SARS-CoV-2 Spike triggers barrier dysfunction and vascular leak via integrins and TGF-β signaling

Severe COVID-19 is associated with epithelial and endothelial barrier dysfunction within the lung as well as in distal organs. While it is appreciated that an exaggerated inflammatory response is associated with barrier dysfunction, the triggers of vascular leak are unclear. Here, we report that cell-intrinsic interactions between the Spike (S) glycoprotein of SARS-CoV-2 and epithelial/endothelial cells are sufficient to induce barrier dysfunction in vitro and vascular leak in vivo, independently of viral replication and the ACE2 receptor. We identify an S-triggered transcriptional response associated with extracellular matrix reorganization and TGF-β signaling. Using genetic knockouts and specific inhibitors, we demonstrate that glycosaminoglycans, integrins, and the TGF-β signaling axis are required for S-mediated barrier dysfunction. Notably, we show that SARS-CoV-2 infection caused leak in vivo, which was reduced by inhibiting integrins. Our findings offer mechanistic insight into SARS-CoV-2-triggered vascular leak, providing a starting point for development of therapies targeting COVID-19

The biogenesis of dengue virus replication organelles requires the ATPase activity of valosin-containing protein

The dengue virus (DENV) causes the most prevalent arthropod-borne viral disease worldwide. While its incidence is increasing in many countries, there is no approved antiviral therapy currently available. In infected cells, the DENV induces extensive morphological alterations of the endoplasmic reticulum (ER) to generate viral replication organelles (vRO), which include convoluted membranes (CM) and vesicle packets (VP) hosting viral RNA replication. The viral non-structural protein NS4B localizes to vROs and is absolutely required for viral replication through poorly defined mechanisms, which might involve cellular protein partners. Previous interactomic studies identified the ATPase valosin-containing protein (VCP) as a DENV NS4B-interacting host factor in infected cells. Using both pharmacological and dominant-negative inhibition approaches, we show, in this study, that VCP ATPase activity is required for efficient DENV replication. VCP associates with NS4B when expressed in the absence of other viral proteins while in infected cells, both proteins colocalize within large DENV-induced cytoplasmic structures previously demonstrated to be CMs. Consistently, VCP inhibition dramatically reduces the abundance of DENV CMs in infected cells. Most importantly, using a recently reported replication-independent plasmid-based vRO induction system, we show that de novo VP biogenesis is dependent on VCP ATPase activity. Overall, our data demonstrate that VCP ATPase activity is required for vRO morphogenesis and/or stability. Considering that VCP was shown to be required for the replication of other flaviviruses, our results argue that VCP is a pan-flaviviral host dependency factor. Given that new generation VCP-targeting drugs are currently evaluated in clinical trials for cancer treatment, VCP may constitute an attractive broad-spectrum antiviral target in drug repurposing approaches

Enhanced SARS-CoV-2 entry via UPR-dependent AMPK-related kinase NUAK2

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remodels the endoplasmic reticulum (ER) to form replication organelles, leading to ER stress and unfolded protein response (UPR). However, the role of specific UPR pathways in infection remains unclear. Here, we found that SARS-CoV-2 infection causes marginal activation of signaling sensor IRE1α leading to its phosphorylation, clustering in the form of dense ER-membrane rearrangements with embedded membrane openings, and XBP1 splicing. By investigating the factors regulated by IRE1α-XBP1 during SARS-CoV-2 infection, we identified stress-activated kinase NUAK2 as a novel host-dependency factor for SARS-CoV-2, HCoV-229E, and MERS-CoV entry. Reducing NUAK2 abundance or kinase activity impaired SARS-CoV-2 particle binding and internalization by decreasing cell surface levels of viral receptors and viral trafficking likely by modulating the actin cytoskeleton. IRE1α-dependent NUAK2 levels were elevated in SARS-CoV-2-infected and bystander non-infected cells, promoting viral spread by maintaining ACE2 cell surface levels and facilitating virion binding to bystander cells

Numerical values used for generation of figures.

Dengue virus (DENV) is a medically important flavivirus causing an estimated 50–100 million dengue cases annually, some of whom progress to severe disease. DENV non-structural protein 1 (NS1) is secreted from infected cells and has been implicated as a major driver of dengue pathogenesis by inducing endothelial barrier dysfunction. However, less is known about how DENV NS1 interacts with immune cells and what role these interactions play. Here we report that DENV NS1 can trigger activation of inflammasomes, a family of cytosolic innate immune sensors that respond to infectious and noxious stimuli, in mouse and human macrophages. DENV NS1 induces the release of IL-1β in a caspase-1 dependent manner. Additionally, we find that DENV NS1-induced inflammasome activation is independent of the NLRP3, Pyrin, and AIM2 inflammasome pathways, but requires CD14. Intriguingly, DENV NS1-induced inflammasome activation does not induce pyroptosis and rapid cell death; instead, macrophages maintain cellular viability while releasing IL-1β. Lastly, we show that caspase-1/11-deficient, but not NLRP3-deficient, mice are more susceptible to lethal DENV infection. Together, these results indicate that the inflammasome pathway acts as a sensor of DENV NS1 and plays a protective role during infection.</div

DENV NS1-induced gasdermin-D cleavage is dependent on caspase-1.

Representative Western blots of cell lysates from BMDMs nucleofected with Cas9-gRNA ribonuclear protein complexes targeting Casp1. Nucleofected BMDMs were primed with PAM3CSK4 (1μg/mL) for 17h and then treated for 48h with 10ug/mL DENV2 NS1 or left untreated for 48h without NS1 treatment. A non-targeting guide (NTG) was used as a control. (PDF)</p

CRISPR-Cas9 targeting of inflammasome pathways results in functional knockouts.

(A) BMDMs were nucleofected with Cas9-gRNA ribonuclear protein complexes to knock out the indicated genes. Two gRNAs per gene were used per nucleofection. Knockout BMDMs were primed with PAM3CSK4 (1μg/mL) for 17h and treated with nigericin (5uM) or left untreated for 2h. Supernatants were then collected, and IL-1β levels were measured by ELISA. (B-C) Same as in A, except cells were primed with LPS (5ug/mL) for 4 hours and then treated with TcdB (5ug/mL) (B) or Poly(dA:dT) / LyoVec (5ug/mL) (C) for 24h before collecting supernatant. *p 0.05. Statistical significance was determined using two-way ANOVA with Holm-Sidak’s multiple comparisons test. The data are shown as the mean ± SD of least 2 biological replicates per guide. NTG, non-targeting guide. (PDF)</p

DENV NS1-induced inflammasome activation is NLPR3-independent.

(A) WT and Nlrp3 -/- BMDMs were primed with PAM3CSK4 (1μg/mL) for 17h and then treated with DENV2 NS1 at indicated concentrations, nigericin (5μM), or medium (PAM only). IL-1β levels in supernatant 2h (nigericin) or 24h (NS1 and PAM only) were measured by ELISA. Statistical significance was determined using two-way ANOVA with Holm-Sidak’s multiple comparisons test. (B) Representative Western blots of cell lysates from WT and Nlrp3-/- BMDMs after priming with PAM3CSK4 (1μg/mL) for 17h and treatment with DENV2 NS1 (10 or 5 μg/mL), treatment with nigericin (5μM), or no treatment for 24h. (C) BMDMs were primed with PAM3CSK4 (1μg/mL) for 17h and then pre-treated with MCC950 at the indicated concentrations before addition of DENV2 NS1 (10μg/mL), nigericin (5μM), or medium (Inhibitor only). IL-1β levels in the supernatant after 2h (Nigericin) or 24h (NS1 and PAM only) were measured by ELISA. (D) Representative Western blots of cell lysates from BMDMs nucleofected with Cas9-gRNA ribonuclear protein complexes to knock out the indicated genes. Two gRNAs per gene were used per nucleofection. NTG = non-targeting guide. (E) Knockout BMDMs from (D) were primed with PAM3CSK4 (1μg/mL) for 17h and treated with DENV2 NS1 (10μg/mL) or left untreated for 48h. Statistical significance was determined using two-way ANOVA followed by with Holm-Sidak’s multiple comparisons test. The data are shown as the mean ± SD of 3 biological replicates (A,C), a representative image taken from 2 biological replicates (B,D), or data pooled from 8 independent experiments with at least 3 biological replicates per guide (E). *p 0.05.</p

CRISPR-Cas9 targeting of CD14 does not result in off-target inhibition of inflammasome activation.

(A-B) BMDMs from Fig 3D were primed with PAM3CSK4 (1μg/mL) for 17h and treated with 5μM nigericin or no treatment for 24h. IL-1β levels in supernatant were measured by ELISA (A). TNF-α levels were measured in supernatants 17h post-priming with PAM3CSK4 (B). (PDF)</p