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2.
Cell Res ; 32(1): 24-37, 2022 01.
Article in English | MEDLINE | ID: covidwho-1537308

ABSTRACT

Host cellular receptors play key roles in the determination of virus tropism and pathogenesis. However, little is known about SARS-CoV-2 host receptors with the exception of ACE2. Furthermore, ACE2 alone cannot explain the multi-organ tropism of SARS-CoV-2 nor the clinical differences between SARS-CoV-2 and SARS-CoV, suggesting the involvement of other receptor(s). Here, we performed genomic receptor profiling to screen 5054 human membrane proteins individually for interaction with the SARS-CoV-2 capsid spike (S) protein. Twelve proteins, including ACE2, ASGR1, and KREMEN1, were identified with diverse S-binding affinities and patterns. ASGR1 or KREMEN1 is sufficient for the entry of SARS-CoV-2 but not SARS-CoV in vitro and in vivo. SARS-CoV-2 utilizes distinct ACE2/ASGR1/KREMEN1 (ASK) receptor combinations to enter different cell types, and the expression of ASK together displays a markedly stronger correlation with virus susceptibility than that of any individual receptor at both the cell and tissue levels. The cocktail of ASK-related neutralizing antibodies provides the most substantial blockage of SARS-CoV-2 infection in human lung organoids when compared to individual antibodies. Our study revealed an interacting host receptome of SARS-CoV-2, and identified ASGR1 and KREMEN1 as alternative functional receptors that play essential roles in ACE2-independent virus entry, providing insight into SARS-CoV-2 tropism and pathogenesis, as well as a community resource and potential therapeutic strategies for further COVID-19 investigations.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Asialoglycoprotein Receptor , Humans , Membrane Proteins , Protein Binding , Receptors, Virus/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
3.
J Virol ; 96(1): e0149221, 2022 01 12.
Article in English | MEDLINE | ID: covidwho-1476391

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in more than 235 million cases worldwide and 4.8 million deaths (October 2021), with various incidences and mortalities among regions/ethnicities. The coronaviruses SARS-CoV, SARS-CoV-2, and HCoV-NL63 utilize the angiotensin-converting enzyme 2 (ACE2) as the receptor to enter cells. We hypothesized that the genetic variability in ACE2 may contribute to the variable clinical outcomes of COVID-19. To test this hypothesis, we first conducted an in silico investigation of single-nucleotide polymorphisms (SNPs) in the coding region of ACE2. We then applied an integrated approach of genetics, biochemistry, and virology to explore the capacity of select ACE2 variants to bind coronavirus spike proteins and mediate viral entry. We identified the ACE2 D355N variant that restricts the spike protein-ACE2 interaction and consequently limits infection both in vitro and in vivo. In conclusion, ACE2 polymorphisms could modulate susceptibility to SARS-CoV-2, which may lead to variable disease severity. IMPORTANCE There is considerable variation in disease severity among patients infected with SARS-CoV-2, the virus that causes COVID-19. Human genetic variation can affect disease outcome, and the coronaviruses SARS-CoV, SARS-CoV-2, and HCoV-NL63 utilize human ACE2 as the receptor to enter cells. We found that several missense ACE2 single-nucleotide variants (SNVs) that showed significantly altered binding with the spike proteins of SARS-CoV, SARS-CoV-2, and NL63-HCoV. We identified an ACE2 SNP, D355N, that restricts the spike protein-ACE2 interaction and consequently has the potential to protect individuals against SARS-CoV-2 infection. Our study highlights that ACE2 polymorphisms could impact human susceptibility to SARS-CoV-2, which may contribute to ethnic and geographical differences in SARS-CoV-2 spread and pathogenicity.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/genetics , Genetic Predisposition to Disease/genetics , Angiotensin-Converting Enzyme 2/metabolism , Genetic Variation , Humans , Polymorphism, Single Nucleotide , Protein Binding , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
4.
Front Cell Dev Biol ; 9: 716208, 2021.
Article in English | MEDLINE | ID: covidwho-1354835

ABSTRACT

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the causative agent for the coronavirus disease 2019 (COVID-19) pandemic and there is an urgent need to understand the cellular response to SARS-CoV-2 infection. Beclin 1 is an essential scaffold autophagy protein that forms two distinct subcomplexes with modulators Atg14 and UVRAG, responsible for autophagosome formation and maturation, respectively. In the present study, we found that SARS-CoV-2 infection triggers an incomplete autophagy response, elevated autophagosome formation but impaired autophagosome maturation, and declined autophagy by genetic knockout of essential autophagic genes reduces SARS-CoV-2 replication efficiency. By screening 26 viral proteins of SARS-CoV-2, we demonstrated that expression of ORF3a alone is sufficient to induce incomplete autophagy. Mechanistically, SARS-CoV-2 ORF3a interacts with autophagy regulator UVRAG to facilitate PI3KC3-C1 (Beclin-1-Vps34-Atg14) but selectively inhibit PI3KC3-C2 (Beclin-1-Vps34-UVRAG). Interestingly, although SARS-CoV ORF3a shares 72.7% amino acid identity with the SARS-CoV-2 ORF3a, the former had no effect on cellular autophagy response. Thus, our findings provide the mechanistic evidence of possible takeover of host autophagy machinery by ORF3a to facilitate SARS-CoV-2 replication and raise the possibility of targeting the autophagic pathway for the treatment of COVID-19.

5.
Nat Commun ; 12(1): 2114, 2021 04 09.
Article in English | MEDLINE | ID: covidwho-1174670

ABSTRACT

Lack of detailed knowledge of SARS-CoV-2 infection has been hampering the development of treatments for coronavirus disease 2019 (COVID-19). Here, we report that RNA triggers the liquid-liquid phase separation (LLPS) of the SARS-CoV-2 nucleocapsid protein, N. By analyzing all 29 proteins of SARS-CoV-2, we find that only N is predicted as an LLPS protein. We further confirm the LLPS of N during SARS-CoV-2 infection. Among the 100,849 genome variants of SARS-CoV-2 in the GISAID database, we identify that ~37% (36,941) of the genomes contain a specific trio-nucleotide polymorphism (GGG-to-AAC) in the coding sequence of N, which leads to the amino acid substitutions, R203K/G204R. Interestingly, NR203K/G204R exhibits a higher propensity to undergo LLPS and a greater effect on IFN inhibition. By screening the chemicals known to interfere with N-RNA binding in other viruses, we find that (-)-gallocatechin gallate (GCG), a polyphenol from green tea, disrupts the LLPS of N and inhibits SARS-CoV-2 replication. Thus, our study reveals that targeting N-RNA condensation with GCG could be a potential treatment for COVID-19.


Subject(s)
Amino Acid Substitution/drug effects , COVID-19/prevention & control , Catechin/analogs & derivatives , Nucleocapsid Proteins/genetics , SARS-CoV-2/drug effects , Virus Replication/drug effects , COVID-19/virology , Catechin/pharmacology , Genome, Viral/genetics , Humans , Liquid-Liquid Extraction , Nucleocapsid Proteins/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , SARS-CoV-2/genetics , Virus Replication/genetics
6.
PLoS Pathog ; 17(3): e1009392, 2021 03.
Article in English | MEDLINE | ID: covidwho-1148252

ABSTRACT

Coronavirus interaction with its viral receptor is a primary genetic determinant of host range and tissue tropism. SARS-CoV-2 utilizes ACE2 as the receptor to enter host cell in a species-specific manner. We and others have previously shown that ACE2 orthologs from New World monkey, koala and mouse cannot interact with SARS-CoV-2 to mediate viral entry, and this defect can be restored by humanization of the restrictive residues in New World monkey ACE2. To better understand the genetic determinants behind the ability of ACE2 orthologs to support viral entry, we compared koala and mouse ACE2 sequences with that of human and identified the key residues in koala and mouse ACE2 that restrict viral receptor activity. Humanization of these critical residues rendered both koala and mouse ACE2 capable of binding the spike protein and facilitating viral entry. Our study shed more lights into the genetic determinants of ACE2 as the functional receptor of SARS-CoV-2, which facilitates our understanding of viral entry.


Subject(s)
COVID-19/enzymology , COVID-19/genetics , Peptidyl-Dipeptidase A/genetics , Receptors, Virus/genetics , SARS-CoV-2/physiology , Animals , Base Sequence , COVID-19/virology , Host Specificity , Humans , Mice/genetics , Mice/virology , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Phascolarctidae/genetics , Phascolarctidae/virology , Receptors, Virus/metabolism , SARS-CoV-2/genetics , Sequence Alignment , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
7.
PLoS Pathog ; 17(3): e1009439, 2021 03.
Article in English | MEDLINE | ID: covidwho-1133695

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the global pandemic of COVID-19. SARS-CoV-2 is classified as a biosafety level-3 (BSL-3) agent, impeding the basic research into its biology and the development of effective antivirals. Here, we developed a biosafety level-2 (BSL-2) cell culture system for production of transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). This trVLP expresses a reporter gene (GFP) replacing viral nucleocapsid gene (N), which is required for viral genome packaging and virion assembly (SARS-CoV-2 GFP/ΔN trVLP). The complete viral life cycle can be achieved and exclusively confined in the cells ectopically expressing SARS-CoV or SARS-CoV-2 N proteins, but not MERS-CoV N. Genetic recombination of N supplied in trans into viral genome was not detected, as evidenced by sequence analysis after one-month serial passages in the N-expressing cells. Moreover, intein-mediated protein trans-splicing approach was utilized to split the viral N gene into two independent vectors, and the ligated viral N protein could function in trans to recapitulate entire viral life cycle, further securing the biosafety of this cell culture model. Based on this BSL-2 SARS-CoV-2 cell culture model, we developed a 96-well format high throughput screening for antivirals discovery. We identified salinomycin, tubeimoside I, monensin sodium, lycorine chloride and nigericin sodium as potent antivirals against SARS-CoV-2 infection. Collectively, we developed a convenient and efficient SARS-CoV-2 reverse genetics tool to dissect the virus life cycle under a BSL-2 condition. This powerful tool should accelerate our understanding of SARS-CoV-2 biology and its antiviral development.


Subject(s)
COVID-19/virology , Cell Culture Techniques/methods , SARS-CoV-2/physiology , Antiviral Agents/pharmacology , Containment of Biohazards , Genome, Viral/drug effects , High-Throughput Screening Assays , Humans , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , SARS-CoV-2/growth & development , Virus Replication/drug effects
8.
Proc Natl Acad Sci U S A ; 118(12)2021 03 23.
Article in English | MEDLINE | ID: covidwho-1117490

ABSTRACT

The pandemic of COVID-19, caused by SARS-CoV-2, is a major global health threat. Epidemiological studies suggest that bats (Rhinolophus affinis) are the natural zoonotic reservoir for SARS-CoV-2. However, the host range of SARS-CoV-2 and intermediate hosts that facilitate its transmission to humans remain unknown. The interaction of coronavirus with its host receptor is a key genetic determinant of host range and cross-species transmission. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as the receptor to enter host cells in a species-dependent manner. In this study, we characterized the ability of ACE2 from diverse species to support viral entry. By analyzing the conservation of five residues in two virus-binding hotspots of ACE2 (hotspot 31Lys and hotspot 353Lys), we predicted 80 ACE2 proteins from mammals that could potentially mediate SARS-CoV-2 entry. We chose 48 ACE2 orthologs among them for functional analysis, and showed that 44 of these orthologs-including domestic animals, pets, livestock, and animals commonly found in zoos and aquaria-could bind the SARS-CoV-2 spike protein and support viral entry. In contrast, New World monkey ACE2 orthologs could not bind the SARS-CoV-2 spike protein and support viral entry. We further identified the genetic determinant of New World monkey ACE2 that restricts viral entry using genetic and functional analyses. These findings highlight a potentially broad host tropism of SARS-CoV-2 and suggest that SARS-CoV-2 might be distributed much more widely than previously recognized, underscoring the necessity to monitor susceptible hosts to prevent future outbreaks.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/veterinary , Receptors, Virus/genetics , SARS-CoV-2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/genetics , COVID-19/metabolism , COVID-19/virology , Host Specificity , Humans , Pandemics/prevention & control , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Phylogeny , Protein Binding , Receptors, Virus/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Viral Tropism , Viral Zoonoses/genetics , Viral Zoonoses/prevention & control , Viral Zoonoses/virology , Virus Attachment , Virus Internalization
9.
Nat Commun ; 12(1): 961, 2021 02 11.
Article in English | MEDLINE | ID: covidwho-1078585

ABSTRACT

The global spread of SARS-CoV-2 is posing major public health challenges. One feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site. Here, we find that the virus with intact spike (Sfull) preferentially enters cells via fusion at the plasma membrane, whereas a clone (Sdel) with deletion disrupting the multi-basic S1/S2 site utilizes an endosomal entry pathway. Using Sdel as model, we perform a genome-wide CRISPR screen and identify several endosomal entry-specific regulators. Experimental validation of hits from the CRISPR screen shows that host factors regulating the surface expression of angiotensin-converting enzyme 2 (ACE2) affect entry of Sfull virus. Animal-to-animal transmission with the Sdel virus is reduced compared to Sfull in the hamster model. These findings highlight the critical role of the S1/S2 boundary of SARS-CoV-2 spike protein in modulating virus entry and transmission and provide insights into entry of coronaviruses.


Subject(s)
COVID-19/virology , CRISPR-Cas Systems , Genome-Wide Association Study , Host-Pathogen Interactions , SARS-CoV-2/physiology , Virus Internalization , A549 Cells , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/genetics , Chlorocebus aethiops , Disease Models, Animal , Endosomes/virology , HeLa Cells , Humans , Mesocricetus , Serine Endopeptidases , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
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