Deletion of the non-adjacent genes UL148 and UL148D impairs human cytomegalovirus-mediated TNF receptor 2 surface upregulation.

Human cytomegalovirus (HCMV) is a prototypical ß-herpesvirus which frequently causes morbidity and mortality in individuals with immature, suppressed, or senescent immunity. HCMV is sensed by various pattern recognition receptors, leading to the secretion of pro-inflammatory cytokines including tumor necrosis factor alpha (TNFα). TNFα binds to two distinct trimeric receptors: TNF receptor (TNFR) 1 and TNFR2, which differ in regard to their expression profiles, affinities for soluble and membrane-bound TNFα, and down-stream signaling pathways. While both TNF receptors engage NFκB signaling, only the nearly ubiquitously expressed TNFR1 exhibits a death domain that mediates TRADD/FADD-dependent caspase activation. Under steady-state conditions, TNFR2 expression is mainly restricted to immune cells where it predominantly submits pro-survival, proliferation-stimulating, and immune-regulatory signals. Based on the observation that HCMV-infected cells show enhanced binding of TNFα, we explored the interplay between HCMV and TNFR2. As expected, uninfected fibroblasts did not show detectable levels of TNFR2 on the surface. Intriguingly, however, HCMV infection increased TNFR2 surface levels of fibroblasts. Using HCMV variants and BACmid-derived clones either harboring or lacking the ULb' region, an association between TNFR2 upregulation and the presence of the ULb' genome region became evident. Applying a comprehensive set of ULb' gene block and single gene deletion mutants, we observed that HCMV mutants in which the non-adjacent genes UL148 or UL148D had been deleted show an impaired ability to upregulate TNFR2, coinciding with an inverse regulation of TACE/ADAM17.

Acute cytomegalovirus infection modulates the intestinal microbiota and targets intestinal epithelial cells.

Primary and recurrent cytomegalovirus (CMV) infections frequently cause CMV colitis in immunocompromised as well as inflammatory bowel disease (IBD) patients. Additionally, colitis occasionally occurs upon primary CMV infection in patients who are apparently immunocompetent. In both cases, the underlying pathophysiologic mechanisms are largely elusive - in part due to the lack of adequate access to specimens. We employed the mouse cytomegalovirus (MCMV) model to assess the association between CMV and colitis. During acute primary MCMV infection of immunocompetent mice, the gut microbial composition was affected as manifested by an altered ratio of the Firmicutes to Bacteroidetes phyla. Interestingly, these microbial changes coincided with high-titer MCMV replication in the colon, crypt hyperplasia, increased colonic pro-inflammatory cytokine levels, and a transient increase in the expression of the antimicrobial protein Regenerating islet-derived protein 3 gamma (Reg3γ). Further analyses revealed that murine and human intestinal epithelial cell lines, as well as primary intestinal crypt cells and organoids represent direct targets of CMV infection causing increased cell death. Accordingly, in vivo MCMV infection disrupted the intestinal epithelial barrier and increased apoptosis of intestinal epithelial cells. In summary, our data show that CMV transiently induces colitis in immunocompetent hosts by altering the intestinal homeostasis.

Obatoclax inhibits SARS-CoV-2 entry by altered endosomal acidification and impaired cathepsin and furin activity in vitro.

Coronavirus disease 2019 (COVID-19) caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has set off a global pandemic. There is an urgent unmet need for safe, affordable, and effective therapeutics against COVID-19. In this regard, drug repurposing is considered as a promising approach. We assessed the compounds that affect the endosomal acidic environment by applying human angiotensin-converting enzyme 2 (hACE2)- expressing cells infected with a SARS-CoV-2 spike (S) protein-pseudotyped HIV reporter virus and identified that obatoclax resulted in the strongest inhibition of S protein-mediated virus entry. The potent antiviral activity of obatoclax at nanomolar concentrations was confirmed in different human lung and intestinal cells infected with the SARS-CoV-2 pseudotype system as well as clinical virus isolates. Furthermore, we uncovered that obatoclax executes a double-strike against SARS-CoV-2. It prevented SARS-CoV-2 entry by blocking endocytosis of virions through diminished endosomal acidification and the corresponding inhibition of the enzymatic activity of the endosomal cysteine protease cathepsin L. Additionally, obatoclax impaired the SARS-CoV-2 S-mediated membrane fusion by targeting the MCL-1 protein and reducing furin protease activity. In accordance with these overarching mechanisms, obatoclax blocked the virus entry mediated by different S proteins derived from several SARS-CoV-2 variants of concern such as, Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2). Taken together, our results identified obatoclax as a novel effective antiviral compound that keeps SARS-CoV-2 at bay by blocking both endocytosis and membrane fusion. Our data suggested that obatoclax should be further explored as a clinical drug for the treatment of COVID-19.

Broad and potent antiviral activity of the NAE inhibitor MLN4924.

In terms of infected human individuals, herpesviruses range among the most successful virus families. Subclinical herpesviral infections in healthy individuals contrast with life-threatening syndromes under immunocompromising and immunoimmature conditions. Based on our finding that cytomegaloviruses interact with Cullin Roc ubiquitin ligases (CRLs) in the context of interferon antagonism, we systematically assessed viral dependency on CRLs by utilizing the drug MLN4924. CRL activity is regulated through the conjugation of Cullins with the ubiquitin-like molecule Nedd8. By inhibiting the Nedd8-activating Enzyme (NAE), MLN4924 interferes with Nedd8 conjugation and CRL activity. MLN4924 exhibited pronounced antiviral activity against mouse and human cytomegalovirus, herpes simplex virus (HSV)- 1 (including multi-drug resistant clinical isolates), HSV-2, adeno and influenza viruses. Human cytomegalovirus genome amplification was blocked at nanomolar MLN4924 concentrations. Global proteome analyses revealed that MLN4924 blocks cytomegaloviral replication despite increased IE1 amounts. Expression of dominant negative Cullins assigned this IE regulation to defined Cullin molecules and phenocopied the antiviral effect of MLN4924.

"Activated" STAT proteins: a paradoxical consequence of inhibited JAK-STAT signaling in cytomegalovirus-infected cells.

We have previously characterized mouse CMV (MCMV)-encoded immune-evasive IFN signaling inhibition and identified the viral protein pM27 as inducer of proteasomal degradation of STAT2. Extending our analysis to STAT1 and STAT3, we found that MCMV infection neither destabilizes STAT1 protein nor prevents STAT1 tyrosine Y701 phosphorylation, nuclear translocation, or the capability to bind γ-activated sequence DNA-enhancer elements. Unexpectedly, the analysis of STAT3 revealed an induction of STAT3 Y705 phosphorylation by MCMV. In parallel, we found decreasing STAT3 protein amounts upon MCMV infection, although STAT3 expression normally is positive autoregulative. STAT3 phosphorylation depended on the duration of MCMV infection, the infectious dose, and MCMV gene expression but was independent of IFNAR1, IL-10, IL-6, and JAK2. Although STAT3 phosphorylation did not require MCMV immediate early 1, pM27, and late gene expression, it was restricted to MCMV-infected cells and not transmitted to bystander cells. Despite intact STAT1 Y701 phosphorylation, IFN-γ-induced target gene transcription (e.g., IRF1 and suppressor of cytokine signaling [SOCS] 1) was strongly impaired. Likewise, the induction of STAT3 target genes (e.g., SOCS3) by IL-6 was also abolished, indicating that MCMV antagonizes STAT1 and STAT3 despite the occurrence of tyrosine phosphorylation. Consistent with the lack of SOCS1 induction, STAT1 phosphorylation was prolonged upon IFN-γ treatment. We conclude that the inhibition of canonical STAT1 and STAT3 target gene expression abrogates their intrinsic negative feedback loops, leading to accumulation of phospho-tyrosine-STAT3 and prolonged STAT1 phosphorylation. These findings challenge the generalization of tyrosine-phosphorylated STATs necessarily being transcriptional active and document antagonistic effects of MCMV on STAT1/3-dependent target gene expression.