Alpha-Soluble NSF Attachment Protein Prevents the Cleavage of the SARS-CoV-2 Spike Protein by Functioning as an Interferon-Upregulated Furin Inhibitor.

Loss of the furin cleavage motif in the SARS-CoV-2 spike protein reduces the virulence and transmission of SARS-CoV-2, suggesting that furin is an attractive antiviral drug target. However, lack of understanding of the regulation of furin activity has largely limited the development of furin-based therapeutic strategies. Here, we find that alpha-soluble NSF attachment protein (α-SNAP), an indispensable component of vesicle trafficking machinery, inhibits the cleavage of SARS-CoV-2 spike protein and other furin-dependent virus glycoproteins. SARS-CoV-2 infection increases the expression of α-SNAP, and overexpression of α-SNAP reduces SARS-CoV-2 infection in cells. We further reveal that α-SNAP is an interferon-upregulated furin inhibitor that inhibits furin function by interacting with its P domain. Our study demonstrates that α-SNAP, in addition to its role in vesicle trafficking, plays an important role in the host defense against furin-dependent virus infection and therefore could be a target for the development of therapeutic options for COVID-19. IMPORTANCE Some key mutations of SARS-CoV-2 spike protein, such as D614G and P681R mutations, increase the transmission or pathogenicity by enhancing the cleavage efficacy of spike protein by furin. Loss of the furin cleavage motif of SARS-CoV-2 spike protein reduces the virulence and transmission, suggesting that furin is an attractive antiviral drug target. However, lack of understanding of the regulation of furin activity has largely limited the development of furin-based therapeutic strategies. Here, we found that in addition to its canonical role in vesicle trafficking, alpha-soluble NSF attachment protein (α-SNAP) plays an important role in the host defense against furin-dependent virus infection. we identified that α-SNAP is a novel interferon-upregulated furin inhibitor and inhibits the cleavage of SARS-CoV-2 spike protein and other furin-dependent virus glycoproteins by interacting with P domain of furin. Our study demonstrates that α-SNAP could be a target for the development of therapeutic options for COVID-19.

Transferrin Receptor Protein 1 Cooperates with mGluR2 To Mediate the Internalization of Rabies Virus and SARS-CoV-2.

Identification of bona fide functional receptors and elucidation of the mechanism of receptor-mediated virus entry are important to reveal targets for developing therapeutics against rabies virus (RABV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our previous studies suggest that metabotropic glutamate receptor subtype 2 (mGluR2) functions as an entry receptor for RABV in vitro, and is an important internalization factor for SARS-CoV-2 in vitro and in vivo. Here, we demonstrate that mGluR2 facilitates RABV internalization in vitro and infection in vivo. We found that transferrin receptor 1 (TfR1) interacts with mGluR2 and internalizes with mGluR2 and RABV in the same clathrin-coated pit. Knockdown of TfR1 blocks agonist-triggered internalization of mGluR2. Importantly, TfR1 also interacts with the SARS-CoV-2 spike protein and is important for SARS-CoV-2 internalization. Our findings identify a novel axis (mGluR2-TfR1 axis) used by RABV and SARS-CoV-2 for entry, and reveal TfR1 as a potential target for therapeutics against RABV and SARS-CoV-2. IMPORTANCE We previously found that metabotropic glutamate receptor subtype 2 (mGluR2) is an entry receptor for RABV in vitro, and an important internalization factor for SARS-CoV-2 in vitro and in vivo. However, whether mGluR2 is required for RABV infection in vivo was unknown. In addition, how mGluR2 mediates the internalization of RABV and SARS-CoV-2 needed to be resolved. Here, we found that mGluR2 gene knockout mice survived a lethal challenge with RABV. To our knowledge, mGluR2 is the first host factor to be definitively shown to play an important role in RABV street virus infection in vivo. We further found that transferrin receptor protein 1 (TfR1) directly interacts and cooperates with mGluR2 to regulate the endocytosis of RABV and SARS-CoV-2. Our study identifies a novel axis (mGluR2-TfR1 axis) used by RABV and SARS-CoV-2 for entry and opens a new door for the development of therapeutics against RABV and SARS-CoV-2.

Development of Highly Potent Noncovalent Inhibitors of SARS-CoV-2 3CLpro.

The 3C-like protease (3CLpro) is an essential enzyme for the replication of SARS-CoV-2 and other coronaviruses and thus is a target for coronavirus drug discovery. Nearly all inhibitors of coronavirus 3CLpro reported so far are covalent inhibitors. Here, we report the development of specific, noncovalent inhibitors of 3CLpro. The most potent one, WU-04, effectively blocks SARS-CoV-2 replications in human cells with EC50 values in the 10-nM range. WU-04 also inhibits the 3CLpro of SARS-CoV and MERS-CoV with high potency, indicating that it is a pan-inhibitor of coronavirus 3CLpro. WU-04 showed anti-SARS-CoV-2 activity similar to that of PF-07321332 (Nirmatrelvir) in K18-hACE2 mice when the same dose was administered orally. Thus, WU-04 is a promising drug candidate for coronavirus treatment.

Development of Highly Potent Noncovalent Inhibitors of SARS-CoV-2 3CLpro

The 3C-like protease (3CLpro) is an essential enzyme for the replication of SARS-CoV-2 and other coronaviruses and thus is a target for coronavirus drug discovery. Nearly all inhibitors of coronavirus 3CLpro reported so far are covalent inhibitors. Here, we report the development of specific, noncovalent inhibitors of 3CLpro. The most potent one, WU-04, effectively blocks SARS-CoV-2 replications in human cells with EC50 values in the 10-nM range. WU-04 also inhibits the 3CLpro of SARS-CoV and MERS-CoV with high potency, indicating that it is a pan-inhibitor of coronavirus 3CLpro. WU-04 showed anti-SARS-CoV-2 activity similar to that of PF-07321332 (Nirmatrelvir) in K18-hACE2 mice when the same dose was administered orally. Thus, WU-04 is a promising drug candidate for coronavirus treatment. A novel oral noncovalent inhibitor of 3C-like protease, named WU-04, was developed as a promising drug candidate for COVID-19 treatment.

Gossypol Broadly Inhibits Coronaviruses by Targeting RNA-Dependent RNA Polymerases.

Outbreaks of coronaviruses (CoVs), especially severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have posed serious threats to humans and animals, which urgently calls for effective broad-spectrum antivirals. RNA-dependent RNA polymerase (RdRp) plays an essential role in viral RNA synthesis and is an ideal pan-coronaviral therapeutic target. Herein, based on cryo-electron microscopy and biochemical approaches, gossypol (GOS) is identified from 881 natural products to directly block SARS-CoV-2 RdRp, thus inhibiting SARS-CoV-2 replication in both cellular and mouse infection models. GOS also acts as a potent inhibitor against the SARS-CoV-2 variant of concern (VOC) and exerts same inhibitory effects toward mutated RdRps of VOCs as the RdRp of the original SARS-CoV-2. Moreover, that the RdRp inhibitor GOS has broad-spectrum anti-coronavirus activity against alphacoronaviruses (porcine epidemic diarrhea virus and swine acute diarrhea syndrome coronavirus), betacoronaviruses (SARS-CoV-2), gammacoronaviruses (avian infectious bronchitis virus), and deltacoronaviruses (porcine deltacoronavirus) is showed. The findings demonstrate that GOS may serve as a promising lead compound for combating the ongoing COVID-19 pandemic and other coronavirus outbreaks.

Novel cleavage sites identified in SARS-CoV-2 spike protein reveal mechanism for cathepsin L-facilitated viral infection and treatment strategies.

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.

Diltiazem inhibits SARS-CoV-2 cell attachment and internalization and decreases the viral infection in mouse lung.

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.

SARS-CoV-2 uses metabotropic glutamate receptor subtype 2 as an internalization factor to infect cells.

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.

The preclinical inhibitor GS441524 in combination with GC376 efficaciously inhibited the proliferation of SARS-CoV-2 in the mouse respiratory tract.

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.

Replication, pathogenicity, and transmission of SARS-CoV-2 in minks.

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.

A single dose of an adenovirus-vectored vaccine provides protection against SARS-CoV-2 challenge.

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.

Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS-coronavirus 2.

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.