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1.
J Virol ; 95(24): e0136821, 2021 11 23.
Article in English | MEDLINE | ID: covidwho-1691427

ABSTRACT

Severe cardiovascular complications can occur in coronavirus disease of 2019 (COVID-19) patients. Cardiac damage is attributed mostly to the aberrant host response to acute respiratory infection. However, direct infection of cardiac tissue by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also occurs. We examined here the cardiac tropism of SARS-CoV-2 in spontaneously beating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). These cardiomyocytes express the angiotensin-converting enzyme 2 (ACE2) receptor but not the transmembrane protease serine 2 (TMPRSS2) that mediates spike protein cleavage in the lungs. Nevertheless, SARS-CoV-2 infection of hiPSC-CMs was prolific; viral transcripts accounted for about 88% of total mRNA. In the cytoplasm of infected hiPSC-CMs, smooth-walled exocytic vesicles contained numerous 65- to 90-nm particles with canonical ribonucleocapsid structures, and virus-like particles with knob-like spikes covered the cell surface. To better understand how SARS-CoV-2 spreads in hiPSC-CMs, we engineered an expression vector coding for the spike protein with a monomeric emerald-green fluorescent protein fused to its cytoplasmic tail (S-mEm). Proteolytic processing of S-mEm and the parental spike were equivalent. Live cell imaging tracked spread of S-mEm cell-to-cell and documented formation of syncytia. A cell-permeable, peptide-based molecule that blocks the catalytic site of furin and furin-like proteases abolished cell fusion. A spike mutant with the single amino acid change R682S that disrupts the multibasic furin cleavage motif was fusion inactive. Thus, SARS-CoV-2 replicates efficiently in hiPSC-CMs and furin, and/or furin-like-protease activation of its spike protein is required for fusion-based cytopathology. This hiPSC-CM platform enables target-based drug discovery in cardiac COVID-19. IMPORTANCE Cardiac complications frequently observed in COVID-19 patients are tentatively attributed to systemic inflammation and thrombosis, but viral replication has occasionally been confirmed in cardiac tissue autopsy materials. We developed an in vitro model of SARS-CoV-2 spread in myocardium using induced pluripotent stem cell-derived cardiomyocytes. In these highly differentiated cells, viral transcription levels exceeded those previously documented in permissive transformed cell lines. To better understand the mechanisms of SARS-CoV-2 spread, we expressed a fluorescent version of its spike protein that allowed us to characterize a fusion-based cytopathic effect. A mutant of the spike protein with a single amino acid mutation in the furin/furin-like protease cleavage site lost cytopathic function. Of note, the fusion activities of the spike protein of other coronaviruses correlated with the level of cardiovascular complications observed in infections with the respective viruses. These data indicate that SARS-CoV-2 may cause cardiac damage by fusing cardiomyocytes.


Subject(s)
COVID-19/virology , Myocytes, Cardiac/virology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolism , Animals , Cathepsin B/metabolism , Cell Fusion , Chlorocebus aethiops , Embryonic Stem Cells/metabolism , Exocytosis , Humans , Induced Pluripotent Stem Cells/metabolism , Microscopy, Confocal , Serine Endopeptidases/metabolism , Vero Cells , Viral Proteins/metabolism , Virus Internalization , Virus Replication
2.
J Virol ; 95(24): e0136821, 2021 11 23.
Article in English | MEDLINE | ID: covidwho-1455676

ABSTRACT

Severe cardiovascular complications can occur in coronavirus disease of 2019 (COVID-19) patients. Cardiac damage is attributed mostly to the aberrant host response to acute respiratory infection. However, direct infection of cardiac tissue by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also occurs. We examined here the cardiac tropism of SARS-CoV-2 in spontaneously beating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). These cardiomyocytes express the angiotensin-converting enzyme 2 (ACE2) receptor but not the transmembrane protease serine 2 (TMPRSS2) that mediates spike protein cleavage in the lungs. Nevertheless, SARS-CoV-2 infection of hiPSC-CMs was prolific; viral transcripts accounted for about 88% of total mRNA. In the cytoplasm of infected hiPSC-CMs, smooth-walled exocytic vesicles contained numerous 65- to 90-nm particles with canonical ribonucleocapsid structures, and virus-like particles with knob-like spikes covered the cell surface. To better understand how SARS-CoV-2 spreads in hiPSC-CMs, we engineered an expression vector coding for the spike protein with a monomeric emerald-green fluorescent protein fused to its cytoplasmic tail (S-mEm). Proteolytic processing of S-mEm and the parental spike were equivalent. Live cell imaging tracked spread of S-mEm cell-to-cell and documented formation of syncytia. A cell-permeable, peptide-based molecule that blocks the catalytic site of furin and furin-like proteases abolished cell fusion. A spike mutant with the single amino acid change R682S that disrupts the multibasic furin cleavage motif was fusion inactive. Thus, SARS-CoV-2 replicates efficiently in hiPSC-CMs and furin, and/or furin-like-protease activation of its spike protein is required for fusion-based cytopathology. This hiPSC-CM platform enables target-based drug discovery in cardiac COVID-19. IMPORTANCE Cardiac complications frequently observed in COVID-19 patients are tentatively attributed to systemic inflammation and thrombosis, but viral replication has occasionally been confirmed in cardiac tissue autopsy materials. We developed an in vitro model of SARS-CoV-2 spread in myocardium using induced pluripotent stem cell-derived cardiomyocytes. In these highly differentiated cells, viral transcription levels exceeded those previously documented in permissive transformed cell lines. To better understand the mechanisms of SARS-CoV-2 spread, we expressed a fluorescent version of its spike protein that allowed us to characterize a fusion-based cytopathic effect. A mutant of the spike protein with a single amino acid mutation in the furin/furin-like protease cleavage site lost cytopathic function. Of note, the fusion activities of the spike protein of other coronaviruses correlated with the level of cardiovascular complications observed in infections with the respective viruses. These data indicate that SARS-CoV-2 may cause cardiac damage by fusing cardiomyocytes.


Subject(s)
COVID-19/virology , Myocytes, Cardiac/virology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolism , Animals , Cathepsin B/metabolism , Cell Fusion , Chlorocebus aethiops , Embryonic Stem Cells/metabolism , Exocytosis , Humans , Induced Pluripotent Stem Cells/metabolism , Microscopy, Confocal , Serine Endopeptidases/metabolism , Vero Cells , Viral Proteins/metabolism , Virus Internalization , Virus Replication
3.
ACS Chem Neurosci ; 12(5): 930-944, 2021 03 03.
Article in English | MEDLINE | ID: covidwho-1091527

ABSTRACT

The COVID-19 pandemic caused by SARS-CoV-2 represents a global public health emergency. The entry of SARS-CoV-2 into host cells requires the activation of its spike protein by host cell proteases. The serine protease, TMPRSS2, and cysteine proteases, Cathepsins B/L, activate spike protein and enable SARS-CoV-2 entry to the host cell through two completely different and independent pathways. Therefore, inhibiting either TMPRSS2 or cathepsin B/L may not sufficiently block the virus entry. We here hypothesized that simultaneous targeting of both the entry pathways would be more efficient to block the virus entry rather than targeting the entry pathways individually. To this end, we utilized the network-based drug repurposing analyses to identify the possible common drugs that can target both the entry pathways. This study, for the first time, reports the molecules like cyclosporine, calcitriol, and estradiol as candidate drugs with the binding ability to the host proteases, TMPRSS2, and cathepsin B/L. Next, we analyzed drug-gene and gene-gene interaction networks using 332 human targets of SARS-CoV-2 proteins. The network results indicate that, out of 332 human proteins, cyclosporine interacts with 216 (65%) proteins. Furthermore, we performed molecular docking and all-atom molecular dynamics (MD) simulations to explore the binding of drug with TMPRSS2 and cathepsin L. The molecular docking and MD simulation results showed strong and stable binding of cyclosporine A (CsA) with TMPRSS2 and CTSL genes. The above results indicate cyclosporine as a potential drug molecule, as apart from interacting with SARS-CoV-2 entry receptors, it also interacts with most of SARS-CoV-2 target host genes; thus it could potentially interfere with functions of SARS-CoV-2 proteins in human cells. We here also suggest that these antiviral drugs alone or in combination can simultaneously target both the entry pathways and thus can be considered as a potential treatment option for COVID-19.


Subject(s)
COVID-19/virology , Cyclosporine/pharmacology , Immunosuppressive Agents/pharmacology , SARS-CoV-2/drug effects , Virus Internalization/drug effects , Antiviral Agents/pharmacology , Cathepsin B/metabolism , Cathepsin L/metabolism , Drug Repositioning , Humans , Models, Molecular , Molecular Docking Simulation , Molecular Dynamics Simulation , Pandemics , Serine Endopeptidases/metabolism
4.
Viruses ; 12(7)2020 07 03.
Article in English | MEDLINE | ID: covidwho-636175

ABSTRACT

Chikungunya virus (CHIKV) is an enveloped virus that enters host cells and transits within the endosomes before starting its replication cycle, the precise mechanism of which is yet to be elucidated. Endocytosis and endosome acidification inhibitors inhibit infection by CHIKV, murine leukemia virus (MLV), or SARS-coronavirus, indicating that these viral entries into host cells occur through endosomes and require endosome acidification. Although endosomal cathepsin B protease is necessary for MLV, Ebola virus, and SARS-CoV infections, its role in CHIKV infection is unknown. Our results revealed that endocytosis inhibitors attenuated CHIKV-pseudotyped MLV vector infection in 293T cells but not in TE671 cells. In contrast, macropinocytosis inhibitors attenuated CHIKV-pseudotyped MLV vector infection in TE671 cells but not in 293T cells, suggesting that CHIKV host cell entry occurs via endocytosis or macropinocytosis, depending on the cell lines used. Cathepsin B inhibitor and knockdown by an shRNA suppressed CHIKV-pseudotyped MLV vector infection both in 293T and TE671 cells. These results show that cathepsin B facilitates CHIKV infection regardless of the entry pathway.


Subject(s)
Cathepsin B/metabolism , Chikungunya Fever/pathology , Chikungunya virus/physiology , Viral Envelope Proteins/metabolism , Virus Internalization , Cathepsin B/antagonists & inhibitors , Cell Line, Tumor , Endocytosis/physiology , Endosomes/virology , HEK293 Cells , HeLa Cells , Humans , Leukemia Virus, Murine/physiology , Pinocytosis/physiology , RNA Interference , RNA, Small Interfering/genetics
6.
Signal Transduct Target Ther ; 5(1): 121, 2020 07 08.
Article in English | MEDLINE | ID: covidwho-640304
7.
Int J Antimicrob Agents ; 55(6): 106004, 2020 Jun.
Article in English | MEDLINE | ID: covidwho-155306

ABSTRACT

SARS-coronavirus 2 is the causal agent of the COVID-19 outbreak. SARS-Cov-2 entry into a cell is dependent upon binding of the viral spike (S) protein to cellular receptor and on cleavage of the spike protein by the host cell proteases such as Cathepsin L and Cathepsin B. CTSL/B are crucial elements of lysosomal pathway and both enzymes are almost exclusively located in the lysosomes. CTSL disruption offers potential for CoVID-19 therapies. The mechanisms of disruption include: decreasing expression of CTSL, direct inhibition of CTSL activity and affecting the conditions of CTSL environment (increase pH in the lysosomes). We have conducted a high throughput drug screen gene expression analysis to identify compounds that would downregulate the expression of CTSL/CTSB. One of the top significant results shown to downregulate the expression of the CTSL gene is amantadine (10uM). Amantadine was approved by the US Food and Drug Administration in 1968 as a prophylactic agent for influenza and later for Parkinson's disease. It is available as a generic drug. Amantadine in addition to downregulating CTSL appears to further disrupt lysosomal pathway, hence, interfering with the capacity of the virus to replicate. It acts as a lysosomotropic agent altering the CTSL functional environment.  We hypothesize that amantadine could decrease the viral load in SARS-CoV-2 positive patients and as such it may serve as a potent therapeutic decreasing the replication and infectivity of the virus likely leading to better clinical outcomes. Clinical studies will be needed to examine the therapeutic utility of amantadine in COVID-19 infection.


Subject(s)
Amantadine/therapeutic use , Antiviral Agents/therapeutic use , Betacoronavirus/drug effects , Cathepsin L/metabolism , Coronavirus Infections/drug therapy , Pneumonia, Viral/drug therapy , COVID-19 , Cathepsin B/metabolism , Cathepsin L/genetics , Cell Line , Down-Regulation/drug effects , Humans , Lysosomes/metabolism , Pandemics , SARS-CoV-2
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