Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 20 de 76
Filter
1.
Cell Biosci ; 12(1): 88, 2022 Jun 11.
Article in English | MEDLINE | ID: covidwho-1957070

ABSTRACT

BACKGROUND: An animal model that can mimic the SARS-CoV-2 infection in humans is critical to understanding the rapidly evolving SARS-CoV-2 virus and for development of prophylactic and therapeutic strategies to combat emerging mutants. Studies show that the spike proteins of SARS-CoV and SARS-CoV-2 bind to human angiotensin-converting enzyme 2 (hACE2, a well-recognized, functional receptor for SARS-CoV and SARS-CoV-2) to mediate viral entry. Several hACE2 transgenic (hACE2Tg) mouse models are being widely used, which are clearly invaluable. However, the hACE2Tg mouse model cannot fully explain: (1) low expression of ACE2 observed in human lung and heart, but lung or heart failure occurs frequently in severe COVID-19 patients; (2) low expression of ACE2 on immune cells, but lymphocytopenia occurs frequently in COVID-19 patients; and (3) hACE2Tg mice do not mimic the natural course of SARS-CoV-2 infection in humans. Moreover, one of most outstanding features of coronavirus infection is the diversity of receptor usage, which includes the newly proposed human CD147 (hCD147) as a possible co-receptor for SARS-CoV-2 entry. It is still debatable whether CD147 can serve as a functional receptor for SARS-CoV-2 infection or entry. RESULTS: Here we successfully generated a hCD147 knock-in mouse model (hCD147KI) in the NOD-scid IL2Rgammanull (NSG) background. In this hCD147KI-NSG mouse model, the hCD147 genetic sequence was placed downstream of the endogenous mouse promoter for mouse CD147 (mCD147), which creates an in vivo model that may better recapitulate physiological expression of hCD147 proteins at the molecular level compared to the existing and well-studied K18-hACE2-B6 (JAX) model. In addition, the hCD147KI-NSG mouse model allows further study of SARS-CoV-2 in the immunodeficiency condition which may assist our understanding of this virus in the context of high-risk populations in immunosuppressed states. Our data show (1) the human CD147 protein is expressed in various organs (including bronchiolar epithelial cells) in hCD147KI-NSG mice by immunohistochemical staining and flow cytometry; (2) hCD147KI-NSG mice are marginally sensitive to SARS-CoV-2 infection compared to WT-NSG littermates characterized by increased viral copies by qRT-PCR and moderate body weight decline compared to baseline; (3) a significant increase in leukocytes in the lungs of hCD147KI-NSG mice, compared to infected WT-NSG mice. CONCLUSIONS: hCD147KI-NSG mice are more sensitive to COVID-19 infection compared to WT-NSG mice. The hCD147KI-NSG mouse model can serve as an additional animal model for further interrogation whether CD147 serve as an independent functional receptor or accessory receptor for SARS-CoV-2 entry and immune responses.

2.
BMC Res Notes ; 15(1): 252, 2022 Jul 15.
Article in English | MEDLINE | ID: covidwho-1938343

ABSTRACT

OBJECTIVE: SARS CoV-2, the etiologic agent of coronavirus disease-2019 (COVID-19) is well-known to use ACE2 to begin internalization. Some viruses enter the host cell through the endocytosis process and involve some endocytosis proteins, such as the Rab family. However, the relationship between SARS CoV-2 infection with endocytic mRNA RAB5, RAB7, and RAB11B is unknown. This study aims to compare the expression of RAB5, RAB7, and RAB11B between positive and negative COVID-19 patient groups. RESULTS: Both viral and human epithelial RNA Isolation and RT-PCR were performed from 249 samples. The genes expression was analysed using appropriate statistical tests. We found the Median (inter-quartile range/IQR) of RAB5, RAB7, and RAB11B expression among the COVID-19 patient group was 2.99 (1.88), 0.17 (0.47), 0.47 (1.49), and 1.60 (2.88), 1.05 (2.49), 1.10 (3.96) among control group respectively. We proceeded with Mann Whitney U Test and found that RAB5 expression was significantly increased (P < 0.001), and RAB7 and RAB11B expression was significantly decreased (P < 0.001 and P = 0.036) in the COVID-19 patient group compared to the control group. This first report showed significant differences in RAB5, RAB7, and RAB11B exist between COVID-19 positive and negative patients.


Subject(s)
COVID-19 , rab5 GTP-Binding Proteins , COVID-19/genetics , Endosomes/metabolism , Gene Expression , Humans , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolism , rab5 GTP-Binding Proteins/genetics , rab5 GTP-Binding Proteins/metabolism , rab7 GTP-Binding Proteins
3.
Microbiol Spectr ; : e0087022, 2022 Jul 11.
Article in English | MEDLINE | ID: covidwho-1938015

ABSTRACT

Severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 have a single envelope glycoprotein (S protein) that binds to human angiotensin-converting enzyme 2 (ACE2) on the host cell membrane. Previous mutational scanning studies have suggested that some substitutions corresponding to single nucleotide variants (SNVs) in human ACE2 affect the binding affinity to the receptor binding domain (RBD) of the SARS-CoV-2 S protein. However, the importance of these substitutions in actual virus infection is still unclear. In this study, we investigated the effects of the reported ACE2 SNV substitutions on the entry of SARS-CoV and SARS-CoV-2 into cells, using vesicular stomatitis Indiana virus (VSIV) pseudotyped with S proteins of these coronaviruses (CoVs). HEK293T cells transfected with plasmids expressing ACE2 having each SNV substitution were infected with the pseudotyped VSIVs and relative infectivities were determined compared to the cells expressing wild-type ACE2. We found that some of the SNV substitutions positively or negatively affected the infectivities of the pseudotyped viruses. Particularly, the H505R substitution significantly enhanced the infection with the pseudotyped VSIVs, including those having the substitutions found in the S protein RBD of SARS-CoV-2 variants of concern. Our findings suggest that human ACE2 SNVs may potentially affect cell susceptibilities to SARS-CoV and SARS-CoV-2. IMPORTANCE SARS-CoV and SARS-CoV-2 are known to cause severe pneumonia in humans. The S protein of these CoVs binds to the ACE2 molecule on the plasma membrane and mediates virus entry into cells. The interaction between the S protein and ACE2 is thought to be important for host susceptibility to these CoVs. Although previous studies suggested that some SNV substitutions in ACE2 might affect the binding to the S protein, it remains elusive whether these SNV substitutions actually alter the efficiency of the entry of SARS CoVs into cells. We analyzed the impact of the ACE2 SNVs on the cellular entry of SARS CoVs using pseudotyped VSIVs having the S protein on the viral surface. We found that some of the SNV substitutions positively or negatively affected the infectivities of the viruses. Our data support the notion that genetic polymorphisms of ACE2 may potentially influence cell susceptibilities to SARS CoVs.

4.
Antimicrob Agents Chemother ; : e0008322, 2022 Jul 05.
Article in English | MEDLINE | ID: covidwho-1938003

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the infectious agent that has caused the current coronavirus disease (COVID) pandemic. Viral infection relies on the viral S (spike) protein/cellular receptor ACE2 interaction. Disrupting this interaction would lead to early blockage of viral replication. To identify chemical tools to further study these functional interfaces, 139,146 compounds from different chemical libraries were screened through an S/ACE2 in silico virtual molecular model. The best compounds were selected for further characterization using both cellular and biochemical approaches, reiterating SARS-CoV-2 entry and the S/ACE2 interaction. We report here two selected hits, bis-indolyl pyridine AB-00011778 and triphenylamine AB-00047476. Both of these compounds can block the infectivity of lentiviral vectors pseudotyped with the SARS-CoV-2 S protein as well as wild-type and circulating variant SARS-CoV-2 strains in various human cell lines, including pulmonary cells naturally susceptible to infection. AlphaLISA and biolayer interferometry confirmed a direct inhibitory effect of these drugs on the S/ACE2 association. A specific study of the AB-00011778 inhibitory properties showed that this drug inhibits viral replication with a 50% effective concentration (EC50) between 0.1 and 0.5 µM depending on the cell lines. Molecular docking calculations of the interaction parameters of the molecules within the S/ACE2 complex from both wild-type and circulating variants of the virus showed that the molecules may target multiple sites within the S/ACE2 interface. Our work indicates that AB-00011778 constitutes a good tool for modulating this interface and a strong lead compound for further therapeutic purposes.

5.
Oral Dis ; 2022 Jul 16.
Article in English | MEDLINE | ID: covidwho-1937980

ABSTRACT

OBJECTIVE: This work aims to determine the efficacy of preprocedural oral rinsing with chlorine dioxide solutions to minimize the risk of coronavirus disease 2019 (COVID-19) transmission during high-risk dental procedures. METHODS: The antiviral activity of chlorine-dioxide-based oral rinse (OR) solutions was tested by pre-incubating with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus in a dosage-dependent manner before transducing to human embryonic kidney epithelial (HEK293T-ACE2) cells, which stably expresses ACE-2 receptor. Viral entry was determined by measuring luciferase activity using a luminescence microplate reader. In the cell-to-cell fusion assay, effector Chinese hamster ovary (CHO-K1) cells co-expressing spike glycoprotein of SARS-CoV-2 and T7 RNA polymerase were pre-incubated with the ORs before co-culturing with the target CHO-K1 cells co-expressing human ACE2 receptor and luciferase gene. The luciferase signal was quantified 24 h after mixing the cells. Surface expression of SARS-CoV-2 spike glycoprotein and ACE-2 receptor was confirmed using direct fluorescent imaging and quantitative cell-ELISA. Finally, dosage-dependent cytotoxic effects of ORs were evaluated at two different time points. RESULTS: A dosage-dependent antiviral effect of the ORs was observed against SARS-CoV-2 cell entry and spike glycoprotein mediated cell-to-cell fusion. This demonstrates that ORs can be useful as a preprocedural step to reduce viral infectivity. CONCLUSIONS: Chlorine-dioxide-based ORs have a potential benefit for reducing SARS-CoV-2 entry and spread.

6.
Future Virol ; 2022 Jun.
Article in English | MEDLINE | ID: covidwho-1928385

ABSTRACT

Aim: Considering the present alarming situation of COVID-19 pandemic, we concentrated on evaluating the efficacy of a novel natural antiviral drug-candidate andrographolide against SARS-CoV-2 through an in silico model of study. Materials & methods: Interaction of andrographolide against the major host molecules that are responsible for SARS-CoV-2 pathogenesis were determined using bio-computational tools, in other words, molecular docking, molecular dynamics simulation and pharmacodynamics-pharmacokinetics analysis. Result: Computational findings represent that andrographolide efficiently interacts with the major human-host-associated putative drug-targets of viral-entry points like furin (-10.54 kcal/mol), TMPRSS-2 (-9.50 kcal/mol), ACE2 (-8.99 kcal/mol) and Cathepsin L (-8.98 kcal/mol). Moreover, it also blocks the inflammatory regulators including TLR4-MD2 and IL-6, which promote virus-induced inflammation leading to cytokine storm in the host body. Conclusion: This work elucidates that, the candidature of andrographolide can be utilized as a potent natural agent for the therapeutic intervention of SARS-CoV-2 through host-directed treatment.

7.
Ind Crops Prod ; 187: 115338, 2022 Nov 01.
Article in English | MEDLINE | ID: covidwho-1926548

ABSTRACT

Carlina acaulis plant is a potential target for the industrial production of phytochemicals that display applicability in pharmacy and medicine. The dry roots of C. acaulis contain up to 2 % of essential oil, the main component (up to 99 %) of which is carlina oxide [2-(3-phenylprop-1-ynyl)furan]. This compound shows multidirectional biological activity, including antibacterial and antifungal properties. Here, we evaluated the capacity of carlina oxide to inhibit the interaction between SARS-CoV-2 and its human receptor in vitro and in silico. A bioluminescent immunoassay was used to study the interaction between the receptor binding domain (RBD) of viral spike protein and the human angiotensin-converting enzyme 2 (ACE2), which serves as a receptor for viral entry. A dose-effect relationship was demonstrated, and a concentration of carlina oxide causing half-maximal inhibition (IC50) of the RBD:ACE2 interaction was determined to be equal to 234.2 µg/mL. Molecular docking suggested the presence of carlina oxide binding sites within the RBD and at the interface between RBD and ACE2. Finally, this study expands the list of potential applications of C. acaulis as a crop species.

8.
Emerg Microbes Infect ; 11(1): 1920-1935, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-1908682

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and related sarbecoviruses enter host cells by receptor-recognition and membrane-fusion. An indispensable step in fusion is the formation of 6-helix bundle by viral spike heptad repeats 1 and 2 (HR1 and HR2). Here, we report the construction of 5-helix bundle (5HB) proteins for virus infection inhibition. The optimal construct inhibits SARS-CoV-2 pseudovirus entry with sub-micromolar IC50. Unlike HR2-based peptides that cannot bind spike in the pre-fusion conformation, 5HB features with the capability of binding to pre-fusion spike. Furthermore, 5HB binds viral HR2 at both serological- and endosomal-pH, highlighting its entry-inhibition capacity when SARS-CoV-2 enters via either cell membrane fusion or endosomal route. Finally, we show that 5HB could neutralize S-mediated entry of the predominant SARS-CoV-2 variants and a wide spectrum of sarbecoviruses. These data provide proof-of-concept evidence that 5HB might be developed for the prevention and treatment of SARS-CoV-2 and other emerging sarbecovirus infections.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Hydrogen-Ion Concentration , Membrane Glycoproteins/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Viral Envelope Proteins/metabolism , Virus Internalization
9.
J Virol ; 96(13): e0047422, 2022 07 13.
Article in English | MEDLINE | ID: covidwho-1891736

ABSTRACT

SARS-CoV-2 spike (S) envelope glycoprotein constitutes the main determinant of virus entry and the target of host immune response, thus being of great interest for antiviral research. It is constituted of S1 and S2 subunits, which are involved in ACE2 receptor binding and fusion between the viral envelope and host cell membrane, respectively. Induction of the fusion process requires S cleavage at the S1-S2 junction and the S2' site located upstream of the fusion peptide. Interestingly, the SARS-CoV-2 spike harbors a 4-residue insertion at the S1-S2 junction that is absent in its closest relatives and constitutes a polybasic motif recognized by furin-like proteases. In addition, the S2' site is characterized by the presence of conserved basic residues. Here, we sought to determine the importance of the furin cleavage site (FCS) and the S2' basic residues for S-mediated entry functions. We determined the impact of mutations introduced at these sites on S processing, fusogenic activity, and its ability to mediate entry in different cellular backgrounds. Strikingly, mutation phenotypes were highly dependent on the host cell background. We confirmed that although the FCS was not absolutely required for virus entry, it contributed to extending the fusogenic potential of S. Cleavage site mutations, as well as inhibition of furin protease activity, affected the cell surface expression of S in a host cell-dependent manner. Finally, inhibition of furin activity differentially affected SARS-CoV-2 virus infectivity in the tested host cells, thereby confirming the host cell-dependent effect of spike processing for the viral life cycle. IMPORTANCE SARS-CoV-2 is responsible for the current global pandemic that has resulted in several million deaths. As the key determinant of virus entry into host cells and the main target of host immune response, the spike glycoprotein constitutes an attractive target for therapeutics development. Entry functions of spike rely on its processing at two sites by host cell proteases. While SARS-CoV-2 spike differs from its closest relatives by the insertion of a basic furin cleavage motif at the first site, it harbors conserved basic residues at the second cleavage site. Characterization of the importance of the basic sequences present at the two cleavage sites revealed that they were influencing spike processing, intracellular localization, induction of fusion, and entry in a host cell-dependent manner. Thus, our results revealed a high heterogeneity in spike sequence requirement for entry functions in the different host cells, in agreement with the high adaptability of the SARS-CoV-2 virus.


Subject(s)
COVID-19 , Furin , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Virus Internalization , COVID-19/virology , Furin/metabolism , Humans , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism
10.
Adv Protein Chem Struct Biol ; 131: 261-276, 2022.
Article in English | MEDLINE | ID: covidwho-1866754

ABSTRACT

Numerous viruses have evolved mechanisms to inhibit or alter the host cell's apoptotic response as part of their coevolution with their hosts. The analysis of virus-host protein interactions require an in-depth understanding of both the viral and host protein structures and repertoires, as well as evolutionary mechanisms and pertinent biological facts. Throughout the course of a viral infection, there is constant battle for binding between virus and cellular proteins. Exogenous interfaces facilitating viral-host interactions are well known for constantly targeting and suppressing endogenous interfaces mediating intraspecific interactions, such as viral-viral and host-host connections. In these interactions, the protein-protein interactions (PPIs), are mostly shown as networks (protein interaction networks, PINs), with proteins represented as nodes and their interactions represented as edges. Host proteins with a higher degree of connectivity are more likely to interact with viral proteins. Due to technical advancements, three-dimensional interactions may now be visualized computationally utilizing molecular modeling and cryo-EM approaches. The uniqueness of viral domain repertoires, their evolution, and their activities during viral infection make viruses fascinating models for research. This chapter aims to provide readers a complete picture of the viral hijacking mechanism in protein-protein interactions.


Subject(s)
Host Microbial Interactions , Viral Proteins , Humans , Viral Proteins/chemistry
11.
Int J Mol Sci ; 23(10)2022 May 17.
Article in English | MEDLINE | ID: covidwho-1862811

ABSTRACT

Coronaviruses, including SARS-CoV-2 (the etiological agent of the current COVID-19 pandemic), rely on the surface spike glycoprotein to access the host cells, mainly through the interaction of their receptor-binding domain (RBD) with the human angiotensin-converting enzyme 2 (ACE2). Therefore, molecular entities able to interfere with the binding of the SARS-CoV-2 spike protein to ACE2 have great potential to inhibit viral entry. Starting from the available structural data on the interaction between SARS-CoV-2 spike protein and the host ACE2 receptor, we engineered a set of soluble and stable spike interactors, here denoted as S-plugs. Starting from the prototype S-plug, we adopted a computational approach by combining stability prediction, associated to single-point mutations, with molecular dynamics to enhance both S-plug thermostability and binding affinity to the spike protein. The best developed molecule, S-plug3, possesses a highly stable α-helical con-formation (with melting temperature Tm of 54 °C) and can interact with the spike RBD and S1 domains with similar low nanomolar affinities. Importantly, S-plug3 exposes the spike RBD to almost the same interface as the human ACE2 receptor, aimed at the recognition of all ACE2-accessing coronaviruses. Consistently, S-plug3 preserves a low nanomolar dissociation constant with the delta B.1.617.2 variant of SARS-CoV-2 spike protein (KD = 29.2 ± 0.6 nM). Taken together, we provide valid starting data for the development of therapeutical and diagnostic tools against coronaviruses accessing through ACE2.


Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Angiotensin-Converting Enzyme 2/genetics , Humans , Membrane Glycoproteins/metabolism , Pandemics , Peptidyl-Dipeptidase A/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Viral Envelope Proteins/chemistry
12.
Comput Biol Med ; 146: 105625, 2022 07.
Article in English | MEDLINE | ID: covidwho-1850905

ABSTRACT

The outbreak of COVID-19 has resulted in millions of deaths. Despite all attempts that have been made to combat the pandemic, the re-emergence of new variants complicated SARS-CoV-2 eradication. The ongoing global spread of COVID-19 demands the incessant development of novel agents in vaccination, diagnosis, and therapeutics. Targeting receptor-binding domain (RBD) of spike protein by which the virus identifies host receptor, angiotensin-converting enzyme (ACE2), is a promising strategy for curbing viral infection. This study aims to discover novel peptide inhibitors against SARS-CoV-2 entry using computational approaches. The RBD binding domain of ACE2 was extracted and docked against the RBD. MMPBSA calculations revealed the binding energies of each residue in the template. The residues with unfavorable binding energies were considered as mutation spots by OSPREY. Binding energies of the residues in RBD-ACE2 interface was determined by molecular docking. Peptide inhibitors were designed by the mutation of RBD residues in the virus-receptors complex which had unfavorable energies. Peptide tendency for RBD binding, safety, and allergenicity were the criteria based on which the final hits were screened among the initial library. Molecular dynamics simulations also provided information on the mechanisms of inhibitory action in peptides. The results were finally validated by molecular docking simulations to make sure the peptides are capable of hindering virus-host interaction. Our results introduce three peptides P7 (RAWTFLDKFNHEAEDLRYQSSLASWN), P13 (RASTFLDKFNHEAEDLRYQSSLASWN), and P19 (RADTFLDKFNHEAEDLRYQSSLASWN) as potential effective inhibitors of SARS-CoV-2 entry which could be considered in drug development for COVID-19 treatment.


Subject(s)
COVID-19 , SARS-CoV-2 , Angiotensin-Converting Enzyme 2 , Binding Sites , COVID-19/drug therapy , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Mutation , Peptides/metabolism , Peptides/pharmacology , Protein Binding , Spike Glycoprotein, Coronavirus/chemistry
13.
Front Microbiol ; 13: 884034, 2022.
Article in English | MEDLINE | ID: covidwho-1847188

ABSTRACT

Since the outbreak of the coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), public health worldwide has been greatly threatened. The development of an effective treatment for this infection is crucial and urgent but is hampered by the incomplete understanding of the viral infection mechanisms and the lack of specific antiviral agents. We previously reported that teicoplanin, a glycopeptide antibiotic that has been commonly used in the clinic to treat bacterial infection, significantly restrained the cell entry of Ebola virus, SARS-CoV, and MERS-CoV by specifically inhibiting the activity of cathepsin L (CTSL). Here, we found that the cleavage sites of CTSL on the spike proteins of SARS-CoV-2 were highly conserved among all the variants. The treatment with teicoplanin suppressed the proteolytic activity of CTSL on spike and prevented the cellular infection of different pseudotyped SARS-CoV-2 viruses. Teicoplanin potently prevented the entry of SARS-CoV-2 into the cellular cytoplasm with an IC50 of 2.038 µM for the Wuhan-Hu-1 reference strain and an IC50 of 2.116 µM for the SARS-CoV-2 (D614G) variant. The pre-treatment of teicoplanin also prevented SARS-CoV-2 infection in hACE2 mice. In summary, our data reveal that CTSL is required for both SARS-CoV-2 and SARS-CoV infection and demonstrate the therapeutic potential of teicoplanin for universal anti-CoVs intervention.

14.
Cell Rep ; 39(7): 110829, 2022 May 17.
Article in English | MEDLINE | ID: covidwho-1814236

ABSTRACT

We report that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta spike mutation P681R plays a key role in the Alpha-to-Delta variant replacement during the coronavirus disease 2019 (COVID-19) pandemic. Delta SARS-CoV-2 efficiently outcompetes the Alpha variant in human lung epithelial cells and primary human airway tissues. The Delta spike mutation P681R is located at a furin cleavage site that separates the spike 1 (S1) and S2 subunits. Reverting the P681R mutation to wild-type P681 significantly reduces the replication of the Delta variant to a level lower than the Alpha variant. Mechanistically, the Delta P681R mutation enhances the cleavage of the full-length spike to S1 and S2, which could improve cell-surface-mediated virus entry. In contrast, the Alpha spike also has a mutation at the same amino acid (P681H), but the cleavage of the Alpha spike is reduced compared with the Delta spike. Our results suggest P681R as a key mutation in enhancing Delta-variant replication via increased S1/S2 cleavage.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/genetics , Humans , Mutation/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
15.
Adv Exp Med Biol ; 1366: 137-153, 2022.
Article in English | MEDLINE | ID: covidwho-1782744

ABSTRACT

With the increasing global human population, travel, and socioeconomic activities, more and more novel pathogenic viruses will emerge or re-emerge. While more than 260 viruses are known to infect humans, only a small minority of these viral diseases are treatable by clinically approved antiviral drugs. Apart from these identified viruses, new emerging viruses and drug-resistant viruses are also important challenges to our public health and healthcare systems. The COVID-19 and influenza pandemics remind us the importance of getting broad-spectrum antivirals against emerging and re-emerging respiratory viruses. Broad-spectrum antivirals against different viral families for fighting the currently known viruses and novel emerging viruses are urgently needed. Viral entry is the universal first step for viral infection, and therefore is a promising target for identifying broad-spectrum antivirals. In this chapter, we mainly focus on discussing the risks of respiratory viruses, the challenge of finding broad-spectrum antivirals, the entry processes of respiratory viruses, the current studies on broad-spectrum entry inhibitors for respiratory viruses, and the directions for discovering broad-spectrum antivirals in the future.


Subject(s)
COVID-19 , Virus Diseases , Viruses , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Humans , Virus Diseases/drug therapy , Virus Internalization
16.
Cells ; 11(8)2022 04 12.
Article in English | MEDLINE | ID: covidwho-1785542

ABSTRACT

Salvia miltiorrhiza Bunge, commonly called danshen, is widely used in traditional Chinese medicine for its cardiovascular and neuroprotective effects, which include antioxidative, anti-inflammatory, and antifibrotic properties. The purpose of this study was to evaluate the preclinical potential of S. miltiorrhiza extracts for the treatment of COVID-19. First, the impact of the extract on the binding between SARS-CoV-2 and the cellular ACE2 receptors was assessed using atomic force microscopy (AFM), showing a significant reduction in binding by the extract at concentrations in the µg/mL range. Second, the interference of this extract with the inflammatory response of blood mononuclear cells (PBMCs) was determined, demonstrating potent inhibitory properties in the same concentration range on pro-inflammatory cytokine release and interference with the activation of NFκB signaling. Together, these in vitro data demonstrate the potential of S. miltiorrhiza against COVID-19, consisting first of the blockade of the binding of SARS-CoV-2 to the ACE2 receptor and the mitigation of the inflammatory response from leukocytes by interfering with NFκB signaling. This dataset prompts the launch of a clinical trial to address in vivo the clinical benefits of this promising agent.


Subject(s)
COVID-19 , Salvia miltiorrhiza , Angiotensin-Converting Enzyme 2 , COVID-19/drug therapy , Medicine, Chinese Traditional , NF-kappa B , SARS-CoV-2 , Salvia miltiorrhiza/chemistry
17.
Int J Mol Sci ; 23(7)2022 Apr 06.
Article in English | MEDLINE | ID: covidwho-1776252

ABSTRACT

Entry inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed to control the outbreak of coronavirus disease 2019 (COVID-19). This study developed a robust and straightforward assay that detected the molecular interaction between the receptor-binding domain (RBD) of viral spike protein and the angiotensin-converting enzyme 2 (ACE2) receptor in just 10 min. A drug library of 1068 approved compounds was used to screen for SARS-CoV2 entry inhibition, and 9 active drugs were identified as specific pseudovirus entry inhibitors. A plaque reduction neutralization test using authentic SARS-CoV-2 virus in Vero E6 cells confirmed that 2 of these drugs (Etravirine and Dolutegravir) significantly inhibited the infection of SARS-CoV-2. With molecular docking, we showed that both Etravirine and Dolutegravir are preferentially bound to primary ACE2-interacting residues on the RBD domain, implying that these two drug blocks may prohibit the viral attachment of SARS-CoV-2. We compared the neutralizing activities of these entry inhibitors against different pseudoviruses carrying spike proteins from alpha, beta, gamma, and delta variants. Both Etravirine and Dolutegravir showed similar neutralizing activities against different variants, with EC50 values between 4.5 to 5.8 nM for Etravirine and 10.2 to 22.9 nM for Dolutegravir. These data implied that Etravirine and Dolutegravir may serve as general spike inhibitors against dominant viral variants of SARS-CoV-2.


Subject(s)
COVID-19 , SARS-CoV-2 , Angiotensin-Converting Enzyme 2 , COVID-19/drug therapy , Humans , Molecular Docking Simulation , RNA, Viral , Spike Glycoprotein, Coronavirus/metabolism
18.
J Virol ; 96(8): e0012822, 2022 04 27.
Article in English | MEDLINE | ID: covidwho-1765079

ABSTRACT

The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directs infection of the lungs and other tissues following its binding to the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2'. The "priming" of the surface S protein at S1/S2 (PRRAR685↓) [the underlined basic amino acids refer to critical residues needed for the furin recognition] by furin has been shown to be important for SARS-CoV-2 infectivity in cells and small-animal models. In this study, for the first time we unambiguously identified by proteomics the fusion activation site S2' as KPSKR815↓ (the underlined basic amino acids refer to critical residues needed for the furin recognition) and demonstrated that this cleavage was strongly enhanced by ACE2 engagement with the S protein. Novel pharmacological furin inhibitors (BOS inhibitors) effectively blocked endogenous S protein processing at both sites in HeLa cells, and SARS-CoV-2 infection of lung-derived Calu-3 cells was completely prevented by combined inhibitors of furin (BOS) and type II transmembrane serine protease 2 (TMPRSS2) (camostat). Quantitative analyses of cell-to-cell fusion and S protein processing revealed that ACE2 shedding by TMPRSS2 was required for TMPRSS2-mediated enhancement of fusion in the absence of S1/S2 priming. We further demonstrated that the collectrin dimerization domain of ACE2 was essential for the effect of TMPRSS2 on cell-to-cell fusion. Overall, our results indicate that furin and TMPRSS2 act synergistically in viral entry and infectivity, supporting the combination of furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2. IMPORTANCE SARS-CoV-2, the etiological agent of COVID-19, has so far resulted in >6.1 million deaths worldwide. The spike protein (S) of the virus directs infection of the lungs and other tissues by binding the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2'. Cleavage at S1/S2 induces a conformational change favoring the S protein recognition by ACE2. The S2' cleavage is critical for triggering membrane fusion and virus entry into host cells. Our study highlights the complex dynamics of interaction between the S protein, ACE2, and the host proteases furin and TMPRSS2 during SARS-CoV-2 entry and suggests that the combination of a nontoxic furin inhibitor with a TMPRSS2 inhibitor significantly reduces viral entry in lung cells, as evidenced by an average synergistic ∼95% reduction of viral infection. This represents a powerful novel antiviral approach to reduce viral spread in individuals infected by SARS-CoV-2 or future related coronaviruses.


Subject(s)
COVID-19 , Furin , SARS-CoV-2 , Serine Endopeptidases , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/pathology , COVID-19/virology , Furin/metabolism , HeLa Cells , Humans , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
19.
Virology ; 569: 13-28, 2022 04.
Article in English | MEDLINE | ID: covidwho-1740261

ABSTRACT

Emerging mutations in the SARS-CoV-2 genome pose a challenge for vaccine development and antiviral therapy. The antiviral efficacy of Azadirachta indica bark extract (NBE) was assessed against SARS-CoV-2 and m-CoV-RSA59 infection. Effects of in vivo intranasal or oral NBE administration on viral load, inflammatory response, and histopathological changes were assessed in m-CoV-RSA59-infection. NBE administered inhibits SARS-CoV-2 and m-CoV-RSA59 infection and replication in vitro, reducing Envelope and Nucleocapsid gene expression. NBE ameliorates neuroinflammation and hepatitis in vivo by restricting viral replication and spread. Isolated fractions of NBE enriched in Nimbin isomers shows potent inhibition of m-CoV-RSA59 infection in vitro. In silico studies revealed that NBE could target Spike and RdRp of m-CoV and SARS-CoV-2 with high affinity. NBE has a triterpenoids origin that may allow them to competitively target panoply of viral proteins to inhibit mouse and different strains of human coronavirus infections, suggesting its potential as an antiviral against pan-ß-Coronaviruses.


Subject(s)
Azadirachta , COVID-19 , Animals , Antiviral Agents/pharmacology , COVID-19/drug therapy , Limonins , Mice , Plant Bark , Plant Extracts/pharmacology , SARS-CoV-2 , Virus Replication
20.
mBio ; 13(2): e0300221, 2022 04 26.
Article in English | MEDLINE | ID: covidwho-1714353

ABSTRACT

Emerging zoonotic viral pathogens threaten global health, and there is an urgent need to discover host and viral determinants influencing infection. We performed a loss-of-function genome-wide CRISPR screen in a human lung cell line using HCoV-OC43, a human betacoronavirus. One candidate gene, VPS29, a component of the retromer complex, was required for infection by HCoV-OC43, SARS-CoV-2, other endemic- and pandemic-threat coronaviruses, as well as ebolavirus. Notably, we observed a heightened requirement for VPS29 by the recently described Omicron variant of SARS-CoV-2 compared to the ancestral variant. However, VPS29 deficiency had no effect on certain other viruses that enter cells via endosomes and had an opposing, enhancing effect on influenza A virus infection. Deficiency in VPS29 or other retromer components caused changes in endosome morphology and acidity and attenuated the activity of endosomal proteases. These changes in endosome properties caused incoming coronavirus, but not influenza virus particles, to become entrapped therein. Overall, these data show how host regulation of endosome characteristics can influence cellular susceptibility to viral infection and identify a host pathway that could serve as a pharmaceutical target for intervention in zoonotic viral diseases. IMPORTANCE These data identify a host pathway by which VPS29 and associated factors control the endosomal environment in a manner that influences susceptibility to viral infection. This pathway could serve as a pharmaceutical target for intervention in zoonotic viral diseases, including those caused by coronaviruses, influenza viruses, and filoviruses, all of which are pandemic threats. Our findings show how host regulation of endosome characteristics can influence viral susceptibility in both a positive and negative manner.


Subject(s)
COVID-19 , Coronavirus OC43, Human , Influenza A virus , Humans , Influenza A virus/physiology , Pharmaceutical Preparations , SARS-CoV-2 , Vesicular Transport Proteins , Virus Internalization
SELECTION OF CITATIONS
SEARCH DETAIL