Repurposing niclosamide as a novel anti-SARS-Cov-2 drug by restricting entry protein CD147.

Background The burst of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing the global COVID-19 pandemic. But until today only limited numbers of drugs are discovered to treat COVID-19 patients. Even worse, the rapid mutations of SARS-CoV-2 compromise the effectiveness of existing vaccines and neutralizing antibodies due to the increased viral transmissibility and immune escape. CD147-spike protein, one of the entries of SRAR-CoV-2 into host cells, has been reported as a promising therapeutic target for developing drugs against COVID-19. Methods CRISPR-Cas9 induced gene knockout, western blotting, tet-off protein overexpression, ribonucleoprotein IP and RNA-IP were used to confirm the regulation of HuR on mRNA of CD147. Regulation of niclosamide on HuR nucleo-translocation was assessed by immunofluorescence staining of cell lines, IHC staining of tissue of mouse model and western blotting. Finally, the suppression of niclosamide on SARS-CoV-2 infection induced CD147 was evaluated by ACE2-expressing A549 cells and western blotting. Results We first discovered a novel regulation mechanism of CD147 via the RNA-binding protein HuR. We found that HuR regulates CD147 post-transcription by directly bound to its 3'-UTR. The loss of HuR reduced CD147 in multiple cell lines. Niclosamide inhibited CD147 function by blocking HuR cytoplasmic translocation and diminishing CD147 glycosylation. SARS-CoV-2 infection induced CD147 in ACE2-expressing A549 cells, which could be neutralized by niclosamide in a dose-dependent manner. Conclusion Together, our study reveals a novel regulation mechanism of CD147 and niclosamide can be repurposed as an effective COVID-19 drug by targeting the virus entry, CD147-spike protein.

Identification of AXL as a co-receptor for human parvovirus B19 infection of human erythroid progenitors.

Parvovirus B19 (B19V) infects human erythroid progenitor cells (EPCs) and causes several hematological disorders and fetal hydrops. Amino acid (aa) 5-68 of minor capsid protein VP1 (VP1u5-68aa) is the minimal receptor binding domain for B19V to enter EPCs. Here, we carried out a genome-wide CRISPR-Cas9 guide RNA screen and identified tyrosine protein kinase receptor UFO (AXL) as a proteinaceous receptor for B19V infection of EPCs. AXL gene silencing in ex vivo expanded EPCs remarkably decreased B19V internalization and replication. Additions of the recombinant AXL extracellular domain or a polyclonal antibody against it upon infection efficiently inhibited B19V infection of ex vivo expanded EPCs. Moreover, B19V VP1u interacted with the recombinant AXL extracellular domain in vitro at a relatively high affinity (KD = 103 nM). Collectively, we provide evidence that AXL is a co-receptor for B19V infection of EPCs.

A Methyltransferase-Defective Vesicular Stomatitis Virus-Based SARS-CoV-2 Vaccine Candidate Provides Complete Protection against SARS-CoV-2 Infection in Hamsters.

The current pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to dramatic economic and health burdens. Although the worldwide SARS-CoV-2 vaccination campaign has begun, exploration of other vaccine candidates is needed due to uncertainties with the current approved vaccines, such as durability of protection, cross-protection against variant strains, and costs of long-term production and storage. In this study, we developed a methyltransferase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidate. We generated mtdVSVs expressing SARS-CoV-2 full-length spike (S) protein, S1, or its receptor-binding domain (RBD). All of these recombinant viruses grew to high titers in mammalian cells despite high attenuation in cell culture. The SARS-CoV-2 S protein and its truncations were highly expressed by the mtdVSV vector. These mtdVSV-based vaccine candidates were completely attenuated in both immunocompetent and immunocompromised mice. Among these constructs, mtdVSV-S induced high levels of SARS-CoV-2-specific neutralizing antibodies (NAbs) and Th1-biased T-cell immune responses in mice. In Syrian golden hamsters, the serum levels of SARS-CoV-2-specific NAbs triggered by mtdVSV-S were higher than the levels of NAbs in convalescent plasma from recovered COVID-19 patients. In addition, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 replication in lung and nasal turbinate tissues, cytokine storm, and lung pathology. Collectively, our data demonstrate that mtdVSV expressing SARS-CoV-2 S protein is a safe and highly efficacious vaccine candidate against SARS-CoV-2 infection. IMPORTANCE Viral mRNA cap methyltransferase (MTase) is essential for mRNA stability, protein translation, and innate immune evasion. Thus, viral mRNA cap MTase activity is an excellent target for development of live attenuated or live vectored vaccine candidates. Here, we developed a panel of MTase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidates expressing full-length S, S1, or several versions of the RBD. These mtdVSV-based vaccine candidates grew to high titers in cell culture and were completely attenuated in both immunocompetent and immunocompromised mice. Among these vaccine candidates, mtdVSV-S induces high levels of SARS-CoV-2-specific neutralizing antibodies (Nabs) and Th1-biased immune responses in mice. Syrian golden hamsters immunized with mtdVSV-S triggered SARS-CoV-2-specific NAbs at higher levels than those in convalescent plasma from recovered COVID-19 patients. Furthermore, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 challenge. Thus, mtdVSV is a safe and highly effective vector to deliver SARS-CoV-2 vaccine.

Direct Activation of Endothelial Cells by SARS-CoV-2 Nucleocapsid Protein Is Blocked by Simvastatin.

Emerging evidence suggests that endothelial activation plays a central role in the pathogenesis of acute respiratory distress syndrome (ARDS) and multiorgan failure in patients with coronavirus disease 2019 (COVID-19). However, the molecular mechanisms underlying endothelial activation in COVID-19 patients remain unclear. In this study, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins that potently activate human endothelial cells were screened to elucidate the molecular mechanisms involved in endothelial activation. It was found that nucleocapsid protein (NP) of SARS-CoV-2 significantly activated human endothelial cells through Toll-like receptor 2 (TLR2)/NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways. Moreover, by screening a natural microbial compound library containing 154 natural compounds, simvastatin was identified as a potent inhibitor of NP-induced endothelial activation. Remarkably, though the protein sequences of N proteins from coronaviruses are highly conserved, only NP from SARS-CoV-2 induced endothelial activation. The NPs from other coronaviruses such as SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), HUB1-CoV, and influenza virus H1N1 did not activate endothelial cells. These findings are consistent with the results from clinical investigations showing broad endotheliitis and organ injury in severe COVID-19 patients. In conclusion, the study provides insights on SARS-CoV-2-induced vasculopathy and coagulopathy and suggests that simvastatin, an FDA-approved lipid-lowering drug, may help prevent the pathogenesis and improve the outcome of COVID-19 patients. IMPORTANCE Coronavirus disease 2019 (COVID-19), caused by the betacoronavirus SARS-CoV-2, is a worldwide challenge for health care systems. The leading cause of mortality in patients with COVID-19 is hypoxic respiratory failure from acute respiratory distress syndrome (ARDS). To date, pulmonary endothelial cells (ECs) have been largely overlooked as a therapeutic target in COVID-19, yet emerging evidence suggests that these cells contribute to the initiation and propagation of ARDS by altering vessel barrier integrity, promoting a procoagulative state, inducing vascular inflammation and mediating inflammatory cell infiltration. Therefore, a better mechanistic understanding of the vasculature is of utmost importance. In this study, we screened the SARS-CoV-2 viral proteins that potently activate human endothelial cells and found that nucleocapsid protein (NP) significantly activated human endothelial cells through TLR2/NF-κB and MAPK signaling pathways. Moreover, by screening a natural microbial compound library containing 154 natural compounds, simvastatin was identified as a potent inhibitor of NP-induced endothelial activation. Our results provide insights on SARS-CoV-2-induced vasculopathy and coagulopathy, and suggests that simvastatin, an FDA-approved lipid-lowering drug, may benefit to prevent the pathogenesis and improve the outcome of COVID-19 patients.

Discovery of Small Anti-ACE2 Peptides to Inhibit SARS-CoV-2 Infectivity (Adv. Therap. 7/2021)

SARS-CoV-2 infects human cells by binding its spike protein to the human ACE2 receptor. Using a peptide biopanning strategy, the authors have discovered small anti-ACE2 peptides that can effectively block the SARS-CoV-2/ACE2 interaction. The anti-ACE2 peptides can be potentially used as prophylactic or therapeutic agents for SARS-CoV-2 and other ACE2-mediated viruses. This is reported by Kun Cheng and co-workers in article number 2100087.

The SARS-CoV-2 Transcriptome and the Dynamics of the S Gene Furin Cleavage Site in Primary Human Airway Epithelia.

The spike (S) polypeptide of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) consists of the S1 and S2 subunits and is processed by cellular proteases at the S1/S2 boundary that contains a furin cleavage site (FCS), 682RRAR↓S686 Various deletions surrounding the FCS have been identified in patients. When SARS-CoV-2 propagated in Vero cells, it acquired deletions surrounding the FCS. We studied the viral transcriptome in Vero cell-derived SARS-CoV-2-infected primary human airway epithelia (HAE) cultured at an air-liquid interface (ALI) with an emphasis on the viral genome stability of the FCS. While we found overall the viral transcriptome is similar to that generated from infected Vero cells, we identified a high percentage of mutated viral genome and transcripts in HAE-ALI. Two highly frequent deletions were found at the FCS region: a 12 amino acid deletion (678TNSPRRAR↓SVAS689) that contains the underlined FCS and a 5 amino acid deletion (675QTQTN679) that is two amino acids upstream of the FCS. Further studies on the dynamics of the FCS deletions in apically released virions from 11 infected HAE-ALI cultures of both healthy and lung disease donors revealed that the selective pressure for the FCS maintains the FCS stably in 9 HAE-ALI cultures but with 2 exceptions, in which the FCS deletions are retained at a high rate of >40% after infection of ≥13 days. Our study presents evidence for the role of unique properties of human airway epithelia in the dynamics of the FCS region during infection of human airways, which is likely donor dependent.IMPORTANCE Polarized human airway epithelia at an air-liquid interface (HAE-ALI) are an in vitro model that supports efficient infection of SARS-CoV-2. The spike (S) protein of SARS-CoV-2 contains a furin cleavage site (FCS) at the boundary of the S1 and S2 domains which distinguishes it from SARS-CoV. However, FCS deletion mutants have been identified in patients and in vitro cell cultures, and how the airway epithelial cells maintain the unique FCS remains unknown. We found that HAE-ALI cultures were capable of suppressing two prevalent FCS deletion mutants (Δ678TNSPRRAR↓SVAS689 and Δ675QTQTN679) that were selected during propagation in Vero cells. While such suppression was observed in 9 out of 11 of the tested HAE-ALI cultures derived from independent donors, 2 exceptions that retained a high rate of FCS deletions were also found. Our results present evidence of the donor-dependent properties of human airway epithelia in the evolution of the FCS during infection.

Discovery of Small Anti-ACE2 Peptides to Inhibit SARS-CoV-2 Infectivity.

COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which infects host cells by binding its viral spike protein receptor-binding domain (RBD) to the angiotensin converting enzyme 2 (ACE2) on host cells. Blocking the SARS-CoV-2-RBD/ACE2 interaction is, therefore, a potential strategy to inhibit viral infections. Using a novel biopanning strategy, a small anti-ACE2 peptide is discovered, which shows high affinity and specificity to human ACE2. It blocks not only the SARS-CoV-2-RBD/ACE2 interaction but also the SARS-CoV-1-RBD/ACE2 interaction. Moreover, it inhibits SARS-CoV-2 infection in Vero-E6 cells. The peptide shows negligible cytotoxicity in Vero-E6 cells and Huh7 cells. In vivo short-term lung toxicity study also demonstrates a good safety of the peptide after intratracheal administration. The anti-ACE2 peptide can be potentially used as a prophylactic or therapeutic agent for SARS-CoV-2 or other ACE2-mediated viruses. The strategy used in this study also provides a fast-track platform to discover other antiviral peptides, which will prepare the world for future pandemics.

A safe and highly efficacious measles virus-based vaccine expressing SARS-CoV-2 stabilized prefusion spike.

The current pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights an urgent need to develop a safe, efficacious, and durable vaccine. Using a measles virus (rMeV) vaccine strain as the backbone, we developed a series of recombinant attenuated vaccine candidates expressing various forms of the SARS-CoV-2 spike (S) protein and its receptor binding domain (RBD) and evaluated their efficacy in cotton rat, IFNAR-/-mice, IFNAR-/--hCD46 mice, and golden Syrian hamsters. We found that rMeV expressing stabilized prefusion S protein (rMeV-preS) was more potent in inducing SARS-CoV-2-specific neutralizing antibodies than rMeV expressing full-length S protein (rMeV-S), while the rMeVs expressing different lengths of RBD (rMeV-RBD) were the least potent. Animals immunized with rMeV-preS produced higher levels of neutralizing antibody than found in convalescent sera from COVID-19 patients and a strong Th1-biased T cell response. The rMeV-preS also provided complete protection of hamsters from challenge with SARS-CoV-2, preventing replication in lungs and nasal turbinates, body weight loss, cytokine storm, and lung pathology. These data demonstrate that rMeV-preS is a safe and highly efficacious vaccine candidate, supporting its further development as a SARS-CoV-2 vaccine.

The RNA Architecture of the SARS-CoV-2 3'-Untranslated Region.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic. The 3' untranslated region (UTR) of this ß-CoV contains essential cis-acting RNA elements for the viral genome transcription and replication. These elements include an equilibrium between an extended bulged stem-loop (BSL) and a pseudoknot. The existence of such an equilibrium is supported by reverse genetic studies and phylogenetic covariation analysis and is further proposed as a molecular switch essential for the control of the viral RNA polymerase binding. Here, we report the SARS-CoV-2 3' UTR structures in cells that transcribe the viral UTRs harbored in a minigene plasmid and isolated infectious virions using a chemical probing technique, namely dimethyl sulfate (DMS)-mutational profiling with sequencing (MaPseq). Interestingly, the putative pseudoknotted conformation was not observed, indicating that its abundance in our systems is low in the absence of the viral nonstructural proteins (nsps). Similarly, our results also suggest that another functional cis-acting element, the three-helix junction, cannot stably form. The overall architectures of the viral 3' UTRs in the infectious virions and the minigene-transfected cells are almost identical.

The RNA Architecture of the SARS-CoV-2 3′-Untranslated Region

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic. The 3′untranslated region (UTR) of this β-CoV contains essential cis-acting RNA elements for the viral genome transcription and replication. These elements include an equilibrium between an extended bulged stem-loop (BSL) and a pseudoknot. The existence of such an equilibrium is supported by reverse genetic studies and phylogenetic covariation analysis and is further proposed as a molecular switch essential for the control of the viral RNA polymerase binding. Here, we report the SARS-CoV-2 3′UTR structures in cells that transcribe the viral UTRs harbored in a minigene plasmid and isolated infectious virions using a chemical probing technique, namely dimethyl sulfate (DMS)-mutational profiling with sequencing (MaPseq). Interestingly, the putative pseudoknotted conformation was not observed, indicating that its abundance in our systems is low in the absence of the viral nonstructural proteins (nsps). Similarly, our results also suggest that another functional cis-acting element, the three-helix junction, cannot stably form. The overall architectures of the viral 3′UTRs in the infectious virions and the minigene-transfected cells are almost identical.

Long-Term Modeling of SARS-CoV-2 Infection of In Vitro Cultured Polarized Human Airway Epithelium.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates throughout human airways. The polarized human airway epithelium (HAE) cultured at an airway-liquid interface (HAE-ALI) is an in vitro model mimicking the in vivo human mucociliary airway epithelium and supports the replication of SARS-CoV-2. Prior studies characterized only short-period SARS-CoV-2 infection in HAE. In this study, continuously monitoring the SARS-CoV-2 infection in HAE-ALI cultures for a long period of up to 51 days revealed that SARS-CoV-2 infection was long lasting with recurrent replication peaks appearing between an interval of approximately 7 to 10 days, which was consistent in all the tested HAE-ALI cultures derived from 4 lung bronchi of independent donors. We also identified that SARS-CoV-2 does not infect HAE from the basolateral side, and the dominant SARS-CoV-2 permissive epithelial cells are ciliated cells and goblet cells, whereas virus replication in basal cells and club cells was not detected. Notably, virus infection immediately damaged the HAE, which is demonstrated by dispersed zonula occludens-1 (ZO-1) expression without clear tight junctions and partial loss of cilia. Importantly, we identified that SARS-CoV-2 productive infection of HAE requires a high viral load of >2.5 × 105 virions per cm2 of epithelium. Thus, our studies highlight the importance of a high viral load and that epithelial renewal initiates and maintains a recurrent infection of HAE with SARS-CoV-2.IMPORTANCE The pandemic of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to >35 million confirmed cases and >1 million fatalities worldwide. SARS-CoV-2 mainly replicates in human airway epithelia in COVID-19 patients. In this study, we used in vitro cultures of polarized human bronchial airway epithelium to model SARS-CoV-2 replication for a period of 21 to 51 days. We discovered that in vitro airway epithelial cultures endure a long-lasting SARS-CoV-2 propagation with recurrent peaks of progeny virus release at an interval of approximately 7 to 10 days. Our study also revealed that SARS-CoV-2 infection causes airway epithelia damage with disruption of tight junction function and loss of cilia. Importantly, SARS-CoV-2 exhibits a polarity of infection in airway epithelium only from the apical membrane; it infects ciliated and goblet cells but not basal and club cells. Furthermore, the productive infection of SARS-CoV-2 requires a high viral load of over 2.5 × 105 virions per cm2 of epithelium. Our study highlights that the proliferation of airway basal cells and regeneration of airway epithelium may contribute to the recurrent infections.

Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates throughout human airways. The polarized human airway epithelium (HAE) cultured at an airway-liquid interface (HAE-ALI) is an in vitro model mimicking the in vivo human mucociliary airway epithelium and supports the replication of SARS-CoV-2. However, previous studies only characterized short-period SARS-CoV-2 infection in HAE. In this study, continuously monitoring the SARS-CoV-2 infection in HAE-ALI cultures for a long period of up to 51 days revealed that SARS-CoV-2 infection was long lasting with recurrent replication peaks appearing between an interval of approximately 7-10 days, which was consistent in all the tested HAE-ALI cultures derived from 4 lung bronchi of independent donors. We also identified that SARS-CoV-2 does not infect HAE from the basolateral side, and the dominant SARS-CoV-2 permissive epithelial cells are ciliated cells and goblet cells, whereas virus replication in basal cells and club cells was not detectable. Notably, virus infection immediately damaged the HAE, which is demonstrated by dispersed Zonula occludens-1 (ZO-1) expression without clear tight junctions and partial loss of cilia. Importantly, we identified that SARS-CoV-2 productive infection of HAE requires a high viral load of 2.5 × 10 5 virions per cm 2 of epithelium. Thus, our studies highlight the importance of a high viral load and that epithelial renewal initiates and maintains a recurrent infection of HAE with SARS-CoV-2.