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Abstract: Cytomegalovirus (CMV) disease occurs occasionally before allogeneic hematopoietic cell transplantation (HCT) and is associated with poor post-HCT outcomes; however, the impact of pre-HCT CMV reactivation is unknown. Pre-HCT CMV reactivation was assessed in HCT candidates from the preemptive antiviral therapy (2007-2017) and letermovir prophylaxis (2018-2021) eras. CMV DNA polymerase chain reaction (PCR) surveillance was routinely performed during the pre-HCT workup period, and antiviral therapy was recommended according to risk of progression to CMV disease. Risk factors for pre-HCT CMV reactivation were characterized, and the associations of pre-HCT CMV reactivation with post-HCT outcomes were examined using logistic regression and Cox proportional hazard models, respectively. A total of 1694 patients were identified, and 11% had pre-HCT CMV reactivation 14 days (median; interquartile range [IQR], 6-23) before HCT. Lymphopenia (≤0.3 × 103/μL) was the strongest risk factor for pre-HCT CMV reactivation at multiple PCR levels. In the preemptive therapy era, patients with pre-HCT CMV reactivation had a significantly increased risk of CMV reactivation by day 100 as well as CMV disease and death by 1 year after HCT. Clearance of pre-HCT CMV reactivation was associated with a lower risk of post-HCT CMV reactivation. Similar associations with post-HCT CMV end points were observed in a cohort of patients receiving letermovir prophylaxis. Pre-HCT CMV reactivation can be routinely detected in high-risk HCT candidates and is a significant risk factor for post-HCT CMV reactivation and disease. Pre-HCT CMV DNA PCR surveillance is recommended in high-risk HCT candidates, and antiviral therapy may be indicated to prevent post-HCT CMV reactivation., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

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., (© 2024. The Author(s).)

G-quadruplex (G4) structures are found in eukaryotic cell replication origins, but their role in origin function remains unclear. In this study G4 motifs are found in the lytic DNA replication origin (oriLyt) of human cytomegalovirus (HCMV) and recombinant viruses show that a G4 motif in oriLyt essential region I (ER-I) is necessary for viral growth. Replication assays of oriLyt-containing plasmids and biochemical/biophysical analyses show that G4 formation in ER-I is crucial for viral DNA replication. G4 pull-down analysis identifies viral DNA replication factors, such as IE2, UL84, and UL44, as G4-binding proteins. In enzyme-linked immunosorbent assays, specific G4-binding ligands inhibit G4 binding by the viral proteins. The Epstein-Barr virus oriLyt core element also forms a stable G4 that could substitute for the oriLyt ER-I G4 in HCMV. These results demonstrate that viral G4s in replication origins represent an essential structural element in recruiting replication factors and might be a therapeutic target against viral infections., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

Cytomegalovirus infection contributes to 10-30% of congenital hearing loss in children. Vertebrate peripheral auditory organs include the outer, middle, and inner ear. Their development is regulated by multiple signaling pathways. However, most ear diseases due to viral infections are due to congenital infections and reactivation and affect healthy adults to a lesser extent. This may be due to the fact that viral infections affect signaling pathways that are important for the development of peripheral hearing organs. Therefore, an in-depth understanding of the relationship between viral infections and the signaling pathways involved in the development of peripheral hearing organs is important for the prevention and treatment of ear diseases. In this review, we summarize the effects of viruses on signaling pathways and signaling molecules in the development of peripheral auditory organs.

There are no licensed vaccines for human cytomegalovirus (HCMV), and current antiviral drugs that target viral proteins are toxic and prone to resistance. Targeting host pathways essential for virus replication provides an alternate strategy that may reduce opportunities for drug resistance to occur. Oxidative stress is triggered by numerous viruses including HCMV. Peroxynitrite is a reactive nitrogen species that is formed during oxidative stress. Herein, we identified that HCMV rapidly induces the generation of intracellular peroxynitrite upon infection in a manner partially dependent upon xanthine oxidase generation. Peroxynitrite promoted HCMV infection in both cell-free and cell-associated infection systems in multiple cell types. Inhibiting peroxynitrite within the first 24 hours of infection prevented HCMV replication and peroxynitrite promoted cell entry and pp65 translocation into the host cell nuclei. Furthermore, using the murine cytomegalovirus model, we demonstrated that antagonizing peroxynitrite significantly reduces cytomegalovirus replication and pathogenesis in vivo . Overall, our study highlights a proviral role for peroxynitrite in CMV infection and implies that RNS and/or the mechanisms that induce their production could be targeted as a novel strategy to inhibit HCMV infection., Importance: Human cytomegalovirus (HCMV) causes significant disease in individuals with impaired or immature immune systems, such as transplant patients and after congenital infection. Antiviral drugs that target the virus directly are toxic and are susceptible to antiviral drug resistance due to virus mutations. An alternate strategy is to target processes within host cells that are required by the virus for replication. Herein, we show that HCMV infection triggers a highly reactive molecule, peroxynitrite, during the initial stages of infection. Peroxynitrite was required for the initial entry of the virus into the cell and promotes virus replication in multiple cell types, suggesting a broad pro-viral function. Importantly, targeting peroxynitrite dramatically inhibited cytomegalovirus replication in cells in the laboratory and in mice, suggesting that therapeutic targeting of this molecule and/or the cellular functions it regulates could represent a novel strategy to inhibit HCMV infection., Competing Interests: The authors declare no conflict of interest.

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.

Viruses are obligate intracellular pathogens as their replication depends on the metabolism of the host cell. The induction of cellular suicide, known as programmed cell death (PCD), has the potential to hinder viral replication and act as a first line of defense against viral pathogens. Apoptosis, necroptosis, and pyroptosis are three important PCD modalities. Different signaling pathways are involved in their execution, and they also differ in their ability to cause inflammation. Cytomegaloviruses (CMV), beta-herpesviruses with large double-stranded DNA genomes, encode a great variety of immune evasion genes, including several cell death suppressors. While CMV inhibitors of apoptosis and necroptosis have been known and studied for years, the first pyroptosis inhibitor has been identified and characterized only recently. Here, we describe how human and murine CMV interfere with apoptosis, necroptosis, and pyroptosis signaling pathways. We also discuss the importance of the different PCD forms and their viral inhibitors for the containment of viral replication and spread in vivo.

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: Cytomegalovirus (CMV) reactivation is a major complication among seropositive allogeneic hematopoietic cell transplantation recipients; however, data on CMV reactivation after chimeric antigen receptor (CAR) T-cell therapy are limited. We report the incidence and outcomes of 95 adult CMV-seropositive patients who received CAR T-cell therapy between February 2018 and February 2023. CMV outcomes were CMV reactivation (any viremia) and clinically significant CMV infection (cs-CMV). Thirty-one patients (33%) had evidence of CMV reactivation (any viremia), and 10 patients (11%) had cs-CMV. The median time from CAR T-cell infusion to CMV reactivation was 19 days (interquartile range [IQR], 9-31). The cumulative incidence of CMV (any viremia) was significantly higher among patients with grade 3 to 4 cytokine release syndrome (67 vs 28%; P = .01), and those who received corticosteroids (39 vs 21%; P = .03), anakinra (56 vs 28%; P = .02), or ≥2 immunosuppressants (41 vs 21%; P = .02). Receipt of corticosteroids (18 vs 0%; P = .004), tocilizumab (14 vs 0%; P = .04), anakinra (33 vs 7%; P = .008), and ≥2 immunosuppressants (20 vs 0%; P = .001) were all associated with cs-CMV. Receiving ≥2 immunosuppressants was associated with a twofold increase in CMV reactivation in multivariate analyses (adjusted odds ratio [aOR], 2.27; 95% confidence interval, 1.1-4.8; P = .03). Overall, the 1-year mortality was significantly higher in those with CMV reactivation (57% vs 23%; P = .001). Immunosuppression, particularly with corticosteroids, for the management of CAR T-cell toxicities, is a major risk factor for CMV reactivation., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

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., Competing Interests: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Human cytomegalovirus (HCMV), a widely prevalent human beta-herpesvirus, establishes lifelong persistence in the host following primary infection. In healthy individuals, the virus is effectively controlled by HCMV-specific T cells and typically exhibits asymptomatic. The T cell immune response plays a pivotal role in combating HCMV infection, while HCMV employs various strategies to counteract it within the host. Previously, we reported that UL23, a tegument protein of HCMV, facilitates viral immune evasion from interferon-gamma (IFN-γ) responses, and it is well known that IFN-γ is mainly derived from T cells. However, the involvement of UL23 in viral immune evasion from T cell-mediated immunity remains unclear. Herein, we present compelling evidence that UL23 significantly enhances viral resistance against T cell-mediated cytotoxicity during HCMV infection from the co-culture assays of HCMV-infected cells with T cells. We found that IFN-γ plays a major role in regulating T cell cytotoxicity mediated by UL23. More interestingly, we demonstrated that UL23 not only regulates the IFN-γ downstream responses but also modulates the IFN-γ secretion by regulating T cell activities. Further experiments indicate that UL23 upregulates the expression and signaling of programmed death ligand 1 (PD-L1), which is responsible for inhibiting multiple aspects of T cell activities, including activation, apoptosis, and IFN-γ secretion, as determined through RNA-seq analysis and inhibitor-blocking experiments, ultimately facilitating viral replication and spread. Our findings highlight the potential role of UL23 as an alternative antagonist in suppressing T cell cytotoxicity and unveil a novel strategy for HCMV to evade T cell immunity., Importance: T cell immunity is pivotal in controlling primary human cytomegalovirus (HCMV) infection, restricting periodic reactivation, and preventing HCMV-associated diseases. Despite inducing a robust T cell immune response, HCMV has developed sophisticated immune evasion mechanisms that specifically target T cell responses. Although numerous studies have been conducted on HCMV-specific T cells, the primary focus has been on the impact of HCMV on T cell recognition via major histocompatibility complex molecules. Our studies show for the first time that HCMV exploits the programmed death ligand 1 (PD-L1) inhibitory signaling pathway to evade T cell immunity by modulating the activities of T cells and thereby blocking the secretion of IFN-γ, which is directly mediated by HCMV-encoded tegument protein UL23. While PD-L1 has been extensively studied in the context of tumors and viruses, its involvement in HCMV infection and viral immune evasion is rarely reported. We observed an upregulation of PD-L1 in normal cells during HCMV infection and provided strong evidence supporting its critical role in UL23-induced inhibition of T cell-mediated cytotoxicity. The novel strategy employed by HCMV to manipulate the inhibitory signaling pathway of T cell immune activation for viral evasion through its encoded protein offers valuable insights for the understanding of HCMV-mediated T cell immunomodulation and developing innovative antiviral treatment strategies., Competing Interests: The authors declare no conflict of interest.

Despite the significant progress made, CMV infection is one of the most frequent infectious complications in transplant recipients. CMV infections that become refractory or resistant (R/R) to the available antiviral drugs constitute a clinical challenge and are associated with increased morbidity and mortality. Novel anti-CMV therapies have been recently developed and introduced in clinical practice, which may improve the treatment of these infections. In this review, we summarize the treatment options for R/R CMV infections in adult hematopoietic cell transplant and solid organ transplant recipients, with a special focus on newly available antiviral agents with anti-CMV activity, including maribavir and letermovir.

Background: Viral reactivations are frequent in hematologial patients due to their cancer-related and drug-induced immunosuppressive status. Daratumumab, an anti-CD38 monoclonal antibody, is used for multiple myeloma (MM) treatment, and causes immunosuppression by targeting CD38-expressing normal lymphocytes. In this single-center two-arm real-life experience, we evaluated incidence of cytomegalovirus (CMV) reactivation in MM patients treated with daratumumab-based regimens as first- or second-line therapy., Methods: A total of 101 consecutive MM patients were included in this study and were divided into two cohorts: daratumumab and nondaratumumab-based (control) regimens. Patients treated with >2 lines of therapies were excluded to reduce the confounding factor of multi-treated cases. Primary endpoint was the CMV reactivation rate., Results: CMV reactivation rate was significantly higher in the daratumumab cohort compared to control group (33% vs. 4%; p < 0.001), also with higher CMV-DNA levels (>1000 UI/mL in 12% of cases; p < 0.05). However, only one subject developed a CMV disease with severe pneumonia, while 12% of patients were successfully treated with preemptive therapy with valganciclovir. No subjects in the control cohort required anti-CMV agents (p = 0.02)., Conclusion: Our single-center retrospective experience showed that daratumumab might significantly increase the risk of CMV reactivation in MM, while currently underestimated and related to morbility and mortality in MM patients under treatments. However, further validation on larger and prospective clinical trials are required., (© 2024 The Author(s). Cancer Medicine published by John Wiley & Sons Ltd.)

During the birch pollen season an enhanced incidence of virus infections is noticed, raising the question whether pollen can affect anti-viral responses independent of allergic reactions. We previously showed that birch pollen-treatment of monocyte-derived dendritic cells (moDC) enhances human cytomegalovirus (HCMV) infection. Here we addressed how in moDC the relatively weak pollen response can affect the comparably strong response to HCMV. To this end, moDC were stimulated with aqueous birch pollen extract (APE), HCMV, and APE with HCMV, and transcriptomic signatures were determined after 6 and 24 h of incubation. Infection was monitored upon exposure of moDC to GFP expressing HCMV by flow cytometric analysis of GFP expressing cells. Principle component analysis of RNA sequencing data revealed close clustering of mock and APE treated moDC, whereas HCMV as well as APE with HCMV treated moDC clustered separately after 6 and 24 h of incubation, respectively. Communally induced genes were detected in APE, HCMV and APE with HCMV treated moDC. In APE with HCMV treated moDC, the comparably weak APE induced signatures were maintained after HCMV exposure. In particular, NF-κB/RELA and PI3K/AKT/MAPK signaling were altered upon APE with HCMV exposure. Earlier, we discovered that NF-κB inhibition alleviated APE induced enhancement of HCMV infection. Here we additionally found that impairment of PI3K signaling reduced HCMV infection in HCMV and APE with HCMV treated moDC. APE treated moDC that were exposed to HCMV show a unique host gene signature, which to a large extent is regulated by NF-κB activation and PI3K/AKT/MAPK signaling., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Butt et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Herpesvirus genomes are maintained as extrachromosomal plasmids within the nuclei of infected cells. Some herpesviruses persist within dividing cells, putting the viral genome at risk of being lost to the cytoplasm during mitosis because karyokinesis (nuclear division) requires nuclear envelope breakdown. Oncogenic herpesviruses (and papillomaviruses) avoid genome loss during mitosis by tethering their genomes to cellular chromosomes, thereby ensuring viral genome uptake into newly formed nuclei. These viruses use viral proteins with DNA- and chromatin-binding capabilities to physically link viral and cellular genomes together in a process called tethering. The known viral tethering proteins of human papillomavirus (E2), Epstein-Barr virus (EBNA1), and Kaposi's sarcoma-associated herpesvirus (LANA) each contain two independent domains required for genome tethering, one that binds sequence specifically to the viral genome and another that binds to cellular chromatin. This latter domain is called a chromatin tethering domain (CTD). The human cytomegalovirus UL123 gene encodes a CTD that is required for the virus to productively infect dividing fibroblast cells within the S phase of the cell cycle, presumably by tethering the viral genome to cellular chromosomes during mitosis. The CTD-containing UL123 gene product that supports S-phase infections is the IE19 protein. Here, we define two motifs in IE19 required for S-phase infections: an N-terminal triple lysine motif and a C-terminal nucleosome-binding motif within the CTD.IMPORTANCEThe IE19 protein encoded by human cytomegalovirus (HCMV) is required for S-phase infection of dividing cells, likely because it tethers the viral genome to cellular chromosomes, thereby allowing them to survive mitosis. The mechanism through which IE19 tethers viral genomes to cellular chromosomes is not understood. For human papillomavirus, Epstein-Barr virus, and Kaposi's sarcoma-associated herpesvirus, viral genome tethering is required for persistence (latency) and pathogenesis (oncogenesis). Like these viruses, HCMV also achieves latency, and it modulates the properties of glioblastoma multiforme tumors. Therefore, defining the mechanism through which IE19 tethers viral genomes to cellular chromosomes may help us understand, and ultimately combat or control, HCMV latency and oncomodulation., Competing Interests: The authors declare no conflict of interest.

Background: Cytomegalovirus (CMV) reactivation is a significant concern following allogeneic stem cell transplantation. While previous research has highlighted the anti-CMV reactivation effect of γδ T cells in immunocompromised transplant patients, their characterization in recipients at high risk of CMV reactivation remains limited., Methods: This study focused on D+/R+ recipients (where both donor and recipient are CMV seropositive) at high risk of CMV reactivation. We analyzed 28 patients who experienced CMV recurrence within 100 days post-allogeneic hematopoietic stem cell transplantation, along with 36 matched recipients who did not experience CMV recurrence. Clinical data from both groups were compared, and risk factors for CMV reactivation were identified. Additionally, CMV viral load was measured, and flow cytometric analysis was conducted to assess changes in peripheral blood γδ T cell proportions, subpopulation distribution, and differentiation status. We also analyzed the CDR3 repertoire of the TCR δ chain in different γδ T cell subsets. Functional analysis was performed by measuring the lysis of CMV-infected cells upon stimulation., Results: CMV reactivation post-transplantation was associated with acute graft-versus-host disease (aGvHD) and reactivation of non-CMV herpesviruses. Notably, CMV reactivation led to sustained expansion of γδ T cells, primarily within the Vδ2 neg γδ T cell subpopulation, with a trend toward differentiation from Naive to effector memory cells. Analysis of the δ chain CDR3 repertoire revealed a delay in the reconstitution of clonal diversity in Vδ2 neg γδ T cells following CMV reactivation, while Vδ2 pos T cells remained unaffected. Upon stimulation with CMV-infected MRC5 cells, the Vδ2 neg γδ T cell subpopulation emerged as the primary effector cell group producing IFN-γ and capable of lysing CMV-infected cells. Moreover, our findings suggest that NKG2D is not necessary involved in Vδ2 neg γδ T cell-mediated anti-CMV cytotoxicity., Conclusion: This study provides novel insights into the role of γδ T cells in the immune response to CMV reactivation in transplantation recipients at high risk of CMV infection. Specifically, the Vδ2 neg γδ T cell subpopulation appears to be closely associated with CMV reactivation, underscoring their potential role in controlling infection and reflecting CMV reactivation in HSCT patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Huang, Jiang, Zhu, Lin, Mao, Yin and Cai.)

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.

Genetic screens have transformed our ability to interrogate cellular factor requirements for viral infections 1,2 , but most current approaches are limited in their sensitivity, biased towards early stages of infection and provide only simplistic phenotypic information that is often based on survival of infected cells 2-4 . Here, by engineering human cytomegalovirus to express single guide RNA libraries directly from the viral genome, we developed virus-encoded CRISPR-based direct readout screening (VECOS), a sensitive, versatile, viral-centric approach that enables profiling of different stages of viral infection in a pooled format. Using this approach, we identified hundreds of host dependency and restriction factors and quantified their direct effects on viral genome replication, viral particle secretion and infectiousness of secreted particles, providing a multi-dimensional perspective on virus-host interactions. These high-resolution measurements reveal that perturbations altering late stages in the life cycle of human cytomegalovirus (HCMV) mostly regulate viral particle quality rather than quantity, establishing correct virion assembly as a critical stage that is heavily reliant on virus-host interactions. Overall, VECOS facilitates systematic high-resolution dissection of the role of human proteins during the infection cycle, providing a roadmap for in-depth study of host-herpesvirus interactions., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

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.

Backgrounds: In critically ill patients with COVID-19, secondary infections are potentially life-threatening complications. This study aimed to determine the prevalence, clinical characteristics, and risk factors of CMV reactivation among critically ill immunocompetent patients with COVID-19 pneumonia., Methods: A retrospective cohort study was conducted among adult patients who were admitted to ICU and screened for quantitative real-time PCR for CMV viral load in a tertiary-care hospital during the third wave of the COVID-19 outbreak in Thailand. Cox regression models were used to identify significant risk factors for developing CMV reactivation., Results: A total of 185 patients were studied; 133 patients (71.9%) in the non-CMV group and 52 patients (28.1%) in the CMV group. Of all, the mean age was 64.7±13.3 years and 101 patients (54.6%) were males. The CMV group had received a significantly higher median cumulative dose of corticosteroids than the non-CMV group (301 vs 177 mg of dexamethasone, p<0.001). Other modalities of treatments for COVID-19 including anti-viral drugs, anti-cytokine drugs and hemoperfusion were not different between the two groups (p>0.05). The 90-day mortality rate for all patients was 29.1%, with a significant difference between the CMV group and the non-CMV group (42.3% vs. 24.1%, p = 0.014). Median length of stay was longer in the CMV group than non-CMV group (43 vs 24 days, p<0.001). The CMV group has detectable CMV DNA load with a median [IQR] of 4,977 [1,365-14,742] IU/mL and 24,570 [3,703-106,642] in plasma and bronchoalveolar fluid, respectively. In multivariate analysis, only a cumulative corticosteroids dose of dexamethasone ≥250 mg (HR = 2.042; 95%CI, 1.130-3.688; p = 0.018) was associated with developing CMV reactivation., Conclusion: In critically ill COVID-19 patients, CMV reactivation is frequent and a high cumulative corticosteroids dose is a significant risk factor for CMV reactivation, which is associated with poor outcomes. Further prospective studies are warranted to determine optimal management., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Tassaneeyasin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

DNA assays for viral load (VL) monitoring are key tools in the management of immunocompromised patients with cytomegalovirus (CMV) or Epstein-Barr virus (EBV) infection. In this study, the analytical and clinical performances of the NeuMoDx™ CMV and EBV Quant Assays were compared with artus CMV and EBV QS-RGQ Kits in a primary hospital testing laboratory. Patient plasma samples previously tested using artus kits were randomly selected for testing by NeuMoDx assays. The NeuMoDx CMV Quant Assay and artus CMV QS-RGQ Kit limits of detection (LoDs) are 20.0 IU/mL and 69.7 IU/mL, respectively; 33/75 (44.0%) samples had CMV DNA levels above the LoD of both assays. The Pearson correlation coefficient was 0.9503; 20 samples (60.6%) had lower NeuMoDx CMV quantification values versus the artus kit. The LoD of the NeuMoDx EBV Quant Assay and artus EBV QS-RGQ Kit are 200 IU/mL and 22.29 IU/mL, respectively; 16/75 (21.3%) samples had EBV DNA levels above the LoD of both assays. The Pearson correlation coefficient was 0.8990. EBV quantification values with the NeuMoDx assay were higher versus the artus kit in 15 samples (93.8%). In conclusion, NeuMoDx CMV and EBV Quant Assays are sensitive and accurate tools for CMV and EBV DNA VL quantification.

Human cytomegalovirus (HCMV) is an important human pathogen that regulates host immunity and hijacks host compartments, including lysosomes, to assemble virions. We combined a quantitative proteomic analysis of HCMV infection with a database of proteins involved in vacuolar acidification, revealing Dmx-like protein-1 (DMXL1) as the only protein that acidifies vacuoles yet is degraded by HCMV. Systematic comparison of viral deletion mutants reveals the uncharacterized 7 kDa US33A protein as necessary and sufficient for DMXL1 degradation, which occurs via recruitment of the E3 ubiquitin ligase Kip1 ubiquitination-promoting complex (KPC). US33A-mediated DMXL1 degradation inhibits lysosome acidification and autophagic cargo degradation. Formation of the virion assembly compartment, which requires lysosomes, occurs significantly later with US33A-expressing virus infection, with reduced viral replication. These data thus identify a viral strategy for cellular remodeling, with the potential to employ US33A in therapies for viral infection or rheumatic conditions, in which inhibition of lysosome acidification can attenuate disease., Competing Interests: Declaration of interests D.C.R. is a consultant for Aladdin Healthcare Technologies Ltd., Mindrank AI, Nido Biosciences, Drishti Discoveries, Retro Biosciences and PAQ Therapeutics., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Human cytomegalovirus (CMV) infection is the leading non-genetic aetiology of congenital malformation in developed countries, causing significant fetal neurological injury. This study investigated potential CMV pathogenetic mechanisms of fetal neural malformation using in vitro human cerebral organoids. Cerebral organoids were permissive to CMV replication, and infection dysregulated cellular pluripotency and differentiation pathways. Aberrant expression of dual-specificity tyrosine phosphorylation-regulated kinases (DYRK), sonic hedgehog (SHH), pluripotency, neurodegeneration, axon guidance, hippo signalling and dopaminergic synapse pathways were observed in CMV-infected organoids using immunofluorescence and RNA-sequencing. Infection with CMV resulted in dysregulation of 236 Autism Spectrum Disorder (ASD)-related genes (p = 1.57E-05) and pathways. This notable observation suggests potential links between congenital CMV infection and ASD. Using DisGeNET databases, 103 diseases related to neural malformation or mental disorders were enriched in CMV-infected organoids. Cytomegalovirus infection-related dysregulation of key cerebral cellular pathways potentially provides important, modifiable pathogenetic mechanisms for congenital CMV-induced neural malformation and ASD., (© 2024. The Author(s).)

Human cytomegalovirus (HCMV) is a widespread pathogen that in immunocompromised hosts can cause life-threatening disease. Studying HCMV-exposed monocyte-derived dendritic cells by single-cell RNA sequencing, we observe that most cells are entered by the virus, whereas less than 30% of them initiate viral gene expression. Increased viral gene expression is associated with activation of the stimulator of interferon genes (STING) that usually induces anti-viral interferon responses, and with the induction of several pro- (RHOB, HSP1A1, DNAJB1) and anti-viral (RNF213, TNFSF10, IFI16) genes. Upon progression of infection, interferon-beta but not interferon-lambda transcription is inhibited. Similarly, interferon-stimulated gene expression is initially induced and then shut off, thus further promoting productive infection. Monocyte-derived dendritic cells are composed of 3 subsets, with one being especially susceptible to HCMV. In conclusion, HCMV permissiveness of monocyte-derived dendritic cells depends on complex interactions between virus sensing, regulation of the interferon response, and viral gene expression., (© 2024. The Author(s).)

Human cytomegalovirus (HCMV) utilizes peripheral blood monocytes as a means to systemically disseminate throughout the host. Following viral entry, HCMV stimulates non-canonical Akt signaling leading to the activation of mTORC1 and the subsequent translation of select antiapoptotic proteins within infected monocytes. However, the full extent to which the HCMV-initiated Akt/mTORC1 signaling axis reshapes the monocyte translatome is unclear. We found HCMV entry alone was able to stimulate widescale changes to mRNA translation levels and that inhibition of mTOR, a component of mTORC1, dramatically attenuated HCMV-induced protein synthesis. Although monocytes treated with normal myeloid growth factors also exhibited increased levels of translation, mTOR inhibition had no effect, suggesting HCMV activation of mTOR stimulates the acquisition of a unique translatome within infected monocytes. Indeed, polyribosomal profiling of HCMV-infected monocytes identified distinct prosurvival transcripts that were preferentially loaded with ribosomes when compared to growth factor-treated cells. Sirtuin 1 (SIRT1), a deacetylase that exerts prosurvival effects through regulation of the PI3K/Akt pathway, was found to be highly enriched following HCMV infection in an mTOR-dependent manner. Importantly, SIRT1 inhibition led to the death of HCMV-infected monocytes while having minimal effect on uninfected cells. SIRT1 also supported a positive feedback loop to sustain Akt/mTORC1 signaling following viral entry. Taken together, HCMV profoundly reshapes mRNA translation in an mTOR-dependent manner to enhance the synthesis of select factors necessary for the survival of infected monocytes.IMPORTANCEHuman cytomegalovirus (HCMV) infection is a significant cause of morbidity and mortality among the immunonaïve and immunocompromised. Peripheral blood monocytes are a major cell type responsible for disseminating the virus from the initial site of infection. In order for monocytes to mediate viral spread within the host, HCMV must subvert the naturally short lifespan of these cells. In this study, we performed polysomal profiling analysis, which demonstrated HCMV to globally redirect mRNA translation toward the synthesis of cellular prosurvival factors within infected monocytes. Specifically, HCMV entry into monocytes induced the translation of cellular SIRT1 to generate an antiapoptotic state. Defining the precise mechanisms through which HCMV stimulates survival will provide insight into novel anti-HCMV drugs able to target infected monocytes., Competing Interests: The authors declare no conflict of interest.

Human cytomegalovirus (HCMV) requires the robust expression of two immediate early proteins, IE1 and IE2, immediately upon infection to suppress the antiviral response and promote viral gene expression. While transcriptional control of IE1 and IE2 has been extensively studied, the role of post-transcriptional regulation of IE1 and IE2 expression is relatively unexplored. We previously found that the shared major immediate early 5' untranslated region (MIE 5' UTR) of the mature IE1 and IE2 transcripts plays a critical role in facilitating the translation of the IE1 and IE2 mRNAs. As RNA secondary structure in 5' UTRs can regulate mRNA translation efficiency, we used selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) to identify RNA structures in the shared MIE 5' UTR. We found that the MIE 5' UTR contains three stable stem loop structures. Using a series of recombinant viruses to investigate the role of each stem loop in IE1 and IE2 protein synthesis, we found that the stem loop closest to the 5' end of the MIE 5' UTR (SL1) is both necessary and sufficient for efficient IE1 and IE2 mRNA translation and HCMV replication. The positive effect of SL1 on mRNA translation and virus replication was dependent on its location within the 5' UTR. Surprisingly, a synthetic stem loop with the same free energy as SL1 in its native location also supported wild type levels of IE1 and IE2 mRNA translation and virus replication, suggesting that the presence of RNA structure at a specific location in the 5' UTR, rather than the primary sequence of the RNA, is critical for efficient IE1 and IE2 protein synthesis. These data reveal a novel post-transcriptional regulatory mechanism controlling IE1 and IE2 expression and reinforce the critical role of RNA structure in regulating HCMV protein synthesis and replication.IMPORTANCEThese results reveal a new aspect of immediate early gene regulation controlled by non-coding RNA structures in viral mRNAs. Previous studies have largely focused on understanding viral gene expression at the level of transcriptional control. Our results show that a complete understanding of the control of viral gene expression must include an understanding of viral mRNA translation, which is driven in part by RNA structure(s) in the 5' UTR of viral mRNAs. Our results illustrate the importance of these additional layers of regulation by defining specific 5' UTR RNA structures regulating immediate early gene expression in the context of infection and identify important features of RNA structure that govern viral mRNA translation efficiency. These results may therefore broadly impact current thinking on how viral gene expression is regulated for human cytomegalovirus and other DNA viruses., Competing Interests: The authors declare no conflict of interest.

Programmed necrosis is an integral part of intrinsic immunity, serving to combat invading pathogens and restricting viral dissemination. The orchestration of necroptosis relies on a precise interplay within the necrosome complex, which consists of RIPK1, RIPK3 and MLKL. Human cytomegalovirus (HCMV) has been found to counteract the execution of necroptosis during infection. In this study, we identify the immediate-early 1 (IE1) protein as a key antagonist of necroptosis during HCMV infection. Infection data obtained in a necroptosis-sensitive cell culture system revealed a robust regulation of post-translational modifications (PTMs) of the necrosome complex as well as the importance of IE1 expression for an effective counteraction of necroptosis. Interaction analyses unveiled an association of IE1 and RIPK3, which occurs in an RHIM-domain independent manner. We propose that this interaction manipulates the PTMs of RIPK3 by promoting its ubiquitination. Furthermore, IE1 was found to exert an indirect activity by modulating the levels of MLKL via antagonizing its interferon-mediated upregulation. Overall, we claim that IE1 performs a broad modulation of innate immune signaling to impede the execution of necroptotic cell death, thereby generating a favorable environment for efficient viral replication.

Individuals differ widely in their immune responses, with age, sex and genetic factors having major roles in this inherent variability 1-6 . However, the variables that drive such differences in cytokine secretion-a crucial component of the host response to immune challenges-remain poorly defined. Here we investigated 136 variables and identified smoking, cytomegalovirus latent infection and body mass index as major contributors to variability in cytokine response, with effects of comparable magnitudes with age, sex and genetics. We find that smoking influences both innate and adaptive immune responses. Notably, its effect on innate responses is quickly lost after smoking cessation and is specifically associated with plasma levels of CEACAM6, whereas its effect on adaptive responses persists long after individuals quit smoking and is associated with epigenetic memory. This is supported by the association of the past smoking effect on cytokine responses with DNA methylation at specific signal trans-activators and regulators of metabolism. Our findings identify three novel variables associated with cytokine secretion variability and reveal roles for smoking in the short- and long-term regulation of immune responses. These results have potential clinical implications for the risk of developing infections, cancers or autoimmune diseases., (© 2024. The Author(s).)

Importance: Viruses modulate host cell metabolism to support the mass production of viral progeny. For human cytomegalovirus, we find that the viral U L 38 protein is critical for driving these pro-viral metabolic changes. However, our results indicate that these changes come at a cost, as U L 38 induces an anabolic rigidity that leads to a metabolic vulnerability. We find that U L 38 decouples the link between glucose availability and fatty acid biosynthetic activity. Normal cells respond to glucose limitation by down-regulating fatty acid biosynthesis. Expression of U L 38 results in the inability to modulate fatty acid biosynthesis in response to glucose limitation, which results in cell death. We find this vulnerability in the context of viral infection, but this linkage between fatty acid biosynthesis, glucose availability, and cell death could have broader implications in other contexts or pathologies that rely on glycolytic remodeling, for example, oncogenesis., Competing Interests: The authors declare no conflict of interest.

Human cytomegalovirus (HCMV) is a paradigm of pathogen immune evasion and sustains lifelong persistent infection in the face of exceptionally powerful host immune responses through the concerted action of multiple immune-evasins. These reduce NK cell activation by inhibiting ligands for activating receptors, expressing ligands for inhibitory receptors, or inhibiting synapse formation. However, these functions only inhibit direct interactions with the infected cell. To determine whether the virus also expresses soluble factors that could modulate NK function at a distance, we systematically screened all 170 HCMV canonical protein-coding genes. This revealed that UL4 encodes a secreted and heavily glycosylated protein (gpUL4) that is expressed with late-phase kinetics and is capable of inhibiting NK cell degranulation. Analyses of gpUL4 binding partners by mass spectrometry identified an interaction with TRAIL. gpUL4 bound TRAIL with picomolar affinity and prevented TRAIL from binding its receptor, thus acting as a TRAIL decoy receptor. TRAIL is found in both soluble and membrane-bound forms, with expression of the membrane-bound form strongly up-regulated on NK cells in response to interferon. gpUL4 inhibited apoptosis induced by soluble TRAIL, while also binding to the NK cell surface in a TRAIL-dependent manner, where it blocked NK cell degranulation and cytokine secretion. gpUL4 therefore acts as an immune-evasin by inhibiting both soluble and membrane-bound TRAIL and is a viral-encoded TRAIL decoy receptor. Interestingly, gpUL4 could also suppress NK responses to heterologous viruses, suggesting that it may act as a systemic virally encoded immunosuppressive agent., Competing Interests: Competing interests statement:The authors declare no competing interest.

COVID-19-associated invasive pulmonary aspergillosis (CAPA) is common and is associated with poor outcomes in critically ill patients. This prospective observational study aimed to explore the association between CAPA development and the incidence and prognosis of cytomegalovirus (CMV) reactivation in critically ill COVID-19 patients. We included all consecutive critically ill adult patients with confirmed COVID-19 infection who were admitted to three COVID-19 intensive care units (ICUs) in an Italian hospital from 25 February 2020 to 8 May 2022. A standardized procedure was employed for early detection of CAPA. Risk factors associated with CAPA and CMV reactivation and the association between CMV recurrence and mortality were estimated using adjusted Cox proportional hazard regression models. CAPA occurred in 96 patients (16.6%) of the 579 patients analyzed. Among the CAPA population, 40 (41.7%) patients developed CMV blood reactivation with a median time of 18 days (IQR 7-27). The CAPA+CMV group did not exhibit a significantly higher 90-day mortality rate (62.5% vs. 48.2%) than the CAPA alone group ( p = 0.166). The CAPA+CMV group had a longer ICU stay, fewer ventilation-free days, and a higher rate of secondary bacterial infections than the control group of CAPA alone. In the CAPA population, prior immunosuppression was the only independent risk factor for CMV reactivation (HR 2.33, 95% C.I. 1.21-4.48, p = 0.011). In critically ill COVID-19 patients, CMV reactivation is common in those with a previous CAPA diagnosis. Basal immunosuppression before COVID-19 appeared to be the primary independent variable affecting CMV reactivation in patients with CAPA. Furthermore, the association of CAPA+CMV versus CAPA alone appears to impact ICU length of stay and secondary bacterial infections but not mortality.

Cytomegalovirus (CMV) infection is one of the severe opportunistic infections faced by severely immunocompromised patients. High viral loads cause tissue-invasive disease and expose to death or various indirect effects. Substantial progress was made in monitoring active infection, and antiviral drugs were developed. However, dose-limiting toxicities and genotypic resistance limit therapeutic efficacy and vaccine development is hampered by the complex biology of the virus. In this issue of EMBO Molecular Medicine, Kandalla et al (2023) suggest an innovative strategy using the cytokine macrophage colony-stimulating factor (M-CSF) whose clinical development was left behind two decades ago. By stimulating an endogenous immune defense mechanism, M-CSF promotes viral clearance in a mouse model of hematopoietic stem cell transplantation, without impairing stem cell engraftment. These results reactivate the interest in the potential therapeutic use of this cytokine., (© 2023 The Author. Published under the terms of the CC BY 4.0 license.)

Importance: CD34 + hematopoietic progenitor cells (HPCs) are an important cellular reservoir for latent human cytomegalovirus (HCMV). Several HCMV genes are expressed during latency that are involved with the maintenance of the viral genome in CD34 + HPC. However, little is known about the process of viral reactivation in these cells. Here, we describe a viral protein, pUL8, and its interaction and stabilization with members of the Wnt/β-catenin pathway as an important component of viral reactivation. We further define that pUL8 and β-catenin interact with DVL2 via a PDZ-binding domain, and loss of UL8 interaction with β-catenin-DVL2 restricts viral reactivation. Our findings will be instrumental in understanding the molecular processes involved in HCMV reactivation in order to design new antiviral therapeutics., Competing Interests: The authors declare no conflict of interest.

Importance: Human cytomegalovirus (HCMV) infection is the leading cause of non-heritable birth defects worldwide. HCMV readily infects the early progenitor cell population of the developing brain, and we have found that infection leads to significantly downregulated expression of key neurodevelopmental transcripts. Currently, there are no approved therapies to prevent or mitigate the effects of congenital HCMV infection. Therefore, we used human-induced pluripotent stem cell-derived organoids and neural progenitor cells to elucidate the glycoproteins and receptors used in the viral entry process and whether antibody neutralization was sufficient to block viral entry and prevent disruption of neurodevelopmental gene expression. We found that blocking viral entry alone was insufficient to maintain the expression of key neurodevelopmental genes, but neutralization combined with neurotrophic factor treatment provided robust protection. Together, these studies offer novel insight into mechanisms of HCMV infection in neural tissues, which may aid future therapeutic development., Competing Interests: The authors declare no conflict of interest.

Importance: Human cytomegalovirus (HCMV) requires inactivation of AKT to efficiently replicate, yet how AKT is shut off during HCMV infection has remained unclear. We show that UL38, an HCMV protein that activates mTORC1, is necessary and sufficient to destabilize insulin receptor substrate 1 (IRS1), a model insulin receptor substrate (IRS) protein. Degradation of IRS proteins in settings of excessive mTORC1 activity is an important mechanism for insulin resistance. When IRS proteins are destabilized, PI3K cannot be recruited to growth factor receptor complexes, and hence, AKT membrane recruitment, a rate limiting step in its activation, fails to occur. Despite its penchant for remodeling host cell signaling pathways, our results reveal that HCMV relies upon a cell-intrinsic negative regulatory feedback loop to inactivate AKT. Given that pharmacological inhibition of PI3K/AKT potently induces HCMV reactivation from latency, our findings also imply that the expression of UL38 activity must be tightly regulated within latently infected cells to avoid spontaneous reactivation., Competing Interests: The authors declare no conflict of interest.

Cytomegalovirus (CMV) is the most common congenital infection and cause of birth defects worldwide. Primary CMV infection during pregnancy leads to a higher frequency of congenital CMV (cCMV) than maternal re-infection, suggesting that maternal immunity confers partial protection. However, poorly understood immune correlates of protection against placental transmission contributes to the current lack of an approved vaccine to prevent cCMV. In this study, we characterized the kinetics of maternal plasma rhesus CMV (RhCMV) viral load (VL) and RhCMV-specific antibody binding and functional responses in a group of 12 immunocompetent dams with acute, primary RhCMV infection. We defined cCMV transmission as RhCMV detection in amniotic fluid (AF) by qPCR. We then leveraged a large group of past and current primary RhCMV infection studies in late-first/early-second trimester RhCMV-seronegative rhesus macaque dams, including immunocompetent (n = 15), CD4+ T cell-depleted with (n = 6) and without (n = 6) RhCMV-specific polyclonal IgG infusion before infection to evaluate differences between RhCMV AF-positive and AF-negative dams. During the first 3 weeks after infection, the magnitude of RhCMV VL in maternal plasma was higher in AF-positive dams in the combined cohort, while RhCMV glycoprotein B (gB)- and pentamer-specific binding IgG responses were lower magnitude compared to AF-negative dams. However, these observed differences were driven by the CD4+ T cell-depleted dams, as there were no differences in plasma VL or antibody responses between immunocompetent AF-positive vs AF-negative dams. Overall, these results suggest that levels of neither maternal plasma viremia nor humoral responses are associated with cCMV following primary maternal infection in healthy individuals. We speculate that other factors related to innate immunity are more important in this context as antibody responses to acute infection likely develop too late to influence vertical transmission. Yet, pre-existing CMV glycoprotein-specific and neutralizing IgG may provide protection against cCMV following primary maternal CMV infection even in high-risk, immunocompromised settings., Competing Interests: SRP provides individual consulting services to Moderna, Merck, Dynavax, Pfizer, GlaxoSmithKline (CMV vaccines) and HOOKIPA Biotech GmbH. She has also led sponsored research programs with Merck Vaccines and Moderna. Oregon Health Sciences University, KF and DM have a substantial financial interest in Vir Biotechnology, Inc. a company that may have a commercial interest in the results of this research and technology. KF and DM are co-inventors of several patents licensed to Vir Biotechnology. KF is also consultants to Vir Biotechnology, Inc. All potential conflicts of interest have been reviewed and managed by OHSU. These competing interests will not alter our adherence to PLOS policies on data and material sharing., (Copyright: © 2023 Otero et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Human cytomegalovirus (HCMV) encodes multiple putative G protein-coupled receptors (GPCRs). US28 functions as a viral chemokine receptor and is expressed during both latent and lytic phases of virus infection. US28 actively promotes cellular migration, transformation, and plays a major role in mediating viral latency and reactivation; however, knowledge about the interaction partners involved in these processes is still incomplete. Herein, we utilized a proximity-dependent biotinylating enzyme (TurboID) to characterize the US28 interactome when expressed in isolation, and during both latent (CD34+ hematopoietic progenitor cells) and lytic (fibroblasts) HCMV infection. Our analyses indicate that the US28 signalosome converges with RhoA and EGFR signal transduction pathways, sharing multiple mediators that are major actors in processes such as cellular proliferation and differentiation. Integral members of the US28 signaling complex were validated in functional assays by immunoblot and small-molecule inhibitors. Importantly, we identified RhoGEFs as key US28 signaling intermediaries. In vitro latency and reactivation assays utilizing primary CD34+ hematopoietic progenitor cells (HPCs) treated with the small-molecule inhibitors Rhosin or Y16 indicated that US28 -RhoGEF interactions are required for efficient viral reactivation. These findings were recapitulated in vivo using a humanized mouse model where inhibition of RhoGEFs resulted in a failure of the virus to reactivate. Together, our data identifies multiple new proteins in the US28 interactome that play major roles in viral latency and reactivation, highlights the utility of proximity-sensor labeling to characterize protein interactomes, and provides insight into targets for the development of novel anti-HCMV therapeutics., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Medica et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Humans display substantial interindividual clinical variability after SARS-CoV-2 infection 1-3 , the genetic and immunological basis of which has begun to be deciphered 4 . However, the extent and drivers of population differences in immune responses to SARS-CoV-2 remain unclear. Here we report single-cell RNA-sequencing data for peripheral blood mononuclear cells-from 222 healthy donors of diverse ancestries-that were stimulated with SARS-CoV-2 or influenza A virus. We show that SARS-CoV-2 induces weaker, but more heterogeneous, interferon-stimulated gene activity compared with influenza A virus, and a unique pro-inflammatory signature in myeloid cells. Transcriptional responses to viruses display marked population differences, primarily driven by changes in cell abundance including increased lymphoid differentiation associated with latent cytomegalovirus infection. Expression quantitative trait loci and mediation analyses reveal a broad effect of cell composition on population disparities in immune responses, with genetic variants exerting a strong effect on specific loci. Furthermore, we show that natural selection has increased population differences in immune responses, particularly for variants associated with SARS-CoV-2 response in East Asians, and document the cellular and molecular mechanisms by which Neanderthal introgression has altered immune functions, such as the response of myeloid cells to viruses. Finally, colocalization and transcriptome-wide association analyses reveal an overlap between the genetic basis of immune responses to SARS-CoV-2 and COVID-19 severity, providing insights into the factors contributing to current disparities in COVID-19 risk., (© 2023. The Author(s).)

Human cytomegalovirus (HCMV) is a beta herpesvirus that persists indefinitely in the human host through a latent infection. The polycistronic UL133-UL138 gene locus of HCMV encodes genes regulating latency and reactivation. While UL138 is pro-latency, restricting virus replication in CD34 + hematopoietic progenitor cells (HPCs), UL135 overcomes this restriction and is required for reactivation. By contrast, UL136 is expressed with later kinetics and encodes multiple proteins with differential roles in latency and reactivation. Like UL135 , the largest UL136 isoform, UL136p33, is required for reactivation from latency in HPCs; viruses failing to express either protein are unresponsive to reactivation stimuli. Furthermore, UL136p33 is unstable, and its instability is important for the establishment of latency, and sufficient accumulation of UL136p33 is a checkpoint for reactivation. We hypothesized that stabilizing UL136p33 might overcome the requirement of UL135 for replication. We generated recombinant viruses lacking UL135 that expressed a stabilized variant of UL136p33. Stabilizing UL136p33 did not impact the replication of the UL135 mutant virus in fibroblasts. However, in the context of infection in HPCs, stabilization of UL136p33 strikingly compensated for the loss of UL135, resulting in increased replication in CD34 + HPCs and in humanized NOD- scid IL2Rγ c null (huNSG) mice. This finding suggests that while UL135 is essential for replication in HPCs, it functions largely at steps preceding the accumulation of UL136p33, and that stabilized expression of UL136p33 largely overcomes the requirement for UL135 . Taken together, our genetic evidence indicates an epistatic relationship between UL136p33 and UL135 , whereby UL135 may initiate events early in reactivation that drive the accumulation of UL136p33 to a threshold required for productive reactivation. IMPORTANCE Human cytomegalovirus (HCMV) is one of nine human herpesviruses and a significant human pathogen. While HCMV establishes a lifelong latent infection that is typically asymptomatic in healthy individuals, its reactivation from latency can have devastating consequences in the immunocompromised. Defining viral genes important in the establishment of or reactivation from latency is important to defining the molecular basis of latent and replicative states and in controlling infection and CMV disease. Here we define a genetic relationship between two viral genes in controlling virus reactivation from latency using primary human hematopoietic progenitor cells and humanized mouse models., Competing Interests: The authors declare no conflict of interest.

Human cytomegalovirus (HCMV) is a betaherpesvirus that establishes lifelong infection in its host and can cause severe comorbidities in individuals with suppressed or compromised immune systems. The lifecycle of HCMV consists of lytic and latent phases, largely dependent upon the cell type infected and whether transcription from the major immediate early locus can ensue. Control of this locus, which acts as a critical "switch" region from where the lytic gene expression cascade originates, as well as regulation of the additional ~235 kilobases of virus genome, occurs through chromatinization with cellular histone proteins after infection. Upon infection of a host cell, an initial intrinsic antiviral response represses gene expression from the incoming genome, which is relieved in permissive cells by viral and host factors in concert. Latency is established in a subset of hematopoietic cells, during which viral transcription is largely repressed while the genome is maintained. As these latently infected cells differentiate, the cellular milieu and epigenetic modifications change, giving rise to the initial stages of virus reactivation from latency. Thus, throughout the cycle of infection, chromatinization, chromatin modifiers, and the recruitment of specific transcription factors influence the expression of genes from the HCMV genome. In this review, we discuss epigenetic regulation of the HCMV genome during the different phases of infection, with an emphasis on recent reports that add to our current perspective., Competing Interests: The authors declare no conflict of interest.

The human cytomegalovirus (HCMV) US12 gene family contributes to virus-host interactions by regulating the virus' cell tropism and its evasion of host innate immune responses. US21, one of the 10 US12 genes (US12-US21), is a descendant of a captured cellular transmembrane BAX inhibitor motif-containing gene. It encodes a 7TMD endoplasmic reticulum (ER)-resident viroporin (pUS21) capable of reducing the Ca 2+ content of ER stores, which, in turn, protects cells against apoptosis. Since regulation of Ca 2+ homeostasis affects a broad range of cellular responses, including cell motility, we investigated whether pUS21 might also interfere with this cytobiological consequence of Ca 2+ signaling. Indeed, deletion of the US21 gene impaired the ability of HCMV-infected cells to migrate, whereas expression of US21 protein stimulated cell migration and adhesion, as well as focal adhesion (FA) dynamics, in a way that depended on its ability to manipulate ER Ca 2+ content. Mechanistic studies revealed pUS21-mediated cell migration to involve calpain 2 activation since its inhibition prevented the viroporin's effects on cell motility. Pertinently, pUS21 expression stimulated a store-operated Ca 2+ entry (SOCE) mechanism that may determine the activation of calpain 2 by promoting Ca 2+ entry. Furthermore, pUS21 was observed to interact with talin-1, a calpain 2 substrate, and crucial protein component of FA complexes. A functional consequence of this interaction was confirmed by talin-1 knockdown, which abrogated the pUS21-mediated increase in cell migration. Together, these results indicate the US21-encoded viroporin to be a viral regulator of cell adhesion and migration in the context of HCMV infection. IMPORTANCE Human cytomegalovirus (HCMV) is an opportunistic pathogen that owes part of its success to the capture, duplication, and tuning of cellular genes to generate modern viral proteins which promote infection and persistence in the host by interfering with many cell biochemical and physiological pathways. The US21 viral protein provides an example of this evolutionary strategy: it is a cellular-derived calcium channel that manipulates intracellular calcium homeostasis to confer edges to HCMV replication. Here, we report on the characterization of a novel function of the US21 protein as a viral regulator of cell migration and adhesion through mechanisms involving its calcium channel activity. Characterization of HCMV multifunctional regulatory proteins, like US21, supports the better understanding of viral pathogenesis and may open avenues for the design of new antiviral strategies that exploit their functions., Competing Interests: The authors declare no conflict of interest.

Human cytomegalovirus (HCMV) is a major human pathogen whose life-long persistence is enabled by its remarkable capacity to systematically subvert host immune defenses. In exploring the finding that HCMV infection up-regulates tumor necrosis factor receptor 2 (TNFR2), a ligand for the pro-inflammatory antiviral cytokine TNFα, we found that the underlying mechanism was due to targeting of the protease, A Disintegrin And Metalloproteinase 17 (ADAM17). ADAM17 is the prototype 'sheddase', a family of proteases that cleaves other membrane-bound proteins to release biologically active ectodomains into the supernatant. HCMV impaired ADAM17 surface expression through the action of two virally-encoded proteins in its U L / b' region, UL148 and UL148D. Proteomic plasma membrane profiling of cells infected with an HCMV double-deletion mutant for UL148 and UL148D with restored ADAM17 expression, combined with ADAM17 functional blockade, showed that HCMV stabilized the surface expression of 114 proteins ( P < 0.05) in an ADAM17-dependent fashion. These included reported substrates of ADAM17 with established immunological functions such as TNFR2 and jagged1, but also numerous unreported host and viral targets, such as nectin1, UL8, and UL144. Regulation of TNFα-induced cytokine responses and NK inhibition during HCMV infection were dependent on this impairment of ADAM17. We therefore identify a viral immunoregulatory mechanism in which targeting a single sheddase enables broad regulation of multiple critical surface receptors, revealing a paradigm for viral-encoded immunomodulation.

Cytomegalovirus (CMV) infection is a major cause of morbidity and mortality after hematopoietic stem cell transplantation (HSCT). Limited data are available from developing countries regarding the frequency of CMV infection and treatment outcomes. We enrolled 230 consecutive patients undergoing allogeneic HSCT for various hematologic disorders at the Armed Forces Bone Marrow Transplant Center/National Institute of Blood And Marrow Transplant between February 2017 and December 202. CMV reactivation post-HSCT was monitored weekly starting at day +30 and continuing until day +100, and preemptive antiviral therapy was administered to prevent CMV disease in all HSCT recipients with ≥2000 CMV copies/mL. The median age of the study cohort was 9.5 years (range, .6 to 53 years), and the male:female ratio was 2.4:1. The most frequent indication for HSCT was beta thalassemia major (36.1%), followed by aplastic anemia (23.9%). Malignant disorders constituted 20% of all the patients. Pretransplantation CMV seropositivity was 99.1% for the recipients and 99.5% for the donors. CMV infection was seen in 66.1% of the patients, and the median time to CMV DNAemia was 36 days (range, 12 to 95 days). Preemptive antiviral therapy was administered to 140 patients with a CMV viral load ≥2000 copies/mL (61%). In multivariate analysis, patient age >12 years, steroid administration, and use of mycophenolate mofetil with or without post-transplantation cyclophosphamide was associated with the greatest probability of CMV reactivation. Overall survival was 97.4% in patients without CMV reactivation, compared to 80.3% in those with CMV reactivation (P = .001). Event-free survival was 78.7% in the total study cohort, including 89.7% for patients without CMV reactivation and 73% for patients with CMV reactivation (P = .003). Our study is the first from this region to explore the frequency of CMV seropositivity and CMV infection, risk factors for CMV reactivation, and outcomes of antiviral therapy in HSCT recipients., (Copyright © 2023 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Liver X receptor (LXR) signaling broadly restricts virus replication; however, the mechanisms of restriction are poorly defined. Here, we demonstrate that the cellular E3 ligase LXR-inducible degrader of low-density lipoprotein receptor (IDOL) targets the human cytomegalovirus (HMCV) UL136p33 protein for turnover. UL136 encodes multiple proteins that differentially impact latency and reactivation. UL136p33 is a determinant of reactivation. UL136p33 is targeted for rapid turnover by the proteasome, and its stabilization by mutation of lysine residues to arginine results in a failure to quiet replication for latency. We show that IDOL targets UL136p33 for turnover but not the stabilized variant. IDOL is highly expressed in undifferentiated hematopoietic cells where HCMV establishes latency but is sharply downregulated upon differentiation, a stimulus for reactivation. We hypothesize that IDOL maintains low levels of UL136p33 for the establishment of latency. Consistent with this hypothesis, knockdown of IDOL impacts viral gene expression in wild-type (WT) HCMV infection but not in infection where UL136p33 has been stabilized. Furthermore, the induction of LXR signaling restricts WT HCMV reactivation from latency but does not affect the replication of a recombinant virus expressing a stabilized variant of UL136p33. This work establishes the UL136p33-IDOL interaction as a key regulator of the bistable switch between latency and reactivation. It further suggests a model whereby a key viral determinant of HCMV reactivation is regulated by a host E3 ligase and acts as a sensor at the tipping point between the decision to maintain the latent state or exit latency for reactivation. IMPORTANCE Herpesviruses establish lifelong latent infections, which pose an important risk for disease particularly in the immunocompromised. Our work is focused on the betaherpesvirus human cytomegalovirus (HCMV) that latently infects the majority of the population worldwide. Defining the mechanisms by which HCMV establishes latency or reactivates from latency is important for controlling viral disease. Here, we demonstrate that the cellular inducible degrader of low-density lipoprotein receptor (IDOL) targets a HCMV determinant of reactivation for degradation. The instability of this determinant is important for the establishment of latency. This work defines a pivotal virus-host interaction that allows HCMV to sense changes in host biology to navigate decisions to establish latency or to replicate., Competing Interests: The authors declare no conflict of interest.

Human cytomegalovirus (HCMV) is the most common vertically transmitted infection worldwide, yet there are no vaccines or therapeutics to prevent congenital HCMV (cCMV) infection. Emerging evidence indicates that antibody Fc effector functions may be a previously underappreciated component of maternal immunity against HCMV. We recently reported that antibody-dependent cellular phagocytosis (ADCP) and IgG activation of FcγRI/FcγRII were associated with protection against cCMV transmission, leading us to hypothesize that additional Fc-mediated antibody functions may be important. In this same cohort of HCMV-transmitting (n = 41) and nontransmitting (n = 40) mother-infant dyads, we report that higher maternal sera antibody-dependent cellular cytotoxicity (ADCC) activation is also associated with lower risk of cCMV transmission. We investigated the relationship between ADCC and IgG responses against 9 viral antigens and found that ADCC activation correlated most strongly with sera IgG binding to the HCMV immunoevasin protein UL16. Moreover, we determined that higher UL16-specific IgG binding and FcγRIII/CD16 engagement were associated with the greatest risk reduction in cCMV transmission. Our findings indicate that ADCC-activating antibodies against targets such as UL16 may represent an important protective maternal immune response against cCMV infection that can guide future HCMV correlates studies and vaccine or antibody-based therapeutic development.

Cytomegalovirus (CMV) infection is a common and typically benign disease in immunocompetent individuals. However, immunocompromised patients are at a greater risk of reactivation, leading to more severe outcomes. Patients with rheumatic diseases have a particularly high risk of opportunistic infections due to both the inherent immunosuppressive state conveyed by the disease itself and the use of potent immunosuppressant drugs, such as glucocorticoids, cyclophosphamide, and rituximab. Limited data are available regarding prophylactic or preemptive treatment of CMV infection in patients with rheumatic diseases. In this article the authors present two cases of rheumatic conditions complicated by CMV infection. The first case describes a patient with eosinophilic granulomatosis with polyangiitis, previously treated with glucocorticoids and cyclophosphamide, who developed CMV colitis with bowel perforation. The second case involves a woman with systemic lupus erythematosus who was diagnosed with CMV meningitis. Both cases reinforce the importance of establishing guidelines for surveillance and prophylaxis of CMV infection in these patients.

In children, CMV-associated protein-losing enteropathy (PLE) is characterised by a benign course and spontaneous healing but can lead to generalised oedema. Poorly defined, it is diagnosed after unnecessary invasive tests. Children with CMV-associated PLE between 2009 and 2019 in two French hospitals are retrospectively described. Clinical and biological signs, CMV identification, endoscopy and histological findings, disease management and course are analysed. CMV-associated PLE is proven in 21 immunocompetent and 22 immunosuppressed patients, with ages consistent with primo-infection and reactivation, respectively. The digestive symptoms prevail in immunocompetent children, mainly with vomiting (85.7% versus 50%, CI [1.2; 39.2], p = 0.02). Immunocompetent patients show more oedema (61.9% versus 4.5%, CI [3.6; 1502.4], p < 0.001), linked to more severe hypoalbuminemia (21.2 g/L [17.6-25.7] versus 29.6 g/L [24.9-33.9], p = 0.01). A severe course is observed in 23.8% of the immunocompetent patients and 54.5% of the immunosuppressed ones ( p = 0.06). Evidence of CMV infection based on non-invasive methods is found on 88.9% of immunocompetent and 95.5% of immunosuppressed patients ( p = 0.58), while endoscopy was performed on 95.2% and 100% of them, respectively ( p = 0.48), without any therapeutic change. Thus, CMV-associated PLE should be suspected in children with generalised oedema. Not as benign as previously described, it can be confirmed using non-invasive tests.

After allogeneic hematopoietic stem cell transplantation (HSCT), the emergence of circulating cytomegalovirus (CMV)- specific T cells correlates with protection from CMV reactivation, an important risk factor for non-relapse mortality. However, functional assays measuring CMV-specific cells are time-consuming and often inaccurate at early time-points. We report the results of a prospective single-center, non-interventional study that identified the enumeration of Dextramerpositive CMV-specific lymphocytes as a reliable and early predictor of viral reactivation. We longitudinally monitored 75 consecutive patients for 1 year after allogeneic HSCT (n=630 samples). The presence of ≥0.5 CMV-specific CD8+ cells/mL at day +45 was an independent protective factor from subsequent clinically relevant reactivation in univariate (P<0.01) and multivariate (P<0.05) analyses. Dextramer quantification correlated with functional assays measuring interferon-γ production, and allowed earlier identification of high-risk patients. In mismatched transplants, the comparative analysis of lymphocytes restricted by shared, donor- and host-specific HLA revealed the dominant role of thymic-independent CMV-specific reconstitution. Shared and donor-restricted CMV-specific T cells reconstituted with similar kinetics in recipients of CMV-seropositive donors, while donor-restricted T-cell reconstitution from CMV-seronegative grafts was impaired, indicating that in primary immunological responses the emergence of viral-specific T cells is largely sustained by antigen encounter on host infected cells rather than by cross-priming/presentation by non-infected donor-derived antigen-presenting cells. Multiparametric flow cytometry and high-dimensional analysis showed that shared-restricted CMV-specific lymphocytes display a more differentiated phenotype and increased persistence than donor-restricted counterparts. In this study, monitoring CMV-specific cells by Dextramer assay after allogeneic HSCT shed light on mechanisms of immune reconstitution and enabled risk stratification of patients, which could improve the clinical management of post-transplant CMV reactivations.