Interferon signaling in the nasal epithelium distinguishes among lethal and common cold respiratory viruses and is critical for viral clearance (preprint)

All respiratory viruses establish primary infections in the nasal epithelium, where efficient innate immune induction may prevent dissemination to the lower airway and thus minimize pathogenesis. Human coronaviruses (HCoVs) cause a range of pathologies, but the host and viral determinants of disease during common cold versus lethal HCoV infections are poorly understood. We model the initial site of infection using primary nasal epithelial cells cultured at air-liquid interface (ALI). HCoV-229E, HCoV-NL63 and human rhinovirus-16 are common cold-associated viruses that exhibit unique features in this model: early induction of antiviral interferon (IFN) signaling, IFN-mediated viral clearance, and preferential replication at nasal airway temperature (33C) which confers muted host IFN responses. In contrast, lethal SARS-CoV-2 and MERS-CoV encode antagonist proteins that prevent IFN-mediated clearance in nasal cultures. Our study identifies features shared among common cold-associated viruses, highlighting nasal innate immune responses as predictive of infection outcomes and nasally-directed IFNs as potential therapeutics.

Comparison of SARS-CoV-2 variants in primary human nasal cultures indicates Delta as most cytopathic and Omicron as fastest replicating (preprint)

The ongoing SARS-CoV-2 pandemic has been marked with emerging viral variants, some of which were designated as variants of concern (VOCs) due to their selection and rapid circulation in the human population. Here we elucidate functional features of each VOC in patient-derived primary nasal cultures grown at air-liquid-interface (ALI) to model upper-respiratory infection, and human lung epithelial cell lines to model lung infection. All VOCs replicated to higher titers than the ancestral virus, and Omicron reached the higher titer in the upper-respiratory system in both nasal cells and parallel human studies. Delta was most adept at cell-to-cell spread and the most cytopathic to nasal cells by compromising cell-barrier integrity and ciliary beating. All VOCs overcame dsRNA-activated cellular responses including interferon signaling, oligoadenylate ribonuclease L (OAS-RNase L) degradation and protein kinase R (PKR) activation. Our findings highlight the functional differences among VOCs and illuminate distinct mechanisms of pathogenesis in infected individuals.

Infection of primary nasal epithelial cells differentiates among lethal and seasonal human coronaviruses (preprint)

The nasal epithelium is the initial entry portal and primary barrier to infection by all human coronaviruses (HCoVs). We utilize primary nasal epithelial cells grown at air-liquid interface, which recapitulate the heterogeneous cellular population as well as mucociliary clearance functions of the in vivo nasal epithelium, to compare lethal (SARS-CoV-2 and MERS-CoV) and seasonal (HCoV-NL63 and HCoV-229E) HCoVs. All four HCoVs replicate productively in nasal cultures but diverge significantly in terms of cytotoxicity induced following infection, as the seasonal HCoVs as well as SARS-CoV-2 cause cellular cytotoxicity as well as epithelial barrier disruption, while MERS-CoV does not. Treatment of nasal cultures with type 2 cytokine IL-13 to mimic asthmatic airways differentially impacts HCoV replication, enhancing MERS-CoV replication but reducing that of SARS-CoV-2 and HCoV-NL63. This study highlights diversity among HCoVs during infection of the nasal epithelium, which is likely to influence downstream infection outcomes such as disease severity and transmissibility.

Cyclooxgenase-2 is induced by SARS-CoV-2 infection but does not affect viral entry or replication (preprint)

Identifying drugs that regulate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and its symptoms has been a pressing area of investigation during the coronavirus disease 2019 (COVID-19) pandemic. Nonsteroidal anti-inflammatory drugs (NSAIDs), which are frequently used for the relief of pain and inflammation, could modulate both SARS-CoV-2 infection and the host response to the virus. NSAIDs inhibit the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), which mediate the production of prostaglandins (PGs). PGE2, one of the most abundant PGs, has diverse biological roles in homeostasis and inflammatory responses. Previous studies have shown that NSAID treatment or inhibition of PGE2 receptor signaling leads to upregulation of angiotensin-converting enzyme 2 (ACE2), the cell entry receptor for SARS-CoV-2, thus raising concerns that NSAIDs could increase susceptibility to infection. COX/PGE2 signaling has also been shown to regulate the replication of many viruses, but it is not yet known whether it plays a role in SARS-CoV-2 replication. The purpose of this study was to dissect the effect of NSAIDs on COVID-19 in terms of SARS-CoV-2 entry and replication. We found that SARS-CoV-2 infection induced COX-2 upregulation in diverse human cell culture and mouse systems. However, suppression of COX-2/PGE2 signaling by two commonly used NSAIDs, ibuprofen and meloxicam, had no effect on ACE2 expression, viral entry, or viral replication. Our findings suggest that COX-2 signaling driven by SARS-CoV-2 may instead play a role in regulating the lung inflammation and injury observed in COVID-19 patients.

SARS-CoV-2 induces double-stranded RNA-mediated innate immune responses in respiratory epithelial derived cells and cardiomyocytes (preprint)

Coronaviruses are adept at evading host antiviral pathways induced by viral double-stranded RNA, including interferon (IFN) signaling, oligoadenylate synthetase-ribonuclease L (OAS-RNase L), and protein kinase R (PKR). While dysregulated or inadequate IFN responses have been associated with severe coronavirus infection, the extent to which the recently emerged SARS-CoV-2 activates or antagonizes these pathways is relatively unknown. We found that SARS-CoV-2 infects patient-derived nasal epithelial cells, present at the initial site of infection, induced pluripotent stem cell-derived alveolar type 2 cells (iAT2), the major cell type infected in the lung, and cardiomyocytes (iCM), consistent with cardiovascular consequences of COVID-19 disease. Robust activation of IFN or OAS-RNase L is not observed in these cell types, while PKR activation is evident in iAT2 and iCM. In SARS-CoV-2 infected Calu-3 and A549ACE2 lung-derived cell lines, IFN induction remains relatively weak; however activation of OAS-RNase L and PKR is observed. This is in contrast to MERS-CoV, which effectively inhibits IFN signaling as well as OAS-RNase L and PKR pathways, but similar to mutant MERS-CoV lacking innate immune antagonists. Remarkably, both OAS-RNase L and PKR are activated in MAVS knockout A549ACE2 cells, demonstrating that SARS-CoV-2 can induce these host antiviral pathways despite minimal IFN production. Moreover, increased replication and cytopathic effect in RNASEL knockout A549ACE2 cells implicates OAS-RNase L in restricting SARS-CoV-2. Finally, while SARS-CoV-2 fails to antagonize these host defense pathways, which contrasts with other coronaviruses, the IFN signaling response is generally weak. These host-virus interactions may contribute to the unique pathogenesis of SARS-CoV-2. SignificanceSARS-CoV-2 emergence in late 2019 led to the COVID-19 pandemic that has had devastating effects on human health and the economy. Early innate immune responses are essential for protection against virus invasion. While inadequate innate immune responses are associated with severe COVID-19 diseases, understanding of the interaction of SARS-CoV-2 with host antiviral pathways is minimal. We have characterized the innate immune response to SARS-CoV-2 infections in relevant respiratory tract derived cells and cardiomyocytes and found that SARS-CoV-2 activates two antiviral pathways, oligoadenylate synthetase-ribonuclease L (OAS-RNase L), and protein kinase R (PKR), while inducing minimal levels of interferon. This in contrast to MERS-CoV which inhibits all three pathways. Activation of these pathways may contribute to the distinctive pathogenesis of SARS-CoV-2.