Octyl itaconate enhances VSVΔ51 oncolytic virotherapy by multitarget inhibition of antiviral and inflammatory pathways.

The presence of heterogeneity in responses to oncolytic virotherapy poses a barrier to clinical effectiveness, as resistance to this treatment can occur through the inhibition of viral spread within the tumor, potentially leading to treatment failures. Here we show that 4-octyl itaconate (4-OI), a chemical derivative of the Krebs cycle-derived metabolite itaconate, enhances oncolytic virotherapy with VSVΔ51 in various models including human and murine resistant cancer cell lines, three-dimensional (3D) patient-derived colon tumoroids and organotypic brain tumor slices. Furthermore, 4-OI in combination with VSVΔ51 improves therapeutic outcomes in a resistant murine colon tumor model. Mechanistically, we find that 4-OI suppresses antiviral immunity in cancer cells through the modification of cysteine residues in MAVS and IKKß independently of the NRF2/KEAP1 axis. We propose that the combination of a metabolite-derived drug with an oncolytic virus agent can greatly improve anticancer therapeutic outcomes by direct interference with the type I IFN and NF-κB-mediated antiviral responses.

Inhibition of exchange proteins directly activated by cAMP as a strategy for broad-spectrum antiviral development.

The recent SARS-CoV-2 and mpox outbreaks have highlighted the need to expand our arsenal of broad-spectrum antiviral agents for future pandemic preparedness. Host-directed antivirals are an important tool to accomplish this as they typically offer protection against a broader range of viruses than direct-acting antivirals and have a lower susceptibility to viral mutations that cause drug resistance. In this study, we investigate the exchange protein activated by cAMP (EPAC) as a target for broad-spectrum antiviral therapy. We find that the EPAC-selective inhibitor, ESI-09, provides robust protection against a variety of viruses, including SARS-CoV-2 and Vaccinia (VACV)-an orthopox virus from the same family as mpox. We show, using a series of immunofluorescence experiments, that ESI-09 remodels the actin cytoskeleton through Rac1/Cdc42 GTPases and the Arp2/3 complex, impairing internalization of viruses that use clathrin-mediated endocytosis (e.g. VSV) or micropinocytosis (e.g. VACV). Additionally, we find that ESI-09 disrupts syncytia formation and inhibits cell-to-cell transmission of viruses such as measles and VACV. When administered to immune-deficient mice in an intranasal challenge model, ESI-09 protects mice from lethal doses of VACV and prevents formation of pox lesions. Altogether, our finding shows that EPAC antagonists such as ESI-09 are promising candidates for broad-spectrum antiviral therapy that can aid in the fight against ongoing and future viral outbreaks.

VPS34-dependent control of apical membrane function of proximal tubule cells and nutrient recovery by the kidney.

The lipid kinase VPS34 orchestrates autophagy, endocytosis, and metabolism and is implicated in cancer and metabolic disease. The proximal tubule in the kidney is a key metabolic organ that controls reabsorption of nutrients such as fatty acids, amino acids, sugars, and proteins. Here, by combining metabolomics, proteomics, and phosphoproteomics analyses with functional and superresolution imaging assays of mice with an inducible deficiency in proximal tubular cells, we revealed that VPS34 controlled the metabolome of the proximal tubule. In addition to inhibiting pinocytosis and autophagy, VPS34 depletion induced membrane exocytosis and reduced the abundance of the retromer complex necessary for proper membrane recycling and lipid retention, leading to a loss of fuel and biomass. Integration of omics data into a kidney cell metabolomic model demonstrated that VPS34 deficiency increased ß-oxidation, reduced gluconeogenesis, and enhanced the use of glutamine for energy consumption. Furthermore, the omics datasets revealed that VPS34 depletion triggered an antiviral response that included a decrease in the abundance of apically localized virus receptors such as ACE2. VPS34 inhibition abrogated SARS-CoV-2 infection in human kidney organoids and cultured proximal tubule cells in a glutamine-dependent manner. Thus, our results demonstrate that VPS34 adjusts endocytosis, nutrient transport, autophagy, and antiviral responses in proximal tubule cells in the kidney.

Identification of FDA-approved bifonazole as a SARS-CoV-2 blocking agent following a bioreporter drug screen.

We established a split nanoluciferase complementation assay to rapidly screen for inhibitors that interfere with binding of the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein with its target receptor, angiotensin-converting enzyme 2 (ACE2). After a screen of 1,200 US Food and Drug Administration (FDA)-approved compounds, we identified bifonazole, an imidazole-based antifungal agent, as a competitive inhibitor of RBD-ACE2 binding. Mechanistically, bifonazole binds ACE2 around residue K353, which prevents association with the RBD, affecting entry and replication of spike-pseudotyped viruses as well as native SARS-CoV-2 and its variants of concern (VOCs). Intranasal administration of bifonazole reduces lethality in K18-hACE2 mice challenged with vesicular stomatitis virus (VSV)-spike by 40%, with a similar benefit after live SARS-CoV-2 challenge. Our screen identified an antiviral agent that is effective against SARS-CoV-2 and VOCs such as Omicron that employ the same receptor to infect cells and therefore has high potential to be repurposed to control, treat, or prevent coronavirus disease 2019 (COVID-19).

Human SNORA31 variations impair cortical neuron-intrinsic immunity to HSV-1 and underlie herpes simplex encephalitis.

Herpes simplex virus-1 (HSV-1) encephalitis (HSE) is typically sporadic. Inborn errors of TLR3- and DBR1-mediated central nervous system cell-intrinsic immunity can account for forebrain and brainstem HSE, respectively. We report five unrelated patients with forebrain HSE, each heterozygous for one of four rare variants of SNORA31, encoding a small nucleolar RNA of the H/ACA class that are predicted to direct the isomerization of uridine residues to pseudouridine in small nuclear RNA and ribosomal RNA. We show that CRISPR/Cas9-introduced bi- and monoallelic SNORA31 deletions render human pluripotent stem cell (hPSC)-derived cortical neurons susceptible to HSV-1. Accordingly, SNORA31-mutated patient hPSC-derived cortical neurons are susceptible to HSV-1, like those from TLR3- or STAT1-deficient patients. Exogenous interferon (IFN)-ß renders SNORA31- and TLR3- but not STAT1-mutated neurons resistant to HSV-1. Finally, transcriptome analysis of SNORA31-mutated neurons revealed normal responses to TLR3 and IFN-α/ß stimulation but abnormal responses to HSV-1. Human SNORA31 thus controls central nervous system neuron-intrinsic immunity to HSV-1 by a distinctive mechanism.

Mutations in RNA Polymerase III genes and defective DNA sensing in adults with varicella-zoster virus CNS infection.

Recently, deficiency in the cytosolic DNA sensor RNA Polymerase III was described in children with severe primary varicella-zoster virus (VZV) infection in the CNS and lungs. In the present study we examined adult patients with VZV CNS infection caused by viral reactivation. By whole exome sequencing we identified mutations in POL III genes in two of eight patients. These mutations were located in the coding regions of the subunits POLR3A and POLR3E. In functional assays, we found impaired expression of antiviral and inflammatory cytokines in response to the POL III agonist Poly(dA:dT) as well as increased viral replication in patient cells compared to controls. Altogether, this study provides significant extension on the current knowledge on susceptibility to VZV infection by demonstrating mutations in POL III genes associated with impaired immunological sensing of AT-rich DNA in adult patients with VZV CNS infection.

Varicella-zoster virus CNS vasculitis and RNA polymerase III gene mutation in identical twins.

OBJECTIVE: Deficiency in the cytosolic DNA sensor RNA Polymerase III (POL III) was recently described in children with severe varicella-zoster virus (VZV) infection in the CNS or lungs. Here, we describe a pair of monozygotic female twins, who both experienced severe recurrent CNS vasculitis caused by VZV reactivation. The clinical presentation and findings included recurrent episodes of headache, dizziness, and neurologic deficits, CSF with pleocytosis and intrathecal VZV antibody production, and MRI of the brain showing ischemic lesions. METHODS: We performed whole-exome sequencing and identified a rare mutation in the POL III subunit POLR3F. Subsequently, antiviral responses in patient peripheral blood mononuclear cells (PBMCs) were examined and compared with healthy controls. RESULTS: The identified R50W POLR3F mutation is predicted by bioinformatics to be damaging, and when tested in functional assays, patient PBMCs exhibited impaired antiviral and inflammatory responses to the POL III agonist poly(dA:dT) and increased viral replication compared with controls. CONCLUSIONS: Altogether, these cases add genetic and immunologic evidence to the novel association between defects in sensing of AT-rich DNA present in the VZV genome and increased susceptibility to severe manifestations of VZV infection in the CNS in humans.

RNA Polymerase III as a Gatekeeper to Prevent Severe VZV Infections.

In most individuals, varicella zoster virus (VZV) causes varicella upon primary infection and zoster during reactivation. However, in a subset of individuals, VZV may cause severe disease, including encephalitis. Host genetics is believed to be the main determinant of exacerbated disease manifestations. Recent studies have demonstrated that defects in the DNA sensor RNA polymerase III (POL III) confer selective increased susceptibility to VZV infection, thus providing fundamental new insight into VZV immunity. Here we describe the roles of POL III in housekeeping and immune surveillance during VZV infection. We present the latest knowledge on the role of POL III in VZV infection and discuss outstanding questions related to the role of POL III in VZV immunity, and how this insight can be translated into clinical medicine.