Defining Mechanisms of SARS-CoV-2 Entry Inhibitors in the Respiratory Epithelium

Project Summary/Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic and severely impacted public health. SARS-CoV-2 primarily infects respiratory epithelium cells expressing host factors required for viral entry. Infection is initiated when the SARS-CoV-2 Spike glycoprotein binds to the host cell receptor angiotensin converting enzyme 2 (ACE2). The viral glycoprotein must be processed by cellular proteases to enable fusion and two distinct proteases have been shown to process Spike in different contexts. In cells that do not express the plasma-membrane associated serine protease 2 (TMPRSS2), the virus is endocytosed and undergoes membrane fusion in acidified compartments by cathepsin proteases. In contrast, in respiratory cells, TMPRSS2 is a plasma-membrane associated protease thought to process the glycoprotein at the plasma membrane for fusion at the surface. Although viral entry is a critical step of infection and can be targeted by therapeutics, the full spectrum of proteins involved and how they are regulated is incompletely understood. Our lab utilizes the Calu-3 cell line which resembles primary cells in morphology, signaling pathways, and expression of both ACE2 and TMPRSS2. We previously identified ~130 drugs with antiviral activity against SARS-CoV-2 including the canonical TMPRSS2 inhibitor Camostat. Thus, we postulated that additional drugs in this set may block TMPRSS2-dependent entry. To identify drugs that block entry we took advantage of recombinant vesicular stomatitis virus (VSV) expressing endogenous glycoprotein (VSV-G), or the SARS-CoV- 2 glycoprotein Spike (VSV-S). I found that two entry inhibitors, Retro2.1 and Staurosporine, block SARS-CoV-2 infection in diverse cell types utilizing TMPRSS2-dependent and cathepsin-dependent entry, suggesting that they impact ACE2, or another common step in the entry pathway. Retro2.1 is known to impact host protein trafficking through inhibition of the ER exit site protein SEC16A, block entry of several viruses, and block uptake of bacterial toxins. Staurosporine is a broad protein kinase c (PKC) inhibitor, and my preliminary data suggests it blocks viral entry. Given that PKCs are known to impact receptor expression and have been implicated during entry of several viruses including SARS-CoV-2, I tested multiple PKC isozymes and implicated a role for PKCη in viral entry. I hypothesize that Retro2.1 and Staurosporine block ACE2 surface expression through inhibition of SEC16A-dependent trafficking and PKCη-regulated recycling. In Aim 1, I will determine the functional impact of Retro2.1 and SEC16A on SARS-CoV-2 binding and infection as well as the surface expression of ACE2. In Aim 2, I will test the role of PKCη in entry and determine the impact of Staurosporine and PKCη depletion on ACE2 surface expression and recycling. The proposed experiments will provide insight into the molecular mechanisms of ACE2 regulation and SARS-CoV-2 entry and may inform the development of therapeutics against emerging variants and zoonotic coronaviruses.