLA-A molecules evaded downregulation by US10, (ii) tapasin-dependent HLA-B molecules showed impaired maturation and cell surface expression, and (iii) β2m-assembled HLA-C, in particular HLA-C* 05:01 and -C*12:03, and HLA-G were strongly retained in complex with US10 in the endoplasmic reticulum. These genotype-specific effects on HLA-I were confirmed through unbiased HLA-I ligandome analyses. Furthermore, in HCMV-infected fibroblasts inhibition of overlapping US10 and US11 transcription had little effect on HLA-A, but induced HLA-B antigen presentation. Thus, the US10-mediated impact on HLA-I results in multiple geno- and allotypic effects in a so far unparalleled and multimodal manner. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus (CMV) infection is the leading non-genetic cause of congenital malformation in developed countries, causing significant fetal injury, and in some cases fetal death. The pathogenetic mechanisms through which this host-specific virus infects then damages both the placenta and the fetal brain are currently ill-defined. We investigated the CMV modulation of key signaling pathway proteins for these organs including dual-specificity tyrosine phosphorylation-regulated kinases (DYRK) and Sonic Hedgehog (SHH) pathway proteins using human first trimester placental trophoblast (TEV-1) cells, primary human astrocyte (NHA) brain cells, and CMV-infected human placental tissue. Immunofluorescence demonstrated the accumulation and re-localization of SHH proteins in CMV-infected TEV-1 cells with Gli2, Ulk3, and Shh re-localizing to the CMV cytoplasmic virion assembly complex (VAC). In CMV-infected NHA cells, DYRK1A re-localized to the VAC and DYRK1B re-localized to the CMV nuclear replication compartments, and the SHH proteins re-localized with a similar pattern as was observed in TEV-1 cells. Western blot analysis in CMV-infected TEV-1 cells showed the upregulated expression of Rb, Ulk3, and Shh, but not Gli2. In CMV-infected NHA cells, there was an upregulation of DYRK1A, DYRK1B, Gli2, Rb, Ulk3, and Shh. These in vitro monoculture findings are consistent with patterns of protein upregulation and re-localization observed in naturally infected placental tissue and CMV-infected ex vivo placental explant histocultures. This study reveals CMV-induced changes in proteins critical for fetal development, and identifies new potential targets for CMV therapeutic development. [ABSTRACT FROM AUTHOR]

Repression of human cytomegalovirus (HCMV) immediate-early (IE) gene expression is a key regulatory step in the establishment and maintenance of latent reservoirs. Viral IE transcription and protein accumulation can be elevated during latency by treatment with histone deacetylase inhibitors such as valproic acid (VPA), rendering infected cells visible to adaptive immune responses. However, the latency-associated viral protein UL138 inhibits the ability of VPA to enhance IE gene expression during infection of incompletely differentiated myeloid cells that support latency. UL138 also limits the accumulation of IFNβ transcripts by inhibiting the cGAS-STING-TBK1 DNA-sensing pathway. Here, we show that, in the absence of UL138, the cGAS-STING-TBK1 pathway promotes both IFNβ accumulation and VPA-responsive IE gene expression in incompletely differentiated myeloid cells. Inactivation of this pathway by either genetic or pharmacological inhibition phenocopied UL138 expression and reduced VPA-responsive IE transcript and protein accumulation. This work reveals a link between cytoplasmic pathogen sensing and epigenetic control of viral lytic phase transcription and suggests that manipulation of pattern recognition receptor signaling pathways could aid in the refinement of MIEP regulatory strategies to target latent viral reservoirs. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus is a ubiquitous herpesvirus that, while latent in most individuals, poses a great risk to immunocompromised patients. In contrast to directly acting traditional antiviral drugs, such as ganciclovir, we aim to emulate a physiological infection control using T cells. For this, we constructed several bispecific T-cell engager (BiTE) constructs targeting different viral glycoproteins of the murine cytomegalovirus and evaluated them in vitro for their efficacy. To isolate the target specific effect without viral immune evasion, we established stable reporter cell lines expressing the viral target glycoprotein B, and the glycoprotein complexes gN-gM and gH-gL, as well as nano-luciferase (nLuc). First, we evaluated binding capacities using flow cytometry and established killing assays, measuring nLuc-release upon cell lysis. All BiTE constructs proved to be functional mediators for T-cell recruitment and will allow a proof of concept for this treatment option. This might pave the way for strikingly safer immunosuppression in vulnerable patient groups. [ABSTRACT FROM AUTHOR]

Background: HCMV causes severe clinical complications in transplant recipients and may lead to graft rejection. Successful renal transplantation heavily relies on the early prevention and diagnosis of CMV infections, followed by prompt prophylactic treatment before transplantation. Despite the majority of renal rejection cases with acute HCMV infections being asymptomatic and occurring one to two years later, the objective of this research was to comprehend the effect of late HCMV infection on renal rejection by examining specific clinical parameters in the Eastern Indian cohort. Method: In this study, 240 patients were studied for five years following transplantation, and their data were collected from the local metropolitan hospital in Eastern India. Both HCMV-positive and -negative post-transplant patients were investigated using the clinical parameters and viral loads for latent infection. Results: Within the studied population, 79 post-transplant patients were found to be HCMV positive. Among them, 13 (16.45%) patients suffered from renal rejection within less than 2 yrs. of transplantation (early rejection) and 22 (27.84%) patients suffered from renal rejection after 2 yrs. from the operation date (late rejection). Assessment of clinical parameters with respect to HCMV infection revealed that in early rejection cases, fever (p-0.035) and urinary tract infection (p-0.017) were prominent, but in late rejection, hematuria (p-0.032), diabetes (p-0.005), and creatinine level changes (p < 0.001) were significant along with urinary tract infection (p-0.047). Conclusions: This study provides valuable insights into monitoring latent CMV infections and highlights the understanding of reducing renal rejection rates and the need for further research in this field. [ABSTRACT FROM AUTHOR]

Complications due to HCMV infection or reactivation remain a challenging clinical problem in immunocompromised patients, mainly due to insufficient or absent T‐cell functionality. Knowledge of viral targets is crucial to improve monitoring of high‐risk patients and optimise antiviral T‐cell therapy. To expand the epitope spectrum, genetically‐engineered dendritic cells (DCs) and fibroblasts were designed to secrete soluble (s)HLA‐A*11:01 and infected with an HCMV mutant lacking immune evasion molecules (US2‐6 + 11). More than 700 HLA‐A*11:01‐restricted epitopes, including more than 50 epitopes derived from a broad range of HCMV open‐reading‐frames (ORFs) were identified by mass spectrometry and screened for HLA‐A*11:01‐binding using established prediction tools. The immunogenicity of the 24 highest scoring new candidates was evaluated in vitro in healthy HLA‐A*11:01+/HCMV+ donors. Thus, four subdominant epitopes and one immunodominant epitope, derived from the anti‐apoptotic protein UL36 and ORFL101C (A11SAL), were identified. Their HLA‐A*11:01 complex stability was verified in vitro. In depth analyses revealed highly proliferative and cytotoxic memory T‐cell responses against A11SAL, with T‐cell responses comparable to the immunodominant HLA‐A*02:01‐restricted HCMVpp65NLV epitope. A11SAL‐specific T cells were also detectable in vivo in immunosuppressed transplant patients and shown to be effective in an in vitro HCMV‐infection model, suggesting their crucial role in inhibiting viral replication and improvement of patient's outcome. The developed in vitro pipeline is the first to utilise genetically‐engineered DCs to identify naturally presented immunodominant HCMV‐derived epitopes. It therefore offers advantages over in silico predictions, is transferable to other HLA alleles, and will significantly expand the repertoire of viral targets to improve therapeutic options. [ABSTRACT FROM AUTHOR]

Central nervous system (CNS) infections represent a challenge due to the complexities associated with their diagnosis and treatment, resulting in a high incidence rate and mortality. Here, we presented a case of CNS mixed infection involving Candida and human cytomegalovirus (HCMV), successfully diagnosed through macrogenomic next-generation sequencing (mNGS) in China. A comprehensive review and discussion of previously reported cases were also provided. Our study emphasizes the critical role of early pathogen identification facilitated by mNGS, underscoring its significance. Notably, the integration of mNGS with traditional methods significantly enhances the diagnostic accuracy of CNS infections. This integrated approach has the potential to provide valuable insights for clinical practice, facilitating early diagnosis, allowing for treatment adjustments, and ultimately, improving the prognosis for patients with CNS infections. [ABSTRACT FROM AUTHOR]

Breast cancer is the most common malignancy in the female sex; although recent therapies have significantly changed the natural history of this cancer, it remains a significant challenge. In the past decade, evidence has been put forward that some oncogenic viruses may play a role in the development of sporadic breast cancer; however, data are scattered and mostly reported as sparse case series or small case–control studies. In this review, we organize and report current evidence regarding the role of high-risk human papillomavirus, mouse mammary tumor virus, Epstein–Barr virus, cytomegalovirus, bovine leukemia virus, human polyomavirus 2, and Merkel cell polyomavirus in the pathogenesis of breast cancer. [ABSTRACT FROM AUTHOR]

ABSTRACT Cell entry is a crucial step for a virus to infect a host cell. Human cytomegalovirus utilizes glycoprotein B (gB) to fuse the viral and host cell membranes upon receptor binding of gH/gL-containing complexes. Fusion is mediated by major conformational changes of gB from a metastable pre-fusion to a stable post-fusion state whereby the central trimeric coiled-coils, formed by domain (Dom)III α helices, remain structurally nearly unchanged. To better understand the role of the stable core, we individually introduced three potentially helix-breaking or one disulfide bond-breaking mutation in the DIII α3 to study different aspects of the viral behavior upon long-term culturing. Two of the three helix-breaking mutations, gB_Y494P and gB_I495P, were lethal for the virus in either fibroblasts or epithelial cells. The third substitution, gB_G493P, on the other hand, displayed a delayed replication and spread, which was more pronounced in epithelial cells, hinting at an impaired fusion. Interestingly, the disulfide bond-breaker mutation, gB_C507S, performed strikingly differently in the two cell types - lethal in epithelial cells and an atypical phenotype in fibroblasts, respectively. Replication curve analyses paired with the infection efficiency, the spread morphology, and the cell–cell fusogenicity suggest a dysregulated fusion process, which could be reverted by second-site mutations mapping predominantly to gB DomV. Our findings underline the functional importance of a stable DomIII core for a well-regulated DomV rearrangement during fusion.IMPORTANCEHuman cytomegalovirus (HCMV) can establish a lifelong infection. In most people, the infection follows an asymptomatic course; however, it is a major cause of morbidity and mortality in immunocompromised patients or neonates. HCMV has a very broad cell tropism, ranging from fibroblasts to epi- and endothelial cells. The virus uses different entry pathways utilizing the core fusion machinery consisting of glycoprotein complexes gH/gL and glycoprotein B (gB). The fusion protein gB undergoes fundamental rearrangements from a metastable pre-fusion to a stable post-fusion conformation. Here, we characterized the viral behavior after the introduction of four single-point mutations in the gB central core. These led to various cell type-specific atypical phenotypes and the emergence of compensatory mutations, demonstrating an important interaction between domains III and V. We provide a new basis for the development of a structurally and functionally altered gB, which can further serve as a tool for drug and vaccine development.