A model for network-based identification and pharmacological targeting of aberrant, replication-permissive transcriptional programs induced by viral infection.

SARS-CoV-2 hijacks the host cell transcriptional machinery to induce a phenotypic state amenable to its replication. Here we show that analysis of Master Regulator proteins representing mechanistic determinants of the gene expression signature induced by SARS-CoV-2 in infected cells revealed coordinated inactivation of Master Regulators enriched in physical interactions with SARS-CoV-2 proteins, suggesting their mechanistic role in maintaining a host cell state refractory to virus replication. To test their functional relevance, we measured SARS-CoV-2 replication in epithelial cells treated with drugs predicted to activate the entire repertoire of repressed Master Regulators, based on their experimentally elucidated, context-specific mechanism of action. Overall, 15 of the 18 drugs predicted to be effective by this methodology induced significant reduction of SARS-CoV-2 replication, without affecting cell viability. This model for host-directed pharmacological therapy is fully generalizable and can be deployed to identify drugs targeting host cell-based Master Regulator signatures induced by virtually any pathogen.

Increased Sensitivity of SARS-CoV-2 to Type III Interferon in Human Intestinal Epithelial Cells.

The coronavirus SARS-CoV-2 caused the COVID-19 global pandemic leading to 5.3 million deaths worldwide as of December 2021. The human intestine was found to be a major viral target which could have a strong impact on virus spread and pathogenesis since it is one of the largest organs. While type I interferons (IFNs) are key cytokines acting against systemic virus spread, in the human intestine type III IFNs play a major role by restricting virus infection and dissemination without disturbing homeostasis. Recent studies showed that both type I and III IFNs can inhibit SARS-CoV-2 infection, but it is not clear whether one IFN controls SARS-CoV-2 infection of the human intestine better or with a faster kinetics. In this study, we could show that type I and III IFNs both possess antiviral activity against SARS-CoV-2 in human intestinal epithelial cells (hIECs); however, type III IFN is more potent. Shorter type III IFN pretreatment times and lower concentrations were required to efficiently reduce virus load compared to type I IFNs. Moreover, type III IFNs significantly inhibited SARS-CoV-2 even 4 h postinfection and induced a long-lasting antiviral effect in hIECs. Importantly, the sensitivity of SARS-CoV-2 to type III IFNs was virus specific since type III IFN did not control VSV infection as efficiently. Together, these results suggest that type III IFNs have a higher potential for IFN-based treatment of SARS-CoV-2 intestinal infection compared to type I IFNs. IMPORTANCE SARS-CoV-2 infection is not restricted to the respiratory tract and a large number of COVID-19 patients experience gastrointestinal distress. Interferons are key molecules produced by the cell to combat virus infection. Here, we evaluated how two types of interferons (type I and III) can combat SARS-CoV-2 infection of human gut cells. We found that type III interferons were crucial to control SARS-CoV-2 infection when added both before and after infection. Importantly, type III interferons were also able to produce a long-lasting effect, as cells were protected from SARS-CoV-2 infection up to 72 h posttreatment. This study suggested an alternative treatment possibility for SARS-CoV-2 infection.

TMPRSS2 expression dictates the entry route used by SARS-CoV-2 to infect host cells.

SARS-CoV-2 is a newly emerged coronavirus that caused the global COVID-19 outbreak in early 2020. COVID-19 is primarily associated with lung injury, but many other clinical symptoms such as loss of smell and taste demonstrated broad tissue tropism of the virus. Early SARS-CoV-2-host cell interactions and entry mechanisms remain poorly understood. Investigating SARS-CoV-2 infection in tissue culture, we found that the protease TMPRSS2 determines the entry pathway used by the virus. In the presence of TMPRSS2, the proteolytic process of SARS-CoV-2 was completed at the plasma membrane, and the virus rapidly entered the cells within 10 min in a pH-independent manner. When target cells lacked TMPRSS2 expression, the virus was endocytosed and sorted into endolysosomes, from which SARS-CoV-2 entered the cytosol via acid-activated cathepsin L protease 40-60 min post-infection. Overexpression of TMPRSS2 in non-TMPRSS2 expressing cells abolished the dependence of infection on the cathepsin L pathway and restored sensitivity to the TMPRSS2 inhibitors. Together, our results indicate that SARS-CoV-2 infects cells through distinct, mutually exclusive entry routes and highlight the importance of TMPRSS2 for SARS-CoV-2 sorting into either pathway.

Single-cell analyses reveal SARS-CoV-2 interference with intrinsic immune response in the human gut.

Exacerbated pro-inflammatory immune response contributes to COVID-19 pathology. However, despite the mounting evidence about SARS-CoV-2 infecting the human gut, little is known about the antiviral programs triggered in this organ. To address this gap, we performed single-cell transcriptomics of SARS-CoV-2-infected intestinal organoids. We identified a subpopulation of enterocytes as the prime target of SARS-CoV-2 and, interestingly, found the lack of positive correlation between susceptibility to infection and the expression of ACE2. Infected cells activated strong pro-inflammatory programs and produced interferon, while expression of interferon-stimulated genes was limited to bystander cells due to SARS-CoV-2 suppressing the autocrine action of interferon. These findings reveal that SARS-CoV-2 curtails the immune response and highlights the gut as a pro-inflammatory reservoir that should be considered to fully understand SARS-CoV-2 pathogenesis.

Interferons and viruses induce a novel truncated ACE2 isoform and not the full-length SARS-CoV-2 receptor.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes COVID-19, utilizes angiotensin-converting enzyme 2 (ACE2) for entry into target cells. ACE2 has been proposed as an interferon-stimulated gene (ISG). Thus, interferon-induced variability in ACE2 expression levels could be important for susceptibility to COVID-19 or its outcomes. Here, we report the discovery of a novel, transcriptionally independent truncated isoform of ACE2, which we designate as deltaACE2 (dACE2). We demonstrate that dACE2, but not ACE2, is an ISG. In The Cancer Genome Atlas, the expression of dACE2 was enriched in squamous tumors of the respiratory, gastrointestinal and urogenital tracts. In vitro, dACE2, which lacks 356 amino-terminal amino acids, was non-functional in binding the SARS-CoV-2 spike protein and as a carboxypeptidase. Our results suggest that the ISG-type induction of dACE2 in IFN-high conditions created by treatments, an inflammatory tumor microenvironment or viral co-infections is unlikely to increase the cellular entry of SARS-CoV-2 and promote infection.