SARS-CoV-2 takes the bait: Exosomes as endogenous decoys

Writing in PLOS Biology, Ching and colleagues show that ACE2-decorated exosomes are deployed as natural inhibitory decoys against SARS-CoV-2. High decoy levels correlate with improved patient outcomes, suggesting they directly help COVID-19 recovery and supporting the concept of successful future decoy-based therapies

Interferon-Mediated Regulation of RNA Virus Infection

Influenza A viruses (IAVs) and coronaviruses (CoVs) are respiratory pathogens that cause seasonal infections in humans and occasional pandemics, as last seen by the 2009 H1N1 influenza virus (Swine flu) pandemic and the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Both viruses heavily rely on the host cell machinery for replication, highlighting the tight interplay between host and pathogens. While viruses exploit host functions for their own benefit, the host cell in turn triggers effective antiviral signaling pathways, such as the interferon (IFN) response. The IFN pathway is an important barrier to infections and part of the innate immune defense system. The immediate activation upon infection triggers a signaling cascade that leads to the induction of hundreds of IFN-stimulated genes (ISGs), some of which possess profound antiviral activities. Understanding the mechanisms and pathways involved in the interplay between virus and host is a prerequisite in the development of antiviral therapies and vaccines. Previous studies reported that the expression of the cellular receptor of SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2) is elevated by the action of IFN, implicating a possible increased susceptibility for infections either as the body triggers IFN to defend itself or in the context of using IFNs as antiviral therapies, which has recently been started in clinical trials. Thus, elucidation of the IFN-inducibility of ACE2 was of great importance. Using a cell line model, it was observed that treatment with IFNβ increased ACE2 mRNA and surface protein expression, but to a much lesser extent than was found for canonical ISGs. Furthermore, IFNλ1 induced antiviral ISGs, but had limited impact on ACE2 mRNA levels and showed no effect on ACE2 cell surface levels. IFNγ marginally increased canonical ISGs, but clearly induced ACE2 surface expression, similar to IFNβ. Strikingly, all types of IFN limited SARS-CoV-2 infection in a dose-dependent manner, highlighting that the antiviral function of IFNs outweighs any potential promotion of SARS-CoV-2 replication by increased ACE2 expression. The data from this first part of the thesis underscore the potential of IFN, specifically IFNλ1, as possible candidates for antiviral therapies against SARS-CoV-2 without increasing susceptibility to infection. While many proteins have been classified as ISGs over the years, most of them remain uncharacterized. As part of an RNA interference (RNAi) screen targeting 100 putative ISGs, several potential ISGs with previously undescribed contributions to IFN-mediated restriction of IAVs were identified. In the second part of this thesis, I focused on studying the Tre2/Bub2/Cdc16 (TBC) domain-containing protein RAB GTPase-activating protein 1 like (RABGAP1L) as a potent host factor restricting IAV. Surprisingly, its expression itself was not detectably induced by IFN, indicating that RABGAP1L is not a classical highly inducible ISG, while its antiviral activity was greatly potentiated by the action of IFN. RABGAP1L further restricted several RNA viruses known to enter host cells via endocytosis. Truncation mapping and site-directed mutagenesis revealed that a catalytically active TBC domain is required for the antiviral activity. Using proximity labeling proteomics to study the RABGAP1L interactome, we discovered its association with proteins involved in endosomal sorting, maturation and trafficking. In line with this, immunofluorescence-based assays uncovered that RABGAP1L expression disrupts endosomal functions and restricts early IAV entry. The work described in this second part of the thesis reports the discovery of a previously undescribed IAV restriction factor that limits early infection by disruption of the endosomal pathway

Combined computational and cellular screening identifies synergistic inhibition of SARS-CoV-2 by lenvatinib and remdesivir

Rapid repurposing of existing drugs as new therapeutics for COVID-19 has been an important strategy in the management of disease severity during the ongoing SARS-CoV-2 pandemic. Here, we used high-throughput docking to screen 6000 compounds within the DrugBank library for their potential to bind and inhibit the SARS-CoV-2 3 CL main protease, a chymotrypsin-like enzyme that is essential for viral replication. For 19 candidate hits, parallel in vitro fluorescence-based protease-inhibition assays and Vero-CCL81 cell-based SARS-CoV-2 replication-inhibition assays were performed. One hit, diclazuril (an investigational anti-protozoal compound), was validated as a SARS-CoV-2 3 CL main protease inhibitor in vitro (IC$_{50}$ value of 29 µM) and modestly inhibited SARS-CoV-2 replication in Vero-CCL81 cells. Another hit, lenvatinib (approved for use in humans as an anti-cancer treatment), could not be validated as a SARS-CoV-2 3 CL main protease inhibitor in vitro, but serendipitously exhibited a striking functional synergy with the approved nucleoside analogue remdesivir to inhibit SARS-CoV-2 replication, albeit this was specific to Vero-CCL81 cells. Lenvatinib is a broadly-acting host receptor tyrosine kinase (RTK) inhibitor, but the synergistic effect with remdesivir was not observed with other approved RTK inhibitors (such as pazopanib or sunitinib), suggesting that the mechanism-of-action is independent of host RTKs. Furthermore, time-of-addition studies revealed that lenvatinib/remdesivir synergy probably targets SARS-CoV-2 replication subsequent to host-cell entry. Our work shows that combining computational and cellular screening is a means to identify existing drugs with repurposing potential as antiviral compounds. Future studies could be aimed at understanding and optimizing the lenvatinib/remdesivir synergistic mechanism as a therapeutic option

Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

Host interferons (IFNs) powerfully restrict viruses through the action of several hundred IFN-stimulated gene (ISG) products, many of which remain uncharacterized. Here, using RNAi screening, we identify several ISG restriction factors with previously undescribed contributions to IFN-mediated defense. Notably, RABGAP1L, a Tre2/Bub2/Cdc16 (TBC)-domain-containing protein involved in regulation of small membrane-bound GTPases, robustly potentiates IFN action against influenza A viruses (IAVs). Functional studies reveal that the catalytically active TBC domain of RABGAP1L promotes antiviral activity, and the RABGAP1L proximal interactome uncovered its association with proteins involved in endosomal sorting, maturation, and trafficking. In this regard, RABGAP1L overexpression is sufficient to disrupt endosomal function during IAV infection and restricts an early post-attachment, but pre-fusion, stage of IAV cell entry. Other RNA viruses that enter cells primarily via endocytosis are also impaired by RABGAP1L, while entry promiscuous SARS-CoV-2 is resistant. Our data highlight virus endocytosis as a key target for host defenses.ISSN:2666-3864ISSN:2211-124

Antiviral Activity of Type I, II, and III Interferons Counterbalances ACE2 Inducibility and Restricts SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), is a recently emerged respiratory coronavirus that has infected >23 million people worldwide with >800,000 deaths. Few COVID-19 therapeutics are available, and the basis for severe infections is poorly understood. Here, we investigated properties of type I (β), II (γ), and III (λ1) interferons (IFNs), potent immune cytokines that are normally produced during infection and that upregulate IFN-stimulated gene (ISG) effectors to limit virus replication. IFNs are already in clinical trials to treat COVID-19. However, recent studies highlight the potential for IFNs to enhance expression of host angiotensin-converting enzyme 2 (ACE2), suggesting that IFN therapy or natural coinfections could exacerbate COVID-19 by upregulating this critical virus entry receptor. Using a cell line model, we found that beta interferon (IFN-β) strongly upregulated expression of canonical antiviral ISGs, as well as ACE2 at the mRNA and cell surface protein levels. Strikingly, IFN-λ1 upregulated antiviral ISGs, but ACE2 mRNA was only marginally elevated and did not lead to detectably increased ACE2 protein at the cell surface. IFN-γ induced the weakest ISG response but clearly enhanced surface expression of ACE2. Importantly, all IFN types inhibited SARS-CoV-2 replication in a dose-dependent manner, and IFN-β and IFN-λ1 exhibited potent antiviral activity in primary human bronchial epithelial cells. Our data imply that type-specific mechanisms or kinetics shape IFN-enhanced ACE2 transcript and cell surface levels but that the antiviral action of IFNs against SARS-CoV-2 counterbalances any proviral effects of ACE2 induction. These insights should aid in evaluating the benefits of specific IFNs, particularly IFN-λ, as repurposed therapeutics. IMPORTANCE Repurposing existing, clinically approved, antiviral drugs as COVID-19 therapeutics is a rapid way to help combat the SARS-CoV-2 pandemic. Interferons (IFNs) usually form part of the body’s natural innate immune defenses against viruses, and they have been used with partial success to treat previous new viral threats, such as HIV, hepatitis C virus, and Ebola virus. Nevertheless, IFNs can have undesirable side effects, and recent reports indicate that IFNs upregulate the expression of host ACE2 (a critical entry receptor for SARS-CoV-2), raising the possibility that IFN treatments could exacerbate COVID-19. Here, we studied the antiviral- and ACE2-inducing properties of different IFN types in both a human lung cell line model and primary human bronchial epithelial cells. We observed differences between IFNs with respect to their induction of antiviral genes and abilities to enhance the cell surface expression of ACE2. Nevertheless, all the IFNs limited SARS-CoV-2 replication, suggesting that their antiviral actions can counterbalance increased ACE2.ISSN:2150-7511ISSN:2161-212