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Principal Investigator: Jorge Alberto Acuna
Organization: UNIVERSITY OF PENNSYLVANIA
Fiscal Year: 2024
Award: $48,974
Funding agency: National Institute of Allergy and Infectious Diseases
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.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><2019-nCoV S protein><2019-nCoV spike glycoprotein><2019-nCoV spike protein><ACE2><Airway infections><Assay><Bacterial Toxins><Binding><Bioassay><Biological Assay><COVID-19 S protein><COVID-19 infection><COVID-19 spike><COVID-19 spike glycoprotein><COVID-19 spike protein><COVID-19 virus><COVID-19 virus infection><COVID19 infection><COVID19 virus><Calcium Phospholipid-Dependent Protein Kinase><Calcium-Activated Phospholipid-Dependent Kinase><Cathepsins><Cell Body><Cell Function><Cell Line><Cell Physiology><Cell Process><Cell membrane><CellLine><Cells><Cellular Function><Cellular Physiology><Cellular Process><CoV-2><CoV2><Confocal Microscopy><Coronaviridae><Coronaviridae Infections><Coronavirus><Coronavirus Infections><Cytoplasmic Membrane><Data><Disease Outbreaks><Drugs><Epithelial Cells><Epitheliasin Gene><Esteroproteases><Flow Cytofluorometries><Flow Cytofluorometry><Flow Cytometry><Flow Microfluorimetry><Flow Microfluorometry><Glycoproteins><Host Factor><Host Factor Protein><Immunoblotting><Infection><Integration Host Factors><Intervention><Intervention Strategies><Isoenzymes><Isoforms><Isozymes><Kinases><Measures><Medication><Membrane><Membrane Fusion><Molecular><Molecular Interaction><Morphology><Outbreaks><PRSS10><Pathway interactions><Peptidases><Peptide Hydrolases><Pharmaceutical Preparations><Phospholipid-Sensitive Calcium-Dependent Protein Kinase><Phosphorylation><Phosphotransferase Gene><Phosphotransferases><Plasma Membrane><Post-Transcriptional Gene Silencing><Posttranscriptional Gene Silencing><Process><Production><Protease Gene><Proteases><Protein Isoforms><Protein Kinase C><Protein Kinase C Inhibitor><Protein Phosphorylation><Protein Trafficking><Proteinases><Proteins><Proteolytic Enzymes><Public Health><RNA Interference><RNA Silencing><RNAi><Receptor Cell><Receptor Protein><Recombinants><Recycling><Regulation><Respiratory Epithelium><Respiratory Infections><Respiratory Tract Infections><Role><SARS corona virus 2><SARS-CO-V2><SARS-COVID-2><SARS-CoV-2><SARS-CoV-2 S><SARS-CoV-2 S protein><SARS-CoV-2 entry inhibitor><SARS-CoV-2 infection><SARS-CoV-2 inhibitor><SARS-CoV-2 spike><SARS-CoV-2 spike glycoprotein><SARS-CoV-2 spike protein><SARS-CoV2><SARS-CoV2 infection><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Sequence-Specific Posttranscriptional Gene Silencing><Serine Endopeptidases><Serine Protease><Serine Protein Hydrolases><Serine Proteinases><Severe Acute Respiratory Coronavirus 2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome coronavirus 2 S protein><Severe acute respiratory syndrome coronavirus 2 entry inhibitor><Severe acute respiratory syndrome coronavirus 2 infection><Severe acute respiratory syndrome coronavirus 2 inhibitor><Severe acute respiratory syndrome coronavirus 2 spike glycoprotein><Severe acute respiratory syndrome coronavirus 2 spike protein><Severe acute respiratory syndrome related corona virus 2><Short interfering RNA><Signal Pathway><Site><Small Interfering RNA><Staurosporine><Strains Cell Lines><Structure of respiratory epithelium><Subcellular Process><Surface><TMPRSS2><TMPRSS2 gene><Testing><Time><Transmission><Transphosphorylases><VSV><Variant><Variation><Vesicular Stomatitis Virus><Vesicular stomatitis Indiana virus><Viral><Viral Activity><Viral Diseases><Viral Function><Viral Physiology><Virion><Virus><Virus Diseases><Virus Particle><Western Blotting><Western Immunoblotting><Work><Wuhan coronavirus><Zoonoses><Zoonotic><Zoonotic Infection><airway epithelium><angiotensin converting enzyme 2><angiotensin converting enzyme II><block SARS-CoV-2><block SARS-CoV-2 entry><block severe acute respiratory syndrome coronavirus 2><block severe acute respiratory syndrome coronavirus 2 entry><block viral entry><cell type><corona virus><coronavirus disease 2019 S protein><coronavirus disease 2019 infection><coronavirus disease 2019 spike glycoprotein><coronavirus disease 2019 spike protein><coronavirus disease 2019 virus><coronavirus disease-19 virus><cultured cell line><desensitization><drug sensitivity><drug/agent><experiment><experimental research><experimental study><experiments><flow cytophotometry><hCoV19><human disease><infected with COVID-19><infected with COVID19><infected with SARS-CoV-2><infected with SARS-CoV2><infected with coronavirus disease 2019><infected with severe acute respiratory syndrome coronavirus 2><inhibit SARS-CoV-2><inhibit SARS-CoV-2 entry><inhibit severe acute respiratory syndrome coronavirus 2><inhibit severe acute respiratory syndrome coronavirus 2 entry><inhibit viral entry><inhibitor><insight><interventional strategy><knock-down><knockdown><membrane structure><nCoV2><novel><pandemic><pandemic disease><pathway><plasmalemma><protein blotting><protein expression><protein transport><receptor><receptor expression><respiratory><respiratory tract epithelium><siRNA><social role><spike proteins on SARS-CoV-2><targeted drug therapy><targeted drug treatments><targeted therapeutic><targeted therapeutic agents><targeted therapy><targeted treatment><therapeutic agent development><therapeutic development><trafficking><transmission process><uptake><viral RNA><viral entry blocker><viral entry inhibitor><viral infection><virus RNA><virus infection><virus-induced disease><zoonotic CoV><zoonotic coronavirus>