Live-cell microscopy analysis of rotavirus-infection spread and its associated innate immune response in human intestinal epithelial cells

Intestinal epithelial cells (IECs) line the surface of the intestinal epithelium and act as a barrier against commensal microbiota. When enteric viruses infect IECs, they induce the production of two types of cytokines, type-I and type-III interferons (IFNs), which can set an antiviral state in the tissue. One of the main pathogens of the intestines is rotavirus, which was shown to elicit the upregulation of both types of IFNs in mice models and commercial cell lines. Nevertheless, the role that each type of IFN plays during rotavirus spread has not been thoroughly evaluated. Here, I generated a collection of fluorescent tools to evaluate rotavirus infection and spread in different contexts using live cell fluorescence microscopy. I could show that rotavirus efficiently blocks type-I IFN-mediated upregulation of interferon stimulated genes (ISGs) through its NSP1 protein, and only type-III IFNs can be upregulated. Moreover, even in the absence of NSP1, only type-III IFNs were able to rapidly establish an antiviral state in a large number of IECs and prevent the spread of rotavirus. On the contrary, type-I IFNs had a delayed antiviral effect, which allowed infection of IECs by newly produced viruses. Moreover, I could observe that rotavirus infection is strongly dependent on ADP-mediated calcium waves. Live microscopy experiments showed that second rounds of infection seem to take place in areas defined by calcium waves elicited by the first round of infection. Importantly, blocking of ADP signaling through the P2Y1 purinergic receptor prevented new infections from taking place, highlighting the importance of calcium waves during rotavirus infection. In conclusion, my results suggest that only type-III IFNs are able to control rotavirus infection and spread in IECs, and that type-I IFNs do not seem to play a role at least in the antiviral state of the intestinal epithelium. Furthermore, calcium waves generated by the first round of infection likely alter the integrity of IECs to allow viruses to more easily infect the culture. I propose that IECs can efficiently control the spread of enteric viruses due low amounts of type-III IFNs being needed to set an antiviral response in a notably high number of cells, and that rotavirus-induced calcium waves alters the polarized nature of IECs to facilitate infection

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

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

International audienceSARS-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

Abstract 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 primate-specific isoform dACE2 and not the SARS-CoV-2 receptor ACE2

https://kent-islandora.s3.us-east-2.amazonaws.com/node/10691/83863-thumbnail.jpgSevere 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. &nbsp; Preprint available here on biorxiv:&nbsp;https://doi.org/10.1101/2020.07.19.210955</p