The lipopeptide Pam3CSK4 inhibits Rift Valley fever virus infection and protects from encephalitis.

Rift Valley fever virus (RVFV) is an encephalitic bunyavirus that can infect neurons in the brain. There are no approved therapeutics that can protect from RVFV encephalitis. Innate immunity, the first line of defense against infection, canonically antagonizes viruses through interferon signaling. We found that interferons did not efficiently protect primary cortical neurons from RVFV, unlike other cell types. To identify alternative neuronal antiviral pathways, we screened innate immune ligands and discovered that the TLR2 ligand Pam3CSK4 inhibited RVFV infection, and other bunyaviruses. Mechanistically, we found that Pam3CSK4 blocks viral fusion, independent of TLR2. In a mouse model of RVFV encephalitis, Pam3CSK4 treatment protected animals from infection and mortality. Overall, Pam3CSK4 is a bunyavirus fusion inhibitor active in primary neurons and the brain, representing a new approach toward the development of treatments for encephalitic bunyavirus infections.

Type I interferon alters invasive extravillous trophoblast function.

Inappropriate type I interferon (IFN) signaling during embryo implantation and placentation is linked to poor pregnancy outcomes. Here, we evaluated the consequence of elevated type I IFN exposure on implantation using a biomimetic model of human implantation in an organ-on-a-chip device. We found that type I IFN reduced extravillous trophoblast (EVT) invasion capacity. Analyzing single-cell transcriptomes, we uncovered that IFN truncated endovascular EVT emergence in the implantation-on-a-chip device by stunting EVT epithelial-to-mesenchymal transition. Disruptions to the epithelial-to-mesenchymal transition is associated with the pathogenesis of preeclampsia, a life-threatening hypertensive disorder of pregnancy. Strikingly, unwarranted IFN stimulation induced genes associated with increased preeclampsia risk and a preeclamptic gene-like signature in EVTs. These dysregulated EVT phenotypes ultimately reduced EVT-mediated endothelial cell vascular remodeling in the implantation-on-a-chip device. Overall, our work indicates IFN signaling can alter EVT epithelial-to-mesenchymal transition progression which results in diminished EVT-mediated spiral artery remodeling and a preeclampsia gene signature upon sustained stimulation. Our work implicates unwarranted type I IFN as a maternal disturbance that can result in abnormal EVT function that could trigger preeclampsia.

Endothelial SARS-CoV-2 infection is not the underlying cause of COVID-19-associated vascular pathology in mice.

Endothelial damage and vascular pathology have been recognized as major features of COVID-19 since the beginning of the pandemic. Two main theories regarding how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) damages endothelial cells and causes vascular pathology have been proposed: direct viral infection of endothelial cells or indirect damage mediated by circulating inflammatory molecules and immune mechanisms. However, these proposed mechanisms remain largely untested in vivo. In the present study, we utilized a set of new mouse genetic tools developed in our lab to test both the necessity and sufficiency of endothelial human angiotensin-converting enzyme 2 (hACE2) in COVID-19 pathogenesis. Our results demonstrate that endothelial ACE2 and direct infection of vascular endothelial cells do not contribute significantly to the diverse vascular pathology associated with COVID-19.

Serotonin reduction in post-acute sequelae of viral infection.

Post-acute sequelae of COVID-19 (PASC, "Long COVID") pose a significant global health challenge. The pathophysiology is unknown, and no effective treatments have been found to date. Several hypotheses have been formulated to explain the etiology of PASC, including viral persistence, chronic inflammation, hypercoagulability, and autonomic dysfunction. Here, we propose a mechanism that links all four hypotheses in a single pathway and provides actionable insights for therapeutic interventions. We find that PASC are associated with serotonin reduction. Viral infection and type I interferon-driven inflammation reduce serotonin through three mechanisms: diminished intestinal absorption of the serotonin precursor tryptophan; platelet hyperactivation and thrombocytopenia, which impacts serotonin storage; and enhanced MAO-mediated serotonin turnover. Peripheral serotonin reduction, in turn, impedes the activity of the vagus nerve and thereby impairs hippocampal responses and memory. These findings provide a possible explanation for neurocognitive symptoms associated with viral persistence in Long COVID, which may extend to other post-viral syndromes.

Endothelial SARS-CoV-2 infection is not the underlying cause of COVID19-associated vascular pathology in mice.

Endothelial damage and vascular pathology have been recognized as major features of COVID-19 since the beginning of the pandemic. Two main theories regarding how Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) damages endothelial cells and causes vascular pathology have been proposed: direct viral infection of endothelial cells or indirect damage mediated by circulating inflammatory molecules and immune mechanisms. However, these proposed mechanisms remain largely untested in vivo. Here, we utilized a set of new mouse genetic tools 1 developed in our lab to test both the necessity and sufficiency of endothelial human angiotensin-converting enzyme 2 (hACE2) in COVID19 pathogenesis. Our results demonstrate that endothelial ACE2 and direct infection of vascular endothelial cells does not contribute significantly to the diverse vascular pathology associated with COVID-19.

Cell-autonomous requirement for ACE2 across organs in lethal mouse SARS-CoV-2 infection.

Angiotensin-converting enzyme 2 (ACE2) is the cell-surface receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). While its central role in Coronavirus Disease 2019 (COVID-19) pathogenesis is indisputable, there remains significant debate regarding the role of this transmembrane carboxypeptidase in the disease course. These include the role of soluble versus membrane-bound ACE2, as well as ACE2-independent mechanisms that may contribute to viral spread. Testing these roles requires in vivo models. Here, we report humanized ACE2-floxed mice in which hACE2 is expressed from the mouse Ace2 locus in a manner that confers lethal disease and permits cell-specific, Cre-mediated loss of function, and LSL-hACE2 mice in which hACE2 is expressed from the Rosa26 locus enabling cell-specific, Cre-mediated gain of function. Following exposure to SARS-CoV-2, hACE2-floxed mice experienced lethal cachexia, pulmonary infiltrates, intravascular thrombosis and hypoxemia-hallmarks of severe COVID-19. Cre-mediated loss and gain of hACE2 demonstrate that neuronal infection confers lethal cachexia, hypoxemia, and respiratory failure in the absence of lung epithelial infection. In this series of genetic experiments, we demonstrate that ACE2 is absolutely and cell-autonomously required for SARS-CoV-2 infection in the olfactory epithelium, brain, and lung across diverse cell types. Therapies inhibiting or blocking ACE2 at these different sites are likely to be an effective strategy towards preventing severe COVID-19.

Neurotropic RNA Virus Modulation of Immune Responses within the Central Nervous System.

The central nervous system (CNS) necessitates intricately coordinated immune responses to prevent neurological disease. However, the emergence of viruses capable of entering the CNS and infecting neurons threatens this delicate balance. Our CNS is protected from foreign invaders and excess solutes by a semipermeable barrier of endothelial cells called the blood-brain barrier. Thereby, viruses have implemented several strategies to bypass this protective layer and modulate immune responses within the CNS. In this review, we outline these immune regulatory mechanisms and provide perspectives on future questions in this rapidly expanding field.

SARS-CoV-2 infection of olfactory epithelial cells and neurons drives acute lung injury and lethal COVID-19 in mice.

Lethal COVID-19 is associated with respiratory failure that is thought to be caused by acute respiratory distress syndrome (ARDS) secondary to pulmonary infection. To date, the cellular pathogenesis has been inferred from studies describing the expression of ACE2, a transmembrane protein required for SARS-CoV-2 infection, and detection of viral RNA or protein in infected humans, model animals, and cultured cells. To functionally test the cellular mechanisms of COVID-19, we generated hACE2 fl animals in which human ACE2 (hACE2) is expressed from the mouse Ace2 locus in a manner that permits cell-specific, Cre-mediated loss of function. hACE2 fl animals developed lethal weight loss and hypoxemia within 7 days of exposure to SARS-CoV-2 that was associated with pulmonary infiltrates, intravascular thrombosis and patchy viral infection of lung epithelial cells. Deletion of hACE2 in lung epithelial cells prevented viral infection of the lung, but not weight loss, hypoxemia or death. Inhalation of SARS-CoV-2 by hACE2 fl animals resulted in early infection of sustentacular cells with subsequent infection of neurons in the neighboring olfactory bulb and cerebral cortexâ€" events that did not require lung epithelial cell infection. Pharmacologic ablation of the olfactory epithelium or Foxg1 Cre mediated deletion of hACE2 in olfactory epithelial cells and neurons prevented lethality and neuronal infection following SARS-CoV-2 infection. Conversely, transgenic expression of hACE2 specifically in olfactory epithelial cells and neurons in Foxg1 Cre ; LSL- hACE2 mice was sufficient to confer neuronal infection associated with respiratory failure and death. These studies establish mouse loss and gain of function genetic models with which to genetically dissect viral-host interactions and demonstrate that lethal disease due to respiratory failure may arise from extrapulmonary infection of the olfactory epithelium and brain. Future therapeutic efforts focused on preventing olfactory epithelial infection may be an effective means of protecting against severe COVID-19.

SARS-CoV-2 viral proteins NSP1 and NSP13 inhibit interferon activation through distinct mechanisms.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a devastating global pandemic, infecting over 43 million people and claiming over 1 million lives, with these numbers increasing daily. Therefore, there is urgent need to understand the molecular mechanisms governing SARS-CoV-2 pathogenesis, immune evasion, and disease progression. Here, we show that SARS-CoV-2 can block IRF3 and NF-κB activation early during virus infection. We also identify that the SARS-CoV-2 viral proteins NSP1 and NSP13 can block interferon activation via distinct mechanisms. NSP1 antagonizes interferon signaling by suppressing host mRNA translation, while NSP13 downregulates interferon and NF-κB promoter signaling by limiting TBK1 and IRF3 activation, as phospho-TBK1 and phospho-IRF3 protein levels are reduced with increasing levels of NSP13 protein expression. NSP13 can also reduce NF-κB activation by both limiting NF-κB phosphorylation and nuclear translocation. Last, we also show that NSP13 binds to TBK1 and downregulates IFIT1 protein expression. Collectively, these data illustrate that SARS-CoV-2 bypasses multiple innate immune activation pathways through distinct mechanisms.

A Peptide Derived from Lens Epithelium-Derived Growth Factor Stimulates HIV-1 DNA Integration and Facilitates Intasome Structural Studies.

The low solubility and aggregation properties of HIV-1 integrase (IN) are major obstacles for biochemical and structural studies. The lens epithelium-derived growth factor (LEDGF) is a cellular factor that binds IN and tethers preintegration complexes to chromatin before integration. The LEDGF also stimulates HIV-1 IN DNA strand transfer activity and improves its solubility in vitro. We show that these properties are conferred by a short peptide spanning residues 178 to 197 of the LEDGF that encompasses its AT-hook DNA-binding elements. The peptide stimulates HIV-1 IN activity both in trans and in cis. Fusion of the peptide to either the N- or C-terminus of IN results in maximal stimulation of concerted integration activity and greatly improves the solubility of the protein and nucleoprotein complexes of IN with viral DNA ends (intasomes). High-resolution structures of HIV-1 intasomes are required to understand the mechanism of IN strand transfer inhibitors (INSTIs), which are front-line drugs for the treatment of HIV-1, and how the virus can develop resistance to INSTIs. We have previously determined the structure of the HIV-1 strand transfer complex intasome. The improved biophysical properties of intasomes assembled with LEDGF peptide fusion IN have enabled us to determine the structure of the cleaved synaptic complex intasome, which is the direct target of INSTIs.

An Antiviral Branch of the IL-1 Signaling Pathway Restricts Immune-Evasive Virus Replication.

Virulent pathogens often cause the release of host-derived damage-associated molecular patterns (DAMPs) from infected cells. During encounters with immune-evasive viruses that block inflammatory gene expression, preformed DAMPs provide backup inflammatory signals that ensure protective immunity. Whether DAMPs exhibit additional backup defense activities is unknown. Herein, we report that viral infection of barrier epithelia (keratinocytes) elicits the release of preformed interleukin-1 (IL-1) family cytokines, including the DAMP IL-1α. Mechanistic studies revealed that IL-1 acts on skin fibroblasts to induce an interferon (IFN)-like state that restricts viral replication. We identified a branch in the IL-1 signaling pathway that induces IFN-stimulated gene expression in infected cells and found that IL-1 signaling is necessary to restrict viral replication in human skin explants. These activities are most important to control immune-evasive virus replication in fibroblasts and other barrier cell types. These findings highlight IL-1 as an important backup antiviral system to ensure barrier defense.

Type I interferons instigate fetal demise after Zika virus infection.

Zika virus (ZIKV) infection during pregnancy is associated with adverse fetal outcomes, including microcephaly, growth restriction, and fetal demise. Type I interferons (IFNs) are essential for host resistance against ZIKV, and IFN-α/ß receptor (IFNAR)-deficient mice are highly susceptible to ZIKV infection. Severe fetal growth restriction with placental damage and fetal resorption is observed after ZIKV infection of type I IFN receptor knockout (Ifnar1-/-) dams mated with wild-type sires, resulting in fetuses with functional type I IFN signaling. The role of type I IFNs in limiting or mediating ZIKV disease within this congenital infection model remains unknown. In this study, we challenged Ifnar1-/- dams mated with Ifnar1+/- sires with ZIKV. This breeding scheme enabled us to examine pregnant dams that carry a mixture of fetuses that express (Ifnar1+/-) or do not express IFNAR (Ifnar1-/-) within the same uterus. Virus replicated to a higher titer in the placenta of Ifnar1-/- than within the Ifnar1+/- concepti. Yet, rather unexpectedly, we found that only Ifnar1+/- fetuses were resorbed after ZIKV infection during early pregnancy, whereas their Ifnar1-/- littermates continue to develop. Analyses of the fetus and placenta revealed that, after ZIKV infection, IFNAR signaling in the conceptus inhibits development of the placental labyrinth, resulting in abnormal architecture of the maternal-fetal barrier. Exposure of midgestation human chorionic villous explants to type I IFN, but not type III IFNs, altered placental morphology and induced cytoskeletal rearrangements within the villous core. Our results implicate type I IFNs as a possible mediator of pregnancy complications, including spontaneous abortions and growth restriction, in the context of congenital viral infections.

Antiviral CD8 T cells induce Zika-virus-associated paralysis in mice.

Zika virus (ZIKV) is an emerging, mosquito-borne RNA virus. The rapid spread of ZIKV within the Americas has unveiled microcephaly 1 and Guillain-Barré syndrome2,3 as ZIKV-associated neurological complications. Recent reports have also indicated other neurological manifestations to be associated with ZIKV, including myelitis 4 , meningoencephalitis 5 and fatal encephalitis 6 . Here, we investigate the neuropathogenesis of ZIKV infection in type I interferon receptor IFNAR knockout (Ifnar1 -/- ) mice, an infection model that exhibits high viral burden within the central nervous system. We show that systemic spread of ZIKV from the site of infection to the brain requires Ifnar1 deficiency in the haematopoietic compartment. However, spread of ZIKV within the central nervous system is supported by Ifnar1-deficient non-haematopoietic cells. Within this context, ZIKV infection of astrocytes results in breakdown of the blood-brain barrier and a large influx of CD8+ effector T cells. We also find that antiviral activity of CD8+ T cells within the brain markedly limits ZIKV infection of neurons, but, as a consequence, instigates ZIKV-associated paralysis. Taken together, our study uncovers mechanisms underlying ZIKV neuropathogenesis within a susceptible mouse model and suggests blood-brain barrier breakdown and T-cell-mediated neuropathology as potential underpinnings of ZIKV-associated neurological complications in humans.

Nlrp9b inflammasome restricts rotavirus infection in intestinal epithelial cells.

Rotavirus, a leading cause of severe gastroenteritis and diarrhoea in young children, accounts for around 215,000 deaths annually worldwide. Rotavirus specifically infects the intestinal epithelial cells in the host small intestine and has evolved strategies to antagonize interferon and NF-κB signalling, raising the question as to whether other host factors participate in antiviral responses in intestinal mucosa. The mechanism by which enteric viruses are sensed and restricted in vivo, especially by NOD-like receptor (NLR) inflammasomes, is largely unknown. Here we uncover and mechanistically characterize the NLR Nlrp9b that is specifically expressed in intestinal epithelial cells and restricts rotavirus infection. Our data show that, via RNA helicase Dhx9, Nlrp9b recognizes short double-stranded RNA stretches and forms inflammasome complexes with the adaptor proteins Asc and caspase-1 to promote the maturation of interleukin (Il)-18 and gasdermin D (Gsdmd)-induced pyroptosis. Conditional depletion of Nlrp9b or other inflammasome components in the intestine in vivo resulted in enhanced susceptibility of mice to rotavirus replication. Our study highlights an important innate immune signalling pathway that functions in intestinal epithelial cells and may present useful targets in the modulation of host defences against viral pathogens.

Inhibition of HIV-1 Maturation via Small-Molecule Targeting of the Amino-Terminal Domain in the Viral Capsid Protein.

The human immunodeficiency virus type 1 (HIV-1) capsid protein is an attractive therapeutic target, owing to its multifunctionality in virus replication and the high fitness cost of amino acid substitutions in capsids to HIV-1 infectivity. To date, small-molecule inhibitors have been identified that inhibit HIV-1 capsid assembly and/or impair its function in target cells. Here, we describe the mechanism of action of the previously reported capsid-targeting HIV-1 inhibitor, Boehringer-Ingelheim compound 1 (C1). We show that C1 acts during HIV-1 maturation to prevent assembly of a mature viral capsid. However, unlike the maturation inhibitor bevirimat, C1 did not significantly affect the kinetics or fidelity of Gag processing. HIV-1 particles produced in the presence of C1 contained unstable capsids that lacked associated electron density and exhibited impairments in early postentry stages of infection, most notably reverse transcription. C1 inhibited assembly of recombinant HIV-1 CA in vitro and induced aberrant cross-links in mutant HIV-1 particles capable of spontaneous intersubunit disulfide bonds at the interhexamer interface in the capsid lattice. Resistance to C1 was conferred by a single amino acid substitution within the compound-binding site in the N-terminal domain of the CA protein. Our results demonstrate that the binding site for C1 represents a new pharmacological vulnerability in the capsid assembly stage of the HIV-1 life cycle.IMPORTANCE The HIV-1 capsid protein is an attractive but unexploited target for clinical drug development. Prior studies have identified HIV-1 capsid-targeting compounds that display different mechanisms of action, which in part reflects the requirement for capsid function at both the efferent and afferent phases of viral replication. Here, we show that one such compound, compound 1, interferes with assembly of the conical viral capsid during virion maturation and results in perturbations at a specific protein-protein interface in the capsid lattice. We also identify and characterize a mutation in the capsid protein that confers resistance to the inhibitor. This study reveals a novel mechanism by which a capsid-targeting small molecule can inhibit HIV-1 replication.