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1.
Cell Discov ; 8(1): 53, 2022 Jun 06.
Article in English | MEDLINE | ID: covidwho-1878521

ABSTRACT

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important target for vaccine and drug development. However, the rapid emergence of variant strains with mutated S proteins has rendered many treatments ineffective. Cleavage of the S protein by host proteases is essential for viral infection. Here, we discovered that the S protein contains two previously unidentified Cathepsin L (CTSL) cleavage sites (CS-1 and CS-2). Both sites are highly conserved among all known SARS-CoV-2 variants. Our structural studies revealed that CTSL cleavage promoted S to adopt receptor-binding domain (RBD) "up" activated conformations, facilitating receptor-binding and membrane fusion. We confirmed that CTSL cleavage is essential during infection of all emerged SARS-CoV-2 variants (including the recently emerged Omicron variant) by pseudovirus (PsV) infection experiment. Furthermore, we found CTSL-specific inhibitors not only blocked infection of PsV/live virus in cells but also reduced live virus infection of ex vivo lung tissues of both human donors and human ACE2-transgenic mice. Finally, we showed that two CTSL-specific inhibitors exhibited excellent In vivo effects to prevent live virus infection in human ACE2-transgenic mice. Our work demonstrated that inhibition of CTSL cleavage of SARS-CoV-2 S protein is a promising approach for the development of future mutation-resistant therapy.

2.
PLoS Pathog ; 18(2): e1010343, 2022 02.
Article in English | MEDLINE | ID: covidwho-1690680

ABSTRACT

The continuous emergence of severe acute respiratory coronavirus 2 (SARS-CoV-2) variants and the increasing number of breakthrough infection cases among vaccinated people support the urgent need for research and development of antiviral drugs. Viral entry is an intriguing target for antiviral drug development. We found that diltiazem, a blocker of the L-type calcium channel Cav1.2 pore-forming subunit (Cav1.2 α1c) and an FDA-approved drug, inhibits the binding and internalization of SARS-CoV-2, and decreases SARS-CoV-2 infection in cells and mouse lung. Cav1.2 α1c interacts with SARS-CoV-2 spike protein and ACE2, and affects the attachment and internalization of SARS-CoV-2. Our finding suggests that diltiazem has potential as a drug against SARS-CoV-2 infection and that Cav1.2 α1c is a promising target for antiviral drug development for COVID-19.


Subject(s)
COVID-19 , Diltiazem/pharmacology , Lung/drug effects , SARS-CoV-2/drug effects , A549 Cells , Animals , COVID-19/drug therapy , COVID-19/pathology , COVID-19/virology , Cells, Cultured , Chlorocebus aethiops , Diltiazem/therapeutic use , Disease Models, Animal , Female , HEK293 Cells , HeLa Cells , Humans , Lung/pathology , Lung/virology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , SARS-CoV-2/physiology , Vero Cells , Virus Attachment/drug effects , Virus Internalization/drug effects
3.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-324470

ABSTRACT

The spike (S) protein of SARS coronavirus 2 (SARS-CoV-2) is an ideal target for the development of specific vaccines or drugs. However, treatments targeting viruses with mutant S proteins that have recently emerged in many countries are limited. Cleavage of the S protein by host proteases is essential for viral infection. Here, we discovered two novel sites (CS-1 and CS-2) in the S protein for cleavage by the protease Cathepsin L (CTSL). Both sites are highly conserved among all SARS-CoV-2 variants of concern. Cryo-electron microscopy structural studies revealed that CTSL cleavage increases the dynamics of the receptor binding domain of S and induces novel conformations. In our pseudovirus (PsV) infection experiment, alteration of the cleavage site significantly reduced the infection efficiency, and CTSL inhibitors markedly inhibited infection with PsVs of both the wild-type and emerged SARS-CoV-2 variants. Furthermore, six highly efficient CTSL inhibitors were found to effectively inhibit live virus infection in human cells in vitro , and two of these were further confirmed to prevent live virus infection in human ACE2 transgenic mice in vivo . Our work suggested that the CTSL cleavage sites in SARS-CoV-2 S are emerging new but effective targets for the development of mutation-resistant vaccines and drugs.

4.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-324160

ABSTRACT

The unprecedented coronavirus disease 2019 (COVID-19) epidemic has created a worldwide public health emergency, and there is an urgent need to develop an effective vaccine to control this severe infectious disease. Here, we found that a single vaccination with a replication-defective human type 5 adenovirus encoding the SARS-CoV-2 spike protein (Ad5-nCoV) protected mice completely against SARS-CoV-2 infection in the upper and lower respiratory tracts. Additionally, a single vaccination with Ad5-nCoV protected ferrets from SARS-CoV-2 infection in the upper respiratory tract. This study suggested that a combination of intramuscular and mucosal vaccination maybe provide a desirable protective efficacy and different Ad5-nCoV delivery modes are worth further investigation in human clinical trials.

5.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-307411

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative pathogen of novel coronavirus disease 2019 (COVID-19)1. SARS-CoV-2 uses angiotensin converting enzyme 2 (ACE2) as a cellular receptor and enters cells via clathrin-mediated endocytosis (CME)2-4. However, the key molecules involved in internalizing and facilitating CME for virus entry remain unknown. Here, we found metabotropic glutamate receptor subtype 2 (mGluR2) is a key entry receptor for SARS-CoV-2 infection. mGluR2 directly interacts with the SARS-CoV-2 spike protein. Knockdown of mGluR2 decreases endocytosis of SARS-CoV-2 but not cell binding. mGluR2 cooperates with ACE2 to facilitate SARS-CoV-2 entry through CME. Knockout of the mGluR2 gene in mice abolished SARS-CoV-2 infection in the nasal turbinates and significantly reduced viral infection in the lungs. Importantly, mGluR2 also is important for severe acute respiratory syndrome coronavirus spike protein and Middle East respiratory syndrome coronavirus spike protein mediated endocytosis. Our study provides important insights into SARS-CoV-2 infection and reveals an important target for the development of novel approaches to limit coronavirus infection.

6.
Virol Sin ; 37(2): 248-255, 2022 Apr.
Article in English | MEDLINE | ID: covidwho-1616811

ABSTRACT

Severe acute respiratory syndrome (SARS) is a highly contagious zoonotic disease caused by SARS coronavirus (SARS-CoV). Since its outbreak in Guangdong Province of China in 2002, SARS has caused 8096 infections and 774 deaths by December 31st, 2003. Although there have been no more SARS cases reported in human populations since 2004, the recent emergence of a novel coronavirus disease (COVID-19) indicates the potential of the recurrence of SARS and other coronavirus disease among humans. Thus, developing a rapid response SARS vaccine to provide protection for human populations is still needed. Spike (S) protein of SARS-CoV can induce neutralizing antibodies, which is a pivotal immunogenic antigen for vaccine development. Here we constructed a recombinant chimeric vesicular stomatitis virus (VSV) VSVΔG-SARS, in which the glycoprotein (G) gene is replaced with the SARS-CoV S gene. VSVΔG-SARS maintains the bullet-like shape of the native VSV, with the heterogeneous S protein incorporated into its surface instead of G protein. The results of safety trials revealed that VSVΔG-SARS is safe and effective in mice at a dose of 1 â€‹× â€‹106 TCID50. More importantly, only a single-dose immunization of 2 â€‹× â€‹107 TCID50 can provide high-level neutralizing antibodies and robust T cell responses to non-human primate animal models. Thus, our data indicate that VSVΔG-SARS can be used as a rapid response vaccine candidate. Our study on the recombinant VSV-vectored SARS-CoV vaccines can accumulate experience and provide a foundation for the new coronavirus disease in the future.


Subject(s)
COVID-19 , SARS Virus , Animals , Antibodies, Neutralizing , Antibodies, Viral , Immunization , Immunogenicity, Vaccine , Macaca mulatta , Mice , SARS Virus/genetics , Spike Glycoprotein, Coronavirus , Vaccines, Synthetic/genetics , Vesicular stomatitis Indiana virus/genetics , Vesicular stomatitis Indiana virus/metabolism
8.
Cell Discov ; 7(1): 119, 2021 Dec 14.
Article in English | MEDLINE | ID: covidwho-1569245

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses angiotensin-converting enzyme 2 (ACE2) as a binding receptor to enter cells via clathrin-mediated endocytosis (CME). However, receptors involved in other steps of SARS-CoV-2 infection remain largely unknown. Here, we found that metabotropic glutamate receptor subtype 2 (mGluR2) is an internalization factor for SARS-CoV-2. Our results show that mGluR2 directly interacts with the SARS-CoV-2 spike protein and that knockdown of mGluR2 decreases internalization of SARS-CoV-2 but not cell binding. Further, mGluR2 is uncovered to cooperate with ACE2 to facilitate SARS-CoV-2 internalization through CME and mGluR2 knockout in mice abolished SARS-CoV-2 infection in the nasal turbinates and significantly reduced viral infection in the lungs. Notably, mGluR2 is also important for SARS-CoV spike protein- and Middle East respiratory syndrome coronavirus spike protein-mediated internalization. Thus, our study identifies a novel internalization factor used by SARS-CoV-2 and opens a new door for antiviral development against coronavirus infection.

10.
Emerg Microbes Infect ; 10(1): 481-492, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1124537

ABSTRACT

The unprecedented coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a serious threat to global public health. Development of effective therapies against SARS-CoV-2 is urgently needed. Here, we evaluated the antiviral activity of a remdesivir parent nucleotide analog, GS441524, which targets the coronavirus RNA-dependent RNA polymerase enzyme, and a feline coronavirus prodrug, GC376, which targets its main protease, using a mouse-adapted SARS-CoV-2 infected mouse model. Our results showed that GS441524 effectively blocked the proliferation of SARS-CoV-2 in the mouse upper and lower respiratory tracts via combined intranasal (i.n.) and intramuscular (i.m.) treatment. However, the ability of high-dose GC376 (i.m. or i.n. and i.m.) was weaker than GS441524. Notably, low-dose combined application of GS441524 with GC376 could effectively protect mice against SARS-CoV-2 infection via i.n. or i.n. and i.m. treatment. Moreover, we found that the pharmacokinetic properties of GS441524 is better than GC376, and combined application of GC376 and GS441524 had a synergistic effect. Our findings support the further evaluation of the combined application of GC376 and GS441524 in future clinical studies.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Respiratory System/virology , SARS-CoV-2/drug effects , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacokinetics , Cell Proliferation/drug effects , Chlorocebus aethiops , Drug Therapy, Combination , Female , Mice , Mice, Inbred BALB C , Prodrugs/pharmacology , Rats , Rats, Sprague-Dawley , Vero Cells
11.
Zhongguo Yufang Shouyi Xuebao / Chinese Journal of Preventive Veterinary Medicine ; 42(11):1145-1151, 2020.
Article in Chinese | CAB Abstracts | ID: covidwho-1073550

ABSTRACT

Severe Acute Respiratory Syndrome (SARS) is a zoonotic disease that is acute, feverish and accompanied by respiratory system and even multiple organ infections. Although no SARS infection cases have been reported since 2004, the outbreak of new coronavirus pneumonia (COVID-19) in Wuhan, Hubei Province, my country in December 2019 indicates that SARS coronavirus (SARS-CoV) or SARS-like coronavirus (SARSL-CoV) is highly likely to become widespread in the population again. In this study, a full-length cDNA clone pBRN-FL-SARS-CoV-S expressing the SARS-CoV spike protein (S protein) was first constructed, and the Newcastle Disease Virus (NDV) LaSota vaccine strain reverse genetic operating system was used to rescue The recombinant virus rLa-SARS-CoV-S expressing SARS-CoV S protein was identified. After rLa-SARS-CoV-S was infected with BHK-21 cells at a dose of MOI 0.01 for 36 hours, the SARS-CoV S protein was detected by western blot and laser confocal test. The results showed that the S protein was correct in the infected cells. Expressed and accurately located on the cell membrane. After inoculating 10-day-old SPF chicken embryos with rLa-SARS-CoV-S at a dose of 1x104 EID50, allantoic fluid was collected at different time points and the EID50 was determined. The growth kinetic curve of the virus showed that rLa-SARSCoV-S could The chicken embryo grows at high titer, consistent with the parental virus. Dilute the rLa-SARS-CoV-S and parent virus by 10-fold ratio and inoculate 10-day-old SPF chicken embryos and record the death time of each chicken embryo. Calculate the average chicken embryo death time according to the highest dilution of the virus. Lethal time (MDT), the results showed that the MDT of rLa-SARS-CoV-S was 112.8 h and the MDT of NDV LaSota was 96 h, indicating that the recombinant virus still maintains the low pathogenicity characteristics of the NDV LaSota vaccine strain. RLa-SARS-CoV-S and NDV LaSota were injected intramuscularly with 6-week-old BALB/c mice at a dose of 5x106 EID50 and boosted on the 21st day. At the same time, a PBS control group was set up. The results of the mouse safety test showed that all the mice vaccinated with rLa-SARS-CoV-S survived without any clinical symptoms, and the weight gain was consistent with that of the NDV LaSota group and the control group;the mice were treated on 21 d and 42 d after immunization Blood was collected to prepare serum, and the level of IgG antibodies against SARS-CoV S protein in mice was detected by ELISA. The results showed that the recombinant protein can induce mice to produce higher levels of specific IgG antibodies after initial immunization and booster immunization. The level can be maintained for a longer period of time. The results of this study indicate that rLa-SARS-CoV-S has potential value as a SARS-CoV vaccine candidate, and at the same time provides ideas for the development of SARS-CoV-2 vaccines.

12.
Natl Sci Rev ; 8(3): nwaa291, 2021 Mar.
Article in English | MEDLINE | ID: covidwho-977391

ABSTRACT

Minks are raised in many countries and have transmitted severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to humans. However, the biologic properties of SARS-CoV-2 in minks are largely unknown. Here, we investigated and found that SARS-CoV-2 replicates efficiently in both the upper and lower respiratory tracts, and transmits efficiently in minks via respiratory droplets; pulmonary lesions caused by SARS-CoV-2 in minks are similar to those seen in humans with COVID-19. We further found that a spike protein-based subunit vaccine largely prevented SARS-CoV-2 replication and lung damage caused by SARS-CoV-2 infection in minks. Our study indicates that minks are a useful animal model for evaluating the efficacy of drugs or vaccines against COVID-19 and that vaccination is a potential strategy to prevent minks from transmitting SARS-CoV-2.

14.
Nat Commun ; 11(1): 4081, 2020 08 14.
Article in English | MEDLINE | ID: covidwho-717117

ABSTRACT

The unprecedented coronavirus disease 2019 (COVID-19) epidemic has created a worldwide public health emergency, and there is an urgent need to develop an effective vaccine to control this severe infectious disease. Here, we find that a single vaccination with a replication-defective human type 5 adenovirus encoding the SARS-CoV-2 spike protein (Ad5-nCoV) protect mice completely against mouse-adapted SARS-CoV-2 infection in the upper and lower respiratory tracts. Additionally, a single vaccination with Ad5-nCoV protects ferrets from wild-type SARS-CoV-2 infection in the upper respiratory tract. This study suggests that the mucosal vaccination may provide a desirable protective efficacy and this delivery mode is worth further investigation in human clinical trials.


Subject(s)
Betacoronavirus/immunology , Coronavirus Infections/prevention & control , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Viral Vaccines/immunology , Animals , Antibodies, Viral/immunology , COVID-19 , COVID-19 Vaccines , Coronavirus Infections/immunology , Disease Models, Animal , Drug Design , Female , Genetic Vectors , HEK293 Cells , Humans , Mice , Mice, Inbred BALB C , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Viral Vaccines/administration & dosage , Viral Vaccines/genetics
16.
Science ; 368(6494): 1016-1020, 2020 05 29.
Article in English | MEDLINE | ID: covidwho-45712

ABSTRACT

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) causes the infectious disease COVID-19 (coronavirus disease 2019), which was first reported in Wuhan, China, in December 2019. Despite extensive efforts to control the disease, COVID-19 has now spread to more than 100 countries and caused a global pandemic. SARS-CoV-2 is thought to have originated in bats; however, the intermediate animal sources of the virus are unknown. In this study, we investigated the susceptibility of ferrets and animals in close contact with humans to SARS-CoV-2. We found that SARS-CoV-2 replicates poorly in dogs, pigs, chickens, and ducks, but ferrets and cats are permissive to infection. Additionally, cats are susceptible to airborne transmission. Our study provides insights into the animal models for SARS-CoV-2 and animal management for COVID-19 control.


Subject(s)
Animals, Domestic , Betacoronavirus/physiology , Coronavirus Infections , Disease Models, Animal , Disease Susceptibility , Ferrets , Pandemics , Pneumonia, Viral , Animals , Antibodies, Viral/blood , Betacoronavirus/immunology , Betacoronavirus/isolation & purification , COVID-19 , Cats , Chickens , Coronavirus Infections/transmission , Coronavirus Infections/virology , Dogs , Ducks , Feces/virology , Female , Male , Pneumonia, Viral/transmission , Pneumonia, Viral/virology , RNA, Viral/isolation & purification , Respiratory System/virology , SARS-CoV-2 , Species Specificity , Sus scrofa , Virus Attachment , Virus Replication
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