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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.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-320927

ABSTRACT

Viruses spread between hosts through particles, but within hosts, viral genomes can spread from cell to cell through fusion, evading antiviral defenses and obviating costly infectious virion production1-3. Billions of electromechanically coupled cardiomyocytes (CMs) make myocardium inherently vulnerable to pathological electromechanical short circuits caused by intercellular viral spread 4-6. Beyond respiratory illness, COVID-19 affects the heart7 and cardiac injury and arrhythmias are serious public health concerns8-12. By studying myocardium of a young woman who died suddenly, diagnosed postmortem with COVID-19, we discovered highly focal myocardial SARS-CoV-2 infection spreading from one CM to another through intercellular junctions identified by highly concentrated sarcolemmal t-tubule viral spike glycoprotein. SARS-CoV-2 permissively infected beating human induced pluripotent stem cell (hiPSC)-CMs building multinucleated cardiomyotubes (CMTs) through cell type-specific fusion driven by proteolytically-activated spike glycoprotein. Recombinant spike glycoprotein, co-localizing to sarcolemma and sarcoplasmic reticulum, produced multinucleated CMTs with pathological structure, electrophysiology and Ca2+ excitation-contraction coupling. Blocking cleavage, a peptide-based protease inhibitor neutralized SARS-CoV-2 spike glycoprotein pathogenicity. We conclude that SARS-CoV-2 spike glycoprotein, efficiently primed, activated and strategically poised during biosynthesis, can exploit the CM’s inherent membranous connectivities to drive heart damage directly, uncoupling clinically common myocardial injury from lymphocytic myocarditis, often suspected but rarely confirmed in COVID-19.

3.
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
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