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Principal Investigator: Sanghyun Lee
Organization: BROWN UNIVERSITY
Fiscal Year: 2023
Award: $168,651
Funding agency: National Institute of General Medical Sciences
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus
disease 19 (COVID-19). Spike protein is the primary antigenic target for COVID vaccines and interfering
with the interface between RBD (Receptor Binding Domain) of spike and ACE2 is the mechanism of
action for the majority of existing therapeutic antibodies, indicating the importance of RBD and its binding
to the cellular receptor for controlling SARS-CoV-2. It is unclear whether there are any intrinsic cellular
proteins that inhibit viral entry of SARS-CoV-2.
Our new preliminary data reveal that cellular entry of SARS-CoV-2 is inhibited by a novel inhibitory cellular
protein, Leucin-rich repeat containing 15 (LRRC15). We generated a focused CRISPR activation
(CRISPRa) library, named surfaceome, that covers all ~6000 known/predicted surface proteins on the
cellular plasma membrane. The surfaceome CRISPRa screening was performed by staining cells with a
recombinant spike protein to identify SARS-CoV-2 binding factors. Strikingly, LRRC15 inhibits
spike-mediated viral entry not only in the same cells, but also in neighboring cells in trans. Expression of
LRRC15 in ACE2+ cells blocked spike-mediated viral entry in ACE2+LRRC15- cells, providing a unique
concept of viral entry inhibition by an inhibitory factor. This result suggests a protective role of LRRC15 in
a physiological context. Our central hypothesis is that human LRRC15 acts as an inhibitory entry factor for
SARS-CoV-2, acting in trans as a decoy receptor expressed in non-susceptible pathological fibroblasts in
the lung. This proposal will explore the mechanism by which LRRC15 inhibits entry of SARS-CoV-2 in
trans through three subaims of Aim 1.
We will leverage the screening platform to identify cellular receptors for secreted virulence proteins of
SARS-CoV-2, Orf3a, Orf7a, and Orf8. Importantly, the same screening platform is validated to be a
efficient platform for secreted virulent proteins. In a separate screening for norovirus secreted virulence
protein (NS1), the surfaceome screening successfully identified Syndecan-4 as a putative cellular receptor
for norovirus NS1 (Li et al., In preparation). In the Aim 2, we will perform a surfaceome screening for
Orf3a, Orf7a, and Orf8 and will discover putative cellular receptors for the secreted virulence proteins.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><ACE2><Binding><COBRE><COVID><COVID-19><COVID-19 screening><COVID-19 virus><COVID19><COVID19 screening><COVID19 virus><CRISPR activation><CRISPR activator><CRISPR based activation><CRISPR editing screen><CRISPR gene activation><CRISPR screen><CRISPR transcription activation><CRISPR transcriptional activation><CRISPR-Cas-9-mediated gene activation><CRISPR-based gene activation><CRISPR-based screen><CRISPR-dCAS9 Activator><CRISPR-mediated transcriptional activation><CRISPR/CAS9 activation><CRISPR/CAS9 gene activation><CRISPR/Cas9 screen><CRISPR/dCas9 activation><CRISPR/dCas9-based transcriptional activation><CRISPRa><CV-19><CV19><Cell Body><Cell membrane><Cells><Center of Biomedical Research Excellence><Centers of Research Excellence><CoV disease><CoV-2><CoV2><Computational Biology><Cytoplasmic Membrane><Data><Fibroblasts><Human><Libraries><Lung><Lung Respiratory System><Mediating><Membrane Protein Gene><Membrane Proteins><Membrane-Associated Proteins><Modern Man><Molecular Interaction><Names><Norovirus><Norwalk-like Viruses><Pathologic><Physiologic><Physiological><Plasma Membrane><Preparation><Proteins><Receptor Protein><Recombinants><Role><SARS corona virus 2><SARS-CO-V2><SARS-COVID-2><SARS-CoV-2><SARS-CoV-2 entry inhibitor><SARS-CoV-2 screening><SARS-CoV2><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><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 entry inhibitor><Severe acute respiratory syndrome related corona virus 2><Staining method><Stains><Surface Proteins><Therapeutic antibodies><Vaccines><Viral><Virulence><Virulent><Wuhan coronavirus><activating CRISPR technology><angiotensin converting enzyme 2><angiotensin converting enzyme II><block SARS-CoV-2 entry><block severe acute respiratory syndrome coronavirus 2 entry><block viral entry><clustered regularly interspaced short palindromic repeats screen><computer biology><corona virus disease><corona virus disease 2019><coronavirus disease><coronavirus disease 2019><coronavirus disease 2019 screening><coronavirus disease 2019 virus><coronavirus disease-19><coronavirus disease-19 virus><coronavirus infectious disease-19><hCoV19><human disease><inhibit SARS-CoV-2 entry><inhibit severe acute respiratory syndrome coronavirus 2 entry><inhibit viral entry><nCoV2><name><named><naming><novel><plasmalemma><preparations><pulmonary><receptor><receptor binding><receptor bound><screening><screenings><severe acute respiratory syndrome coronavirus 2 screening><social role><syndecan-4><syndecan4><viral entry blocker><viral entry inhibitor>