Type I interferon signaling induces a delayed antiproliferative response in Calu-3 cells during SARS-CoV-2 infection (preprint)

Disease progression during SARS-CoV-2 infection is tightly linked to the fate of lung epithelial cells, with severe cases of COVID-19 characterized by direct injury of the alveolar epithelium and an impairment in its regeneration from progenitor cells. The molecular pathways that govern respiratory epithelial cell death and proliferation during SARS-CoV-2 infection, however, remain poorly understood. We now report a high-throughput CRISPR screen for host genetic modifiers of the fitness of SARS-CoV-2-infected Calu-3 respiratory epithelial cells. The top 4 genes identified in our screen encode components of the same type I interferon signaling complex - IFNAR1, IFNAR2, JAK1, and TYK2. The 5th gene, ACE2, was an expected control encoding the SARS-CoV-2 viral receptor. Surprisingly, despite the antiviral properties of IFN-I signaling, its disruption in our screen was associated with an increase in Calu-3 cell fitness. We validated this effect and found that IFN-I signaling did not sensitize SARS-CoV-2-infected cultures to cell death but rather inhibited the proliferation of surviving cells after the early peak of viral replication and cytopathic effect. We also found that IFN-I signaling alone, in the absence of viral infection, was sufficient to induce this delayed antiproliferative response. Together, these findings highlight a cell autonomous antiproliferative response by respiratory epithelial cells to persistent IFN-I signaling during SARS-CoV-2 infection. This response may contribute to the deficient alveolar regeneration that has been associated with COVID-19 lung injury and represents a promising area for host-targeted therapeutic development.

Identification of ACE2 modifiers by CRISPR screening (preprint)

SARS-CoV-2 infection is initiated by binding of the viral spike protein to its receptor, ACE2, on the surface of host cells. ACE2 expression is heterogeneous both in vivo and in immortalized cell lines, but the molecular pathways that govern ACE2 expression remain unclear. We now report high-throughput CRISPR screens for functional modifiers of ACE2 surface abundance. We identified 35 genes whose disruption was associated with a change in the surface abundance of ACE2 in HuH7 cells. Enriched among these ACE2 regulators were established transcription factors, epigenetic regulators, and functional networks. We further characterized individual cell lines with disruption of SMAD4, EP300, PIAS1, or BAMBI and found these genes to regulate ACE2 at the mRNA level and to influence cellular susceptibility to SARS-CoV-2 infection. Collectively, our findings clarify the host factors involved in SARS-CoV-2 entry and suggest potential targets for therapeutic development.

A Combination Adjuvant for the Induction of Potent Antiviral Immune Responses for a Recombinant SARS-CoV-2 Protein Vaccine (preprint)

Several promising vaccines for SARS-CoV-2 have received emergency use authorization in various countries and are being administered to the general population. However, many issues associated with the vaccines and the protection they provide remain unresolved, including the duration of conferred immunity, whether or not sterilizing immunity is imparted, and the degree of cross-variant protection that is achieved with these vaccines. Early evidence has suggested potentially reduced vaccine efficacy towards certain viral variants in circulation. Development of adjuvants compatible with these vaccine platforms that enhance the immune response and guide the adaptive and cellular immune responses towards the types of responses most effective for broad protection against SARS-CoV-2 will likely be pivotal for complete protection. Natural viral infection stimulates strong immune responses through the activation of three main pathways involving Toll-, RIG-I-, and NOD-like receptors (TLRs, RLRs, NLRs). As induction of appropriate innate responses is crucial for long-lasting adaptive immunity and for shaping the correct types of immune responses, we developed a combination, intranasal, adjuvant integrating a nanoemulsion-based adjuvant (NE) that activates TLRs and NLRP3 with an RNA agonist of RIG-I (IVT DI). This rationally designed combination adjuvant yielded a synergistic immune response with highly robust humoral and cellular responses towards SARS-CoV-2 using a recombinant spike protein S1 subunit antigen. Significantly enhanced virus neutralizing antibody titers were achieved towards both a homologous SARS-CoV-2 virus (IC50 titers of 1:104) and a mouse-adapted variant containing the N501Y mutation present in the B1.1.7 UK and B.1.351 South Africa variants. Importantly, NE/IVT DI dramatically enhanced the TH1-biased cellular response, which is expected to provide more durable and tailored cellular immunity while avoiding potential vaccine enhanced pathology previously associated with TH2-biased responses in some SARS-CoV and MERS-CoV vaccines. Our previous work with the NE/IVT DI adjuvant has demonstrated its compatibility with a broad range of antigen types. Thus, this combined adjuvant approach has strong potential for improving the induced immune profile for a variety of SARS-CoV-2 vaccine candidates such that better protection against future drift variants and prevention of transmission can be achieved.

Amilorides inhibit SARS-CoV-2 replication in vitro by targeting RNA structures (preprint)

The SARS-CoV-2 pandemic, and the likelihood of future coronavirus pandemics, has rendered our understanding of coronavirus biology more essential than ever. Small molecule chemical probes offer to both reveal novel aspects of virus replication and to serve as leads for antiviral therapeutic development. The RNA-biased amiloride scaffold was recently tuned to target a viral RNA structure critical for translation in enterovirus 71, ultimately uncovering a novel mechanism to modulate positive-sense RNA viral translation and replication. Analysis of CoV RNA genomes reveal many conserved RNA structures in the 5-UTR and proximal region critical for viral translation and replication, including several containing bulge-like secondary structures suitable for small molecule targeting. Following phylogenetic conservation analysis of this region, we screened an amiloride-based small molecule library against a less virulent human coronavirus, OC43, to identify lead ligands. Amilorides inhibited OC43 replication as seen in viral plaque assays. Select amilorides also potently inhibited replication competent SARS-CoV-2 as evident in the decreased levels of cell free virions in cell culture supernatants of treated cells. Reporter screens confirmed the importance of RNA structures in the 5-end of the viral genome for small molecule activity. Finally, NMR chemical shift perturbation studies of the first six stem loops of the 5-end revealed specific amiloride interactions with stem loops 4, 5a, and 6, all of which contain bulge like structures and were predicted to be strongly bound by the lead amilorides in retrospective docking studies. Taken together, the use of multiple orthogonal approaches allowed us to identify the first small molecules aimed at targeting RNA structures within the 5-UTR and proximal region of the CoV genome. These molecules will serve as chemical probes to further understand CoV RNA biology and can pave the way for the development of specific CoV RNA-targeted antivirals.