Discovery of a Novel Antiviral Effect of the Restriction Factor SPOC1 against Human Cytomegalovirus.

The chromatin-remodeler SPOC1 (PHF13) is a transcriptional co-regulator and has been identified as a restriction factor against various viruses, including human cytomegalovirus (HCMV). For HCMV, SPOC1 was shown to block the onset of immediate-early (IE) gene expression under low multiplicities of infection (MOI). Here, we demonstrate that SPOC1-mediated restriction of IE expression is neutralized by increasing viral titers. Interestingly, our study reveals that SPOC1 exerts an additional antiviral function beyond the IE phase of HCMV replication. Expression of SPOC1 under conditions of high MOI resulted in severely impaired viral DNA replication and viral particle release, which may be attributed to inefficient viral transcription. With the use of click chemistry, the localization of viral DNA was investigated at late time points after infection. Intriguingly, we detected a co-localization of SPOC1, RNA polymerase II S5P and polycomb repressor complex 2 (PRC2) components in close proximity to viral DNA in areas that are hypothesized to harbor viral transcription sites. We further identified the N-terminal domain of SPOC1 to be responsible for interaction with EZH2, a subunit of the PRC2 complex. With this study, we report a novel and potent antiviral function of SPOC1 against HCMV that is efficient even with unrestricted IE gene expression.

IE1 of Human Cytomegalovirus Inhibits Necroptotic Cell Death via Direct and Indirect Modulation of the Necrosome Complex.

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.

Neurological manifestation of HEV infection: still a rare disease entity?

Hepatitis E virus (HEV) infection is the most common form of viral hepatitis and is reported to cause neurological manifestation in up to 30% of diagnosed infections. We evaluated the medical reports of all patients (n = 29,994) who were discharged from the Department of Neurology of Ulm University between 01.01.2015 and 30.09.2022 to detect neurological manifestations of HEV. In addition, we retrospectively analyzed the serum samples of n = 99 patients representing different neurological diseases possibly related to HEV for anti-HEV-IgM and anti-HEV-IgG. At the time of discharge from hospital, the etiology of neurological symptoms in these patients was unclear. Overall, five cases of extrahepatic neurological manifestation of HEV (defined as anti-HEV-IgM and HEV-IgG positive) could be detected. An increase of both, anti-IgM- and anti-IgG-serum levels was significantly more common in neuralgic amyotrophy/plexus neuritis/radiculitis than in AIDP/CIDP (P = 0.01), meningitis/encephalitis (P = 0.02), idiopathic peripheral facial paralysis (P = 0.02) and tension headache (P = 0.02). In 15% (n = 15 out of 99) of retrospectively analyzed serum samples, conspicuous positive anti-HEV-IgG levels were detected. This finding was most common in AIDP/CIDP. In conclusion, results of this study indicate neurological manifestation of HEV to be a rare but still underestimated course of disease, occurring at any age and gender. Therefore, testing for HEV should be considered in patients with neurological symptoms of unknown origin, especially in those with neuralgic amyotrophy/plexus neuritis.

Quantitative Evaluation of COVID-19 Pneumonia CT Using AI Analysis-Feasibility and Differentiation from Other Common Pneumonia Forms.

PURPOSE: To implement the technical feasibility of an AI-based software prototype optimized for the detection of COVID-19 pneumonia in CT datasets of the lung and the differentiation between other etiologies of pneumonia. METHODS: This single-center retrospective case-control-study consecutively yielded 144 patients (58 female, mean age 57.72 ± 18.25 y) with CT datasets of the lung. Subgroups including confirmed bacterial (n = 24, 16.6%), viral (n = 52, 36.1%), or fungal (n = 25, 16.6%) pneumonia and (n = 43, 30.7%) patients without detected pneumonia (comparison group) were evaluated using the AI-based Pneumonia Analysis prototype. Scoring (extent, etiology) was compared to reader assessment. RESULTS: The software achieved an optimal sensitivity of 80.8% with a specificity of 50% for the detection of COVID-19; however, the human radiologist achieved optimal sensitivity of 80.8% and a specificity of 97.2%. The mean postprocessing time was 7.61 ± 4.22 min. The use of a contrast agent did not influence the results of the software (p = 0.81). The mean evaluated COVID-19 probability is 0.80 ± 0.36 significantly higher in COVID-19 patients than in patients with fungal pneumonia (p < 0.05) and bacterial pneumonia (p < 0.001). The mean percentage of opacity (PO) and percentage of high opacity (PHO ≥ -200 HU) were significantly higher in COVID-19 patients than in healthy patients. However, the total mean HU in COVID-19 patients was -679.57 ± 112.72, which is significantly higher than in the healthy control group (p < 0.001). CONCLUSION: The detection and quantification of pneumonia beyond the primarily trained COVID-19 datasets is possible and shows comparable results for COVID-19 pneumonia to an experienced reader. The advantages are the fast, automated segmentation and quantification of the pneumonia foci.

High antibody levels and reduced cellular response in children up to one year after SARS-CoV-2 infection.

The COVID-19 course and immunity differ in children and adults. We analyzed immune response dynamics in 28 families up to 12 months after mild or asymptomatic infection. Unlike adults, the initial response is plasmablast-driven in children. Four months after infection, children show an enhanced specific antibody response and lower but detectable spike 1 protein (S1)-specific B and T cell responses than their parents. While specific antibodies decline, neutralizing antibody activity and breadth increase in both groups. The frequencies of S1-specific B and T cell responses remain stable. However, in children, one year after infection, an increase in the S1-specific IgA class switch and the expression of CD27 on S1-specific B cells and T cell maturation are observed. These results, together with the enhanced neutralizing potential and breadth of the specific antibodies, suggest a progressive maturation of the S1-specific immune response. Hence, the immune response in children persists over 12 months but dynamically changes in quality, with progressive neutralizing, breadth, and memory maturation. This implies a benefit for booster vaccination in children to consolidate memory formation.

Highly Conserved Interaction Profiles between Clinically Relevant Mutants of the Cytomegalovirus CDK-like Kinase pUL97 and Human Cyclins: Functional Significance of Cyclin H.

The complex host interaction network of human cytomegalovirus (HCMV) involves the regulatory protein kinase pUL97, which represents a viral cyclin-dependent kinase (CDK) ortholog. pUL97 interacts with the three human cyclin types T1, H, and B1, whereby the binding region of cyclin T1 and the pUL97 oligomerization region were both assigned to amino acids 231-280. We further addressed the question of whether HCMVs harboring mutations in ORF-UL97, i.e., short deletions or resistance-conferring point mutations, are affected in the interaction with human cyclins and viral replication. To this end, clinically relevant UL97 drug-resistance-conferring mutants were analyzed by whole-genome sequencing and used for genetic marker transfer experiments. The recombinant HCMVs indicated conservation of pUL97-cyclin interaction, since all viral UL97 point mutants continued to interact with the analyzed cyclin types and exerted wild-type-like replication fitness. In comparison, recombinant HCMVs UL97 Δ231-280 and also the smaller deletion Δ236-275, but not Δ241-270, lost interaction with cyclins T1 and H, showed impaired replication efficiency, and also exhibited reduced kinase activity. Moreover, a cellular knock-out of cyclins B1 or T1 did not alter HCMV replication phenotypes or pUL97 kinase activity, possibly indicating alternative, compensatory pUL97-cyclin interactions. In contrast, however, cyclin H knock-out, similar to virus deletion mutants in the pUL97-cyclin H binding region, exhibited strong defective phenotypes of HCMV replication, as supported by reduced pUL97 kinase activity in a cyclin H-dependent coexpression setting. Thus, cyclin H proved to be a very relevant determinant of pUL97 kinase activity and viral replication efficiency. As a conclusion, the results provide evidence for the functional importance of pUL97-cyclin interaction. High selective pressure on the formation of pUL97-cyclin complexes was identified by the use of clinically relevant mutants.

Promyelocytic Leukemia Protein Potently Restricts Human Cytomegalovirus Infection in Endothelial Cells.

PML nuclear bodies (PML-NBs) are dynamic macromolecular complexes that mediate intrinsic immunity against viruses of different families, including human cytomegalovirus (HCMV). Upon HCMV infection, PML-NBs target viral genomes entering the nucleus and restrict viral immediate-early gene expression by epigenetic silencing. Studies from several groups performed in human fibroblast cells have shown that the major PML-NB components PML, Daxx, Sp100 and ATRX contribute to this repression in a cooperative manner. Their role for HCMV restriction in endothelial cells, however, has not yet been characterized although infected endothelium is thought to play a crucial role for HCMV dissemination and development of vascular disease in vivo. Here, we use conditionally immortalized umbilical vein endothelial cells (HEC-LTT) as a cell culture model to elucidate the impact of PML-NB proteins on lytic HCMV infection. Depletion of individual PML-NB proteins by lentiviral transduction showed a particularly strong antiviral effect of PML in HEC-LTT, compared to human fibroblasts. A closer characterization of this antiviral function revealed that PML may not only effectively inhibit HCMV immediate-early gene expression but also act at later steps of the viral replication cycle. At contrast, we surprisingly noted an antiviral behavior of Daxx in complementary approaches: Depletion of Daxx resulted in decreased viral gene expression, while overexpression of Daxx promoted HCMV infection. In summary, our data demonstrate a cell type-specific effect of PML-NB components on lytic HCMV infection and suggest an important role of PML in the inhibition of HCMV dissemination through infected endothelial cells.

Long COVID symptoms in exposed and infected children, adolescents and their parents one year after SARS-CoV-2 infection: A prospective observational cohort study.

BACKGROUND: Long COVID in children and adolescents remains poorly understood due to a lack of well-controlled studies with long-term follow-up. In particular, the impact of the family context on persistent symptoms following SARS-CoV-2 infection remains unknown. We examined long COVID symptoms in a cohort of infected children, adolescents, and adults and their exposed but non-infected household members approximately 1 year after infection and investigated clustering of persistent symptoms within households. METHODS: 1267 members of 341 households (404 children aged <14 years, 140 adolescents aged 14-18 years and 723 adults) were categorized as having had either a SARS-CoV-2 infection or household exposure to SARS-CoV-2 without infection, based on three serological assays and history of laboratory-confirmed infection. Participants completed questionnaires assessing the presence of long COVID symptoms 11-12 months after infection in the household using online questionnaires. FINDINGS: The prevalence of moderate or severe persistent symptoms was statistically significantly higher in infected than in exposed women (36.4% [95% CI: 30.7-42.4%] vs 14.2% [95% CI: 8.7-21.5%]), infected men (22.9% [95% CI: 17.9-28.5%] vs 10.3% [95% CI: 5.8-16.9%]) and infected adolescent girls (32.1% 95% CI: 17.2-50.5%] vs 8.9% [95%CI: 3.1-19.8%]). However, moderate or severe persistent symptoms were not statistically more common in infected adolescent boys aged 14-18 (9.7% [95% CI: 2.8-23.6%] or in infected children <14 years (girls: 4.3% [95% CI: 1.2-11.0%]; boys: 3.7% [95% CI: 1.1-9.6%]) than in their exposed counterparts (adolescent boys: 0.0% [95% CI: 0.0-6.7%]; girls < 14 years: 2.3% [95% CI: 0·7-6·1%]; boys < 14 years: 0.0% [95% CI: 0.0-2.0%]). The number of persistent symptoms reported by individuals was associated with the number of persistent symptoms reported by their household members (IRR=1·11, p=·005, 95% CI [1.03-1.20]). INTERPRETATION: In this controlled, multi-centre study, infected men, women and adolescent girls were at increased risk of negative outcomes 11-12 months after SARS-CoV-2 infection. Amongst non-infected adults, prevalence of negative outcomes was also high. Prolonged symptoms tended to cluster within families, suggesting family-level interventions for long COVID could prove useful. FUNDING: Ministry of Science, Research and the Arts, Baden-Württemberg, Germany.

The chromatin remodeling protein ATRX positively regulates IRF3-dependent type I interferon production and interferon-induced gene expression.

The chromatin remodeling protein alpha thalassemia/mental retardation syndrome X-linked (ATRX) is a component of promyelocytic leukemia nuclear bodies (PML-NBs) and thereby mediates intrinsic immunity against several viruses including human cytomegalovirus (HCMV). As a consequence, viruses have evolved different mechanisms to antagonize ATRX, such as displacement from PML-NBs or degradation. Here, we show that depletion of ATRX results in an overall impaired antiviral state by decreasing transcription and subsequent secretion of type I IFNs, which is followed by reduced expression of interferon-stimulated genes (ISGs). ATRX interacts with the transcription factor interferon regulatory factor 3 (IRF3) and associates with the IFN-ß promoter to facilitate transcription. Furthermore, whole transcriptome sequencing revealed that ATRX is required for efficient IFN-induced expression of a distinct set of ISGs. Mechanistically, we found that ATRX positively modulates chromatin accessibility specifically upon IFN signaling, thereby affecting promoter regions with recognition motifs for AP-1 family transcription factors. In summary, our study uncovers a novel co-activating function of the chromatin remodeling factor ATRX in innate immunity that regulates chromatin accessibility and subsequent transcription of interferons and ISGs. Consequently, ATRX antagonization by viral proteins and ATRX mutations in tumors represent important strategies to broadly compromise both intrinsic and innate immune responses.

Cross-Species Analysis of Innate Immune Antagonism by Cytomegalovirus IE1 Protein.

The human cytomegalovirus (CMV) immediate early 1 (IE1) protein has evolved as a multifunctional antagonist of intrinsic and innate immune mechanisms. In addition, this protein serves as a transactivator and potential genome maintenance protein. Recently, the crystal structures of the human and rat CMV IE1 (hIE1, rIE1) core domain were solved. Despite low sequence identity, the respective structures display a highly similar, all alpha-helical fold with distinct variations. To elucidate which activities of IE1 are either species-specific or conserved, this study aimed at a comparative analysis of hIE1 and rIE1 functions. To facilitate the quantitative evaluation of interactions between IE1 and cellular proteins, a sensitive NanoBRET assay was established. This confirmed the species-specific interaction of IE1 with the cellular restriction factor promyelocytic leukemia protein (PML) and with the DNA replication factor flap endonuclease 1 (FEN1). To characterize the respective binding surfaces, helix exchange mutants were generated by swapping hIE1 helices with the corresponding rIE1 helices. Interestingly, while all mutants were defective for PML binding, loss of FEN1 interaction was confined to the exchange of helices 1 and 2, suggesting that FEN1 binds to the stalk region of IE1. Furthermore, our data reveal that both hIE1 and rIE1 antagonize human STAT2; however, distinct regions of the respective viral proteins mediated the interaction. Finally, while PML, FEN1, and STAT2 binding were conserved between primate and rodent proteins, we detected that rIE1 lacks a chromatin tethering function suggesting that this activity is dispensable for rat CMV. In conclusion, our study revealed conserved and distinct functions of primate and rodent IE1 proteins, further supporting the concept that IE1 proteins underwent a narrow co-evolution with their respective hosts to maximize their efficacy in antagonizing innate immune mechanisms and supporting viral replication.

Inhibitors of Activin Receptor-like Kinase 5 Interfere with SARS-CoV-2 S-Protein Processing and Spike-Mediated Cell Fusion via Attenuation of Furin Expression.

Screening of a protein kinase inhibitor library identified SB431542, targeting activin receptor-like kinase 5 (ALK5), as a compound interfering with SARS-CoV-2 replication. Since ALK5 is implicated in transforming growth factor ß (TGF-ß) signaling and regulation of the cellular endoprotease furin, we pursued this research to clarify the role of this protein kinase for SARS-CoV-2 infection. We show that TGF-ß1 induces the expression of furin in a broad spectrum of cells including Huh-7 and Calu-3 that are permissive for SARS-CoV-2. The inhibition of ALK5 by incubation with SB431542 revealed a dose-dependent downregulation of both basal and TGF-ß1 induced furin expression. Furthermore, we demonstrate that the ALK5 inhibitors SB431542 and Vactosertib negatively affect the proteolytic processing of the SARS-CoV-2 Spike protein and significantly reduce spike-mediated cell-cell fusion. This correlated with an inhibitory effect of ALK5 inhibition on the production of infectious SARS-CoV-2. Altogether, our study shows that interference with ALK5 signaling attenuates SARS-CoV-2 infectivity and cell-cell spread via downregulation of furin which is most pronounced upon TGF-ß stimulation. Since a TGF-ß dominated cytokine storm is a hallmark of severe COVID-19, ALK5 inhibitors undergoing clinical trials might represent a potential therapy option for COVID-19.

Role of ABO Blood Group in SARS-CoV-2 Infection in Households.

An association between certain ABO/Rh blood groups and susceptibility to SARS-CoV-2 infection has been proposed for adults, although this remains controversial. In children and adolescents, the relationship is unclear due to a lack of robust data. Here, we investigated the association of ABO/Rh blood groups and SARS-CoV-2 in a multi-center study comprising 163 households with 281 children and 355 adults and at least one SARS-CoV-2 seropositive individual as determined by three independent assays as a proxy for previous infection. In line with previous findings, we found a higher frequency of blood group A (+ 6%) and a lower frequency of blood group O (-6%) among the SARS-CoV-2 seropositive adults compared to the seronegative ones. This trend was not seen in children. In contrast, SARS-CoV-2 seropositive children had a significantly lower frequency of Rh-positive blood groups. ABO compatibility did not seem to play a role in SARS-CoV-2 transmission within the families. A correction for family clusters was performed and estimated fixed effects of the blood group on the risk of SARS-CoV-2 seropositivity and symptomatic infection were determined. Although we found a different distribution of blood groups in seropositive individuals compared to the reference population, the risk of SARS-CoV-2 seropositivity or symptomatic infection was not increased in children or in adults with blood group A or AB versus O or B. Increasing age was the only parameter positively correlating with the risk of SARS-CoV-2 infection. In conclusion, specific ABO/Rh blood groups and ABO compatibility appear not to predispose for SARS-CoV-2 susceptibility in children.

Dual signaling via interferon and DNA damage response elicits entrapment by giant PML nuclear bodies.

PML nuclear bodies (PML-NBs) are dynamic interchromosomal macromolecular complexes implicated in epigenetic regulation as well as antiviral defense. During herpesvirus infection, PML-NBs induce epigenetic silencing of viral genomes, however, this defense is antagonized by viral regulatory proteins such as IE1 of human cytomegalovirus (HCMV). Here, we show that PML-NBs undergo a drastic rearrangement into highly enlarged PML cages upon infection with IE1-deficient HCMV. Importantly, our results demonstrate that dual signaling by interferon and DNA damage response is required to elicit giant PML-NBs. DNA labeling revealed that invading HCMV genomes are entrapped inside PML-NBs and remain stably associated with PML cages in a transcriptionally repressed state. Intriguingly, by correlative light and transmission electron microscopy (EM), we observed that PML cages also entrap newly assembled viral capsids demonstrating a second defense layer in cells with incomplete first-line response. Further characterization by 3D EM showed that hundreds of viral capsids are tightly packed into several layers of fibrous PML. Overall, our data indicate that giant PML-NBs arise via combined interferon and DNA damage signaling which triggers entrapment of both nucleic acids and proteinaceous components. This represents a multilayered defense strategy to act in a cytoprotective manner and to combat viral infections.

Robust and durable serological response following pediatric SARS-CoV-2 infection.

The quality and persistence of children's humoral immune response following SARS-CoV-2 infection remains largely unknown but will be crucial to guide pediatric SARS-CoV-2 vaccination programs. Here, we examine 548 children and 717 adults within 328 households with at least one member with a previous laboratory-confirmed SARS-CoV-2 infection. We assess serological response at 3-4 months and 11-12 months after infection using a bead-based multiplex immunoassay for 23 human coronavirus antigens including SARS-CoV-2 and its Variants of Concern (VOC) and endemic human coronaviruses (HCoVs), and additionally by three commercial SARS-CoV-2 antibody assays. Neutralization against wild type SARS-CoV-2 and the Delta VOC are analysed in a pseudotyped virus assay. Children, compared to adults, are five times more likely to be asymptomatic, and have higher specific antibody levels which persist longer (96.2% versus 82.9% still seropositive 11-12 months post infection). Of note, symptomatic and asymptomatic infections induce similar humoral responses in all age groups. SARS-CoV-2 infection occurs independent of HCoV serostatus. Neutralization responses of children and adults are similar, although neutralization is reduced for both against the Delta VOC. Overall, the long-term humoral immune response to SARS-CoV-2 infection in children is of longer duration than in adults even after asymptomatic infection.

Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 in Households with Children, Southwest Germany, May-August 2020.

Resolving the role of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission in households with members from different generations is crucial for containing the current pandemic. We conducted a large-scale, multicenter, cross-sectional seroepidemiologic household transmission study in southwest Germany during May 11-August 1, 2020. We included 1,625 study participants from 405 households that each had ≥1 child and 1 reverse transcription PCR-confirmed SARS-CoV-2-infected index case-patient. The overall secondary attack rate was 31.6% and was significantly higher in exposed adults (37.5%) than in children (24.6%-29.2%; p = <0.015); the rate was also significantly higher when the index case-patient was >60 years of age (72.9%; p = 0.039). Other risk factors for infectiousness of the index case-patient were SARS-CoV-2-seropositivity (odds ratio [OR] 27.8, 95% CI 8.26-93.5), fever (OR 1.93, 95% CI 1.14-3.31), and cough (OR 2.07, 95% CI 1.21-3.53). Secondary infections in household contacts generate a substantial disease burden.

Cytomegalovirus immediate-early 1 proteins form a structurally distinct protein class with adaptations determining cross-species barriers.

Restriction factors are potent antiviral proteins that constitute a first line of intracellular defense by blocking viral replication and spread. During co-evolution, however, viruses have developed antagonistic proteins to modulate or degrade the restriction factors of their host. To ensure the success of lytic replication, the herpesvirus human cytomegalovirus (HCMV) expresses the immediate-early protein IE1, which acts as an antagonist of antiviral, subnuclear structures termed PML nuclear bodies (PML-NBs). IE1 interacts directly with PML, the key protein of PML-NBs, through its core domain and disrupts the dot-like multiprotein complexes thereby abrogating the antiviral effects. Here we present the crystal structures of the human and rat cytomegalovirus core domain (IE1CORE). We found that IE1CORE domains, also including the previously characterized IE1CORE of rhesus CMV, form a distinct class of proteins that are characterized by a highly similar and unique tertiary fold and quaternary assembly. This contrasts to a marked amino acid sequence diversity suggesting that strong positive selection evolved a conserved fold, while immune selection pressure may have fostered sequence divergence of IE1. At the same time, we detected specific differences in the helix arrangements of primate versus rodent IE1CORE structures. Functional characterization revealed a conserved mechanism of PML-NB disruption, however, primate and rodent IE1 proteins were only effective in cells of the natural host species but not during cross-species infection. Remarkably, we observed that expression of HCMV IE1 allows rat cytomegalovirus replication in human cells. We conclude that cytomegaloviruses have evolved a distinct protein tertiary structure of IE1 to effectively bind and inactivate an important cellular restriction factor. Furthermore, our data show that the IE1 fold has been adapted to maximize the efficacy of PML targeting in a species-specific manner and support the concept that the PML-NBs-based intrinsic defense constitutes a barrier to cross-species transmission of HCMV.

Human cytomegalovirus-induced host protein citrullination is crucial for viral replication.

Citrullination is the conversion of arginine-to-citrulline by protein arginine deiminases (PADs), whose dysregulation is implicated in the pathogenesis of various types of cancers and autoimmune diseases. Consistent with the ability of human cytomegalovirus (HCMV) to induce post-translational modifications of cellular proteins to gain a survival advantage, we show that HCMV infection of primary human fibroblasts triggers PAD-mediated citrullination of several host proteins, and that this activity promotes viral fitness. Citrullinome analysis reveals significant changes in deimination levels of both cellular and viral proteins, with interferon (IFN)-inducible protein IFIT1 being among the most heavily deiminated one. As genetic depletion of IFIT1 strongly enhances HCMV growth, and in vitro IFIT1 citrullination impairs its ability to bind to 5'-ppp-RNA, we propose that viral-induced IFIT1 citrullination is a mechanism of HCMV evasion from host antiviral resistance. Overall, our findings point to a crucial role of citrullination in subverting cellular responses to viral infection.

Identification of UL69 Gene and Protein in Cytomegalovirus-Transformed Human Mammary Epithelial Cells.

A growing body of evidence addressing the involvement of human cytomegalovirus (HCMV) in malignancies had directed attention to the oncomodulation paradigm. HCMV-DB infected human mammary epithelial cells (HMECs) in culture showed the emergence of clusters of rapidly proliferating, spheroid-shaped transformed cells named CTH (CMV-Transformed HMECs) cells. CTH cells assessment suggests a direct contribution of HCMV to oncogenesis, from key latent and lytic genes activating oncogenic pathways to fueling tumor evolution. We hypothesized that the presence of HCMV genome in CTH cells is of pivotal importance for determining its oncogenic potential. We previously reported the detection of a long non-coding (lnc) RNA4.9 gene in CTH cells. Therefore, we assessed here the presence of UL69 gene, located nearby and downstream of the lncRNA4.9 gene, in CTH cells. The HCMV UL69 gene in CTH cells was detected using polymerase chain reaction (PCR) and sequencing of UL69 gene was performed using Sanger method. The corresponding amino acid sequence was then blasted against the UL69 sequence derived from HCMV-DB genome using NCBI Protein BLAST tool. A 99% identity was present between the nucleotide sequence present in CTH cells and HCMV-DB genome. UL69 transcript was detected in RNA extracts of CTH cells, using a reverse transcription polymerase chain reaction (RT-PCR) assay, and pUL69 protein was identified in CTH lysates using western blotting. Ganciclovir-treated CTH cells showed a decrease in UL69 gene detection and cellular proliferation. In CTH cells, the knockdown of UL69 with siRNA was assessed by RT-qPCR and western blot to reveal the impact of pUL69 on HCMV replication and CTH cell proliferation. Finally, UL69 gene was detected in breast cancer biopsies. Our results indicate a close link between the UL69 gene detected in the HCMV-DB isolate used to infect HMECs, and the UL69 gene present in transformed CTH cells and tumor biopsies, further highlighting a direct role for HCMV in breast tumor development.

Role of CMV chemokine receptor M33 in airway graft rejection in a mouse transplant model.

BACKGROUND: Cytomegalovirus (CMV) infection is a risk factor for bronchiolitis obliterans (BO), one form of chronic lung allograft dysfunction (CLAD). The viral chemokine receptor M33 is essential for successful spread of murine CMV to host salivary glands. In the present study we investigated the impact of M33 on chronic airway rejection. METHODS: MHC I-mismatched tracheas of C·B10-H2b/LilMcdJ mice were transplanted into BALB/c (H2d) recipients and infected at different dates with wild type (WT) or M33-deleted (delM33) MCMV representing clinical settings of viral recipient (R)-donor (D)-serostatus: (D-/R+) or (D+/R-). Grafts were recovered for gene expression and histological / immunofluorescence analysis, respectively. RESULTS: Evaluations showed significantly increased signs of chronic rejection in WT-infected mice compared to uninfected allografts seen in lower epithelium/lamina propria-ratio (ELR) (ELR 0.46 ± 0.07 [WT post] vs. ELR 0.66 ± 0.10 [non-inf.]; p < 0.05). The rejection in delM33-infected groups was significantly reduced vs. WT-infected groups (0.67 ± 0.04 [delM33 post]; vs. WT post p < 0.05). Furthermore, decreased rejection was observed in WT pre-infected compared to post-infected groups (0.56 ± 0.08 [WT pre]; vs. WT post p < 0.05). CD8+ T cell infiltration was significantly higher in WT-post compared to the delM33 infected or non-infected allografts. CONCLUSIONS: These data support the role of the CMV in accelerating CLAD. The deletion of chemokine receptor M33 leads to attenuated rejection.

Functional regulation of the structure-specific endonuclease FEN1 by the human cytomegalovirus protein IE1 suggests a role for the re-initiation of stalled viral replication forks.

Flap endonuclease 1 (FEN1) is a member of the family of structure-specific endonucleases implicated in regulation of DNA damage response and DNA replication. So far, knowledge on the role of FEN1 during viral infections is limited. Previous publications indicated that poxviruses encode a conserved protein that acts in a manner similar to FEN1 to stimulate homologous recombination, double-strand break (DSB) repair and full-size genome formation. Only recently, cellular FEN1 has been identified as a key component for hepatitis B virus cccDNA formation. Here, we report on a novel functional interaction between Flap endonuclease 1 (FEN1) and the human cytomegalovirus (HCMV) immediate early protein 1 (IE1). Our results provide evidence that IE1 manipulates FEN1 in an unprecedented manner: we observed that direct IE1 binding does not only enhance FEN1 protein stability but also phosphorylation at serine 187. This correlates with nucleolar exclusion of FEN1 stimulating its DSB-generating gap endonuclease activity. Depletion of FEN1 and inhibition of its enzymatic activity during HCMV infection significantly reduced nascent viral DNA synthesis demonstrating a supportive role for efficient HCMV DNA replication. Furthermore, our results indicate that FEN1 is required for the formation of DSBs during HCMV infection suggesting that IE1 acts as viral activator of FEN1 in order to re-initiate stalled replication forks. In summary, we propose a novel mechanism of viral FEN1 activation to overcome replication fork barriers at difficult-to-replicate sites in viral genomes.