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.
Integration is essential for HIV-1 replication, and the viral integrase (IN) protein is an important therapeutic target. Allosteric IN inhibitors (ALLINIs) that engage the IN dimer interface at the ...binding site for the host protein lens epithelium-derived growth factor (LEDGF)/transcriptional coactivator p75 are an emerging class of small molecule antagonists. Consistent with the inhibition of a multivalent drug target, ALLINIs display steep antiviral dose–response curves ex vivo. ALLINIs multimerize IN protein and concordantly block its assembly with viral DNA in vitro, indicating that the disruption of two integration-associated functions, IN catalysis and the IN-LEDGF/p75 interaction, determines the multimode mechanism of ALLINI action. We now demonstrate that ALLINI potency is unexpectedly accounted for during the late phase of HIV-1 replication. The compounds promote virion IN multimerization and, reminiscent of class II IN mutations, block the formation of the electron-dense viral core and inhibit reverse transcription and integration in subsequently infected target cells. Mature virions are recalcitrant to ALLINI treatment, and compound potency during virus production is independent of the level of LEDGF/p75 expression. We conclude that cooperative multimerization of IN by ALLINIs together with the inability for LEDGF/p75 to effectively engage the virus during its egress from cells underscores the multimodal mechanism of ALLINI action. Our results highlight the versatile nature of allosteric inhibitors to primarily inhibit viral replication at a step that is distinct from the catalytic requirement for the target enzyme. The vulnerability of IN to small molecules during the late phase of HIV-1 replication unveils a pharmacological Achilles’ heel for exploitation in clinical ALLINI development.
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.
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.
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.
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•Keratinocytes release IL-1 cytokines in response to PRR-evasive virus infection•IL-1 cytokines induce an antiviral state in human fibroblasts and endothelial cells•This antiviral response is important during encounters with immune-evasive viruses•IL-1 stimulates this antiviral response through an IRF1/gp130/STAT1 signaling axis
Keratinocytes are a barrier cell type that contribute to antiviral host defense. Orzalli et al. report that IL-1 cytokines released from infected keratinocytes induce antiviral responses in stromal cells. Mechanistic studies identified a genetic requirement for an IRF1-dependent signaling axis that regulates antiviral gene expression downstream of the IL-1R.
The DNA cutting and joining reactions of HIV-1 integration are catalyzed by integrase (IN), a viral protein that functions as a tetramer bridging the two viral DNA ends (intasome). Two major ...obstacles for biochemical and structural studies of HIV-1 intasomes are 1) the low efficiency of assembly with oligonucleotide DNA substrates, and 2) the non-specific aggregation of both intasomes and free IN in the reaction mixture. By fusing IN with a small non-specific DNA binding protein, Sulfolobus solfataricus chromosomal protein Sso7d (PDB: 1BNZ), we have engineered a highly soluble and hyperactive IN. Unlike wild-type IN, it efficiently catalyzes intasome assembly and concerted integration with oligonucleotide DNA substrates. The fusion IN protein also functions to integrate viral reverse transcripts during HIV-infection. The hyperactive HIV-1 IN may assist in facilitating future biochemical and structural studies of HIV-1 intasomes. Understanding the mechanistic basis of the Sso7d-IN fusion protein could provide insight into the factors that have hindered biophysical studies of wild-type HIV-1 IN and intasomes.
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.
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•Long COVID is associated with reduced circulating serotonin levels•Serotonin depletion is driven by viral RNA-induced type I interferons (IFNs)•IFNs reduce serotonin through diminished tryptophan uptake and hypercoagulability•Peripheral serotonin deficiency impairs cognition via reduced vagal signaling
Post-viral syndromes are associated with serotonin reduction, which may contribute to the neurological and cognitive symptoms seen in individuals with Long COVID.
During virion maturation, HIV-1 capsid protein assembles into a conical core containing the viral ribonucleoprotein (vRNP) complex, thought to be composed mainly of the viral RNA and nucleocapsid ...protein (NC). After infection, the viral RNA is reverse transcribed into double-stranded DNA, which is then incorporated into host chromosomes by integrase (IN) catalysis. Certain IN mutations (class II) and antiviral drugs (allosteric IN inhibitors ALLINIs) adversely affect maturation, resulting in virions that contain "eccentric condensates," electron-dense aggregates located outside seemingly empty capsids. Here we demonstrate that in addition to this mislocalization of electron density, a class II IN mutation and ALLINIs each increase the fraction of virions with malformed capsids (from ∼ 12% to ∼ 53%). Eccentric condensates have a high NC content, as demonstrated by "tomo-bubblegram" imaging, a novel labeling technique that exploits the susceptibility of NC to radiation damage. Tomo-bubblegrams also localized NC inside wild-type cores and lining the spherical Gag shell in immature virions. We conclude that eccentric condensates represent nonpackaged vRNPs and that either genetic or pharmacological inhibition of IN can impair vRNP incorporation into mature cores. Supplying IN in trans as part of a Vpr-IN fusion protein partially restored the formation of conical cores with internal electron density and the infectivity of a class II IN deletion mutant virus. Moreover, the ability of ALLINIs to induce eccentric condensate formation required both IN and viral RNA. Based on these observations, we propose a role for IN in initiating core morphogenesis and vRNP incorporation into the mature core during HIV-1 maturation.
Maturation, a process essential for HIV-1 infectivity, involves core assembly, whereby the viral ribonucleoprotein (vRNP, composed of vRNA and nucleocapsid protein NC) is packaged into a conical capsid. Allosteric integrase inhibitors (ALLINIs) affect multiple viral processes. We have characterized ALLINIs and integrase mutants that have the same phenotype. First, by comparing the effects of ALLINIs on several steps of the viral cycle, we show that inhibition of maturation accounts for compound potency. Second, by using cryoelectron tomography, we find that ALLINIs impair conical capsid assembly. Third, by developing tomo-bubblegram imaging, which specifically labels NC protein, we find that ALLINIs block vRNP packaging; instead, vRNPs form "eccentric condensates" outside the core. Fourth, malformed cores, typical of integrase-deleted virus, are partially replaced by conical cores when integrase is supplied in trans. Fifth, vRNA is necessary for ALLINI-induced eccentric condensate formation. These observations suggest that integrase is involved in capsid morphogenesis and vRNP packaging.
There is an urgent need for antivirals to treat the newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To identify new candidates, we screen a repurposing library of ∼3,000 ...drugs. Screening in Vero cells finds few antivirals, while screening in human Huh7.5 cells validates 23 diverse antiviral drugs. Extending our studies to lung epithelial cells, we find that there are major differences in drug sensitivity and entry pathways used by SARS-CoV-2 in these cells. Entry in lung epithelial Calu-3 cells is pH independent and requires TMPRSS2, while entry in Vero and Huh7.5 cells requires low pH and triggering by acid-dependent endosomal proteases. Moreover, we find nine drugs are antiviral in respiratory cells, seven of which have been used in humans, and three are US Food and Drug Administration (FDA) approved, including cyclosporine. We find that the antiviral activity of cyclosporine is targeting Cyclophilin rather than calcineurin, revealing essential host targets that have the potential for rapid clinical implementation.
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•3,000 compounds screened in two cell types against SARS-CoV-2•Entry pathways are distinct in hepatocyte Huh7.5 and respiratory Calu-3 cells•Only nine compounds that are active in Huh7.5 cells are active in Calu-3 cells•Cyclosporin and cyclophilin inhibitors block SARS-CoV-2 infection in diverse cells
There is an urgent need for antivirals to treat the newly emerged SARS-CoV-2. Dittmar et al. find nine host-directed drugs are antiviral in respiratory cells, seven of which have been given to humans, and three are FDA approved. We show host targets that have the potential for rapid clinical implementation.
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.
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•Structural studies of the HIV-1 intasomes have been frustrated by aggregation.•Structures of intasomes are needed to understand inhibitor mechanism and resistance.•Fusion of a peptide to HIV-1 integrase (IN) stimulates catalytic activity.•The peptide fusion integrase assembles intasomes with lesser aggregation.•We have used the fusion IN to determine the structure of the HIV-1 cleaved synaptic complex intasome.
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