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1.
J Endocrinol Invest ; 44(12): 2675-2684, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1504521

ABSTRACT

PURPOSE: Due to relevant repercussions on reproductive medicine, we aimed to evaluate feasibility of RT-PCR as a detection method of SARS-CoV-2 RNA in seminal fluid. METHODS: A qualitative determination of the RT-PCR assays in semen was performed through different approaches: (1) efficiency of RNA extraction from sperm and seminal plasma was determined using PRM1 and PRM2 mRNA and a heterologous system as control; (2) samples obtained by diluting viral preparation from a SARS-CoV-2 panel (virus cultured in Vero E6 cell lines) were tested; (3) viral presence in different fractions of seminal fluid (whole sample, seminal plasma and post-centrifugation pellet) was evaluated. Semen samples from mild and recovered COVID-19 subjects were collected by patients referring to the Infectious Disease Department of the Policlinico Umberto I Hospital - "Sapienza" University of Rome. Control subjects were recruited at the Laboratory of Seminology-Sperm Bank "Loredana Gandini'' of the same hospital. RESULTS: The control panel using viral preparations diluted in saline and seminal fluid showed the capability to detect viral RNA presence with Ct values depending on the initial viral concentration. All tested semen samples were negative for SARS-CoV-2, regardless of the nasopharyngeal swab result or seminal fluid fraction. CONCLUSION: These preliminary data show that RT-PCR for SARS-CoV-2 RNA testing appears to be a feasible method for the molecular diagnosis of SARS-CoV-2 in seminal fluid, supported by results of the control panel. The ability to detect SARS-CoV-2 in semen is extremely important for reproductive medicine, especially in assisted reproductive technology and sperm cryopreservation.


Subject(s)
COVID-19/diagnosis , Pathology, Molecular/methods , Semen/virology , Adult , Animals , Chlorocebus aethiops , Feasibility Studies , Humans , Male , RNA, Messenger/chemistry , RNA, Viral/chemistry , Real-Time Polymerase Chain Reaction , Reproductive Techniques , Vero Cells
2.
NPJ Vaccines ; 6(1): 83, 2021 Jun 17.
Article in English | MEDLINE | ID: covidwho-1387359

ABSTRACT

An array of SARS-CoV-2 virus variants have been isolated, propagated and used in in vitro assays, in vivo animal studies and human clinical trials. Observations of working stocks of SARS-CoV-2 suggest that sequential propagation in Vero cells leads to critical changes in the region of the furin cleavage site, which significantly reduce the value of the working stock for critical research studies. Serially propagating SARS-CoV-2 in Vero E6 cells leads to rapid increases in genetic variants while propagation in other cell lines (e.g. Vero/hSLAM) appears to mitigate this risk thereby improving the overall genetic stability of working stocks. From these observations, investigators are urged to monitor genetic variants carefully when propagating SARS-CoV-2 in Vero cells.

3.
Methods Mol Biol ; 2305: 129-140, 2021.
Article in English | MEDLINE | ID: covidwho-1355903

ABSTRACT

The expression of mammalian recombinant proteins in insect cell lines using transient-plasmid-based gene expression enables the production of high-quality protein samples. Here, the procedure for virus-free transient gene expression (TGE) in High Five insect cells is described in detail. The parameters that determine the efficiency and reproducibility of the method are presented in a robust protocol for easy implementation and set-up of the method. The applicability of the TGE method in High Five cells for proteomic, structural, and functional analysis of the expressed proteins is shown.


Subject(s)
Biotechnology/methods , Cloning, Molecular , Insecta/metabolism , Spike Glycoprotein, Coronavirus/biosynthesis , Transfection/methods , Animals , Bioreactors , Cell Culture Techniques/methods , Cell Line , Gene Expression , Glycosylation , Humans , Insecta/cytology , Mammals/genetics , Mammals/metabolism , Plasmids , Proteomics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Reproducibility of Results , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics
4.
Methods Mol Biol ; 2099: 3-8, 2020.
Article in English | MEDLINE | ID: covidwho-1292543

ABSTRACT

Forced viral adaptation is a powerful technique employed to study the ways viruses may overcome various selective pressures that reduce viral replication. Here, we describe methods for in vitro serial passaging of Middle East respiratory syndrome coronavirus (MERS-CoV) to select for mutations which increase replication on semi-permissive cell lines as described in Letko et al., Cell Rep 24, 1730-1737, 2018.


Subject(s)
Adaptation, Physiological/genetics , Coronavirus Infections/virology , Middle East Respiratory Syndrome Coronavirus/genetics , Virus Replication/genetics , Animals , Biological Evolution , Cell Line , Chlorocebus aethiops , Host Specificity , Humans , Middle East Respiratory Syndrome Coronavirus/physiology , Serial Passage , Vero Cells
5.
Am J Pathol ; 191(7): 1193-1208, 2021 07.
Article in English | MEDLINE | ID: covidwho-1283899

ABSTRACT

Pulmonary fibrosis (PF) can arise from unknown causes, as in idiopathic PF, or as a consequence of infections, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current treatments for PF slow, but do not stop, disease progression. We report that treatment with a runt-related transcription factor 1 (RUNX1) inhibitor (Ro24-7429), previously found to be safe, although ineffective, as a Tat inhibitor in patients with HIV, robustly ameliorates lung fibrosis and inflammation in the bleomycin-induced PF mouse model. RUNX1 inhibition blunted fundamental mechanisms downstream pathologic mediators of fibrosis and inflammation, including transforming growth factor-ß1 and tumor necrosis factor-α, in cultured lung epithelial cells, fibroblasts, and vascular endothelial cells, indicating pleiotropic effects. RUNX1 inhibition also reduced the expression of angiotensin-converting enzyme 2 and FES Upstream Region (FURIN), host proteins critical for SARS-CoV-2 infection, in mice and in vitro. A subset of human lungs with SARS-CoV-2 infection overexpress RUNX1. These data suggest that RUNX1 inhibition via repurposing of Ro24-7429 may be beneficial for PF and to battle SARS-CoV-2, by reducing expression of viral mediators and by preventing respiratory complications.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , Core Binding Factor Alpha 2 Subunit/antagonists & inhibitors , Furin/metabolism , Lung/drug effects , Pulmonary Fibrosis/drug therapy , Animals , Bleomycin , Cells, Cultured , Disease Models, Animal , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Female , Lung/metabolism , Lung/pathology , Male , Mice , Pulmonary Fibrosis/chemically induced , Pulmonary Fibrosis/pathology , Treatment Outcome
6.
Biotechniques ; 71(1): 370-375, 2021 07.
Article in English | MEDLINE | ID: covidwho-1278249

ABSTRACT

Inactivation of SARS-CoV-2 virus is necessary to mitigate risk but may interfere with diagnostic assay performance. We examined the effect of heat inactivation on a prototype SARS-CoV-2 antigen immunoassay run on the ARCHITECT automated analyzer. Recombinant full-length SARS-CoV-2 nucleocapsid protein and virus lysate detection was reduced by 66 and 31%, respectively. Several nonionic detergents were assessed as inactivation alternatives based on infectivity in cultured Vero CCL81 cells. Incubation of SARS-CoV-2 in 0.1% Tergitol 15-S-9 for 10 min significantly reduced infectivity and increased the immunoassay signal for cultured lysate and patient specimens. Tergitol 15-S-9 can inactivate SARS-CoV-2 while preserving epitopes on the nucleocapsid protein for enhanced detection by immunoassay antibodies.


Subject(s)
COVID-19 Testing/methods , Poloxalene/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/immunology , Virus Inactivation/drug effects , Animals , Antibodies, Viral/drug effects , Antibodies, Viral/immunology , COVID-19/diagnosis , COVID-19/immunology , COVID-19 Testing/standards , Cells, Cultured , Chlorocebus aethiops , Humans , Immunoassay/methods , Immunoassay/standards , Nucleocapsid/immunology , Surface-Active Agents/pharmacology , Vero Cells
7.
Viruses ; 13(6)2021 05 21.
Article in English | MEDLINE | ID: covidwho-1244140

ABSTRACT

Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19), enters cells through attachment to the human angiotensin converting enzyme 2 (hACE2) via the receptor-binding domain (RBD) in the surface/spike (S) protein. Several pseudotyped viruses expressing SARS-CoV-2 S proteins are available, but many of these can only infect hACE2-overexpressing cell lines. Here, we report the use of a simple, two-plasmid, pseudotyped virus system comprising a SARS-CoV-2 spike-expressing plasmid and an HIV vector with or without vpr to investigate the SARS-CoV-2 entry event in various cell lines. When an HIV vector without vpr was used, pseudotyped SARS-CoV-2 viruses produced in the presence of fetal bovine serum (FBS) were able to infect only engineered hACE2-overexpressing cell lines, whereas viruses produced under serum-free conditions were able to infect a broader range of cells, including cells without hACE2 overexpression. When an HIV vector containing vpr was used, pseudotyped viruses were able to infect a broad spectrum of cell types regardless of whether viruses were produced in the presence or absence of FBS. Infection sensitivities of various cell types did not correlate with mRNA abundance of hACE2, TMPRSS2, or TMPRSS4. Pseudotyped SARS-CoV-2 viruses and replication-competent SARS-CoV-2 virus were equally sensitive to neutralization by an anti-spike RBD antibody in cells with high abundance of hACE2. However, the anti-spike RBD antibody did not block pseudotyped viral entry into cell lines with low abundance of hACE2. We further found that CD147 was involved in viral entry in A549 cells with low abundance of hACE2. Thus, our assay is useful for drug and antibody screening as well as for investigating cellular receptors, including hACE2, CD147, and tyrosine-protein kinase receptor UFO (AXL), for the SARS-CoV-2 entry event in various cell lines.


Subject(s)
HIV/genetics , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/physiology , Virus Internalization , Caco-2 Cells , Cell Line , Genetic Vectors , HEK293 Cells , Humans , Plasmids , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , Transfection , vpr Gene Products, Human Immunodeficiency Virus/metabolism
8.
Am J Pathol ; 191(7): 1193-1208, 2021 07.
Article in English | MEDLINE | ID: covidwho-1242859

ABSTRACT

Pulmonary fibrosis (PF) can arise from unknown causes, as in idiopathic PF, or as a consequence of infections, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current treatments for PF slow, but do not stop, disease progression. We report that treatment with a runt-related transcription factor 1 (RUNX1) inhibitor (Ro24-7429), previously found to be safe, although ineffective, as a Tat inhibitor in patients with HIV, robustly ameliorates lung fibrosis and inflammation in the bleomycin-induced PF mouse model. RUNX1 inhibition blunted fundamental mechanisms downstream pathologic mediators of fibrosis and inflammation, including transforming growth factor-ß1 and tumor necrosis factor-α, in cultured lung epithelial cells, fibroblasts, and vascular endothelial cells, indicating pleiotropic effects. RUNX1 inhibition also reduced the expression of angiotensin-converting enzyme 2 and FES Upstream Region (FURIN), host proteins critical for SARS-CoV-2 infection, in mice and in vitro. A subset of human lungs with SARS-CoV-2 infection overexpress RUNX1. These data suggest that RUNX1 inhibition via repurposing of Ro24-7429 may be beneficial for PF and to battle SARS-CoV-2, by reducing expression of viral mediators and by preventing respiratory complications.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , Core Binding Factor Alpha 2 Subunit/antagonists & inhibitors , Furin/metabolism , Lung/drug effects , Pulmonary Fibrosis/drug therapy , Animals , Bleomycin , Cells, Cultured , Disease Models, Animal , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Female , Lung/metabolism , Lung/pathology , Male , Mice , Pulmonary Fibrosis/chemically induced , Pulmonary Fibrosis/pathology , Treatment Outcome
9.
Cell Discov ; 7(1): 37, 2021 May 24.
Article in English | MEDLINE | ID: covidwho-1241945

ABSTRACT

Treatment options for COVID-19 remain limited, especially during the early or asymptomatic phase. Here, we report a novel SARS-CoV-2 viral replication mechanism mediated by interactions between ACE2 and the epigenetic eraser enzyme LSD1, and its interplay with the nuclear shuttling importin pathway. Recent studies have shown a critical role for the importin pathway in SARS-CoV-2 infection, and many RNA viruses hijack this axis to re-direct host cell transcription. LSD1 colocalized with ACE2 at the cell surface to maintain demethylated SARS-CoV-2 spike receptor-binding domain lysine 31 to promote virus-ACE2 interactions. Two newly developed peptide inhibitors competitively inhibited virus-ACE2 interactions, and demethylase access to significantly inhibit viral replication. Similar to some other predominantly plasma membrane proteins, ACE2 had a novel nuclear function: its cytoplasmic domain harbors a nuclear shuttling domain, which when demethylated by LSD1 promoted importin-α-dependent nuclear ACE2 entry following infection to regulate active transcription. A novel, cell permeable ACE2 peptide inhibitor prevented ACE2 nuclear entry, significantly inhibiting viral replication in SARS-CoV-2-infected cell lines, outperforming other LSD1 inhibitors. These data raise the prospect of post-exposure prophylaxis for SARS-CoV-2, either through repurposed LSD1 inhibitors or new, nuclear-specific ACE2 inhibitors.

10.
mSphere ; 6(3)2021 05 12.
Article in English | MEDLINE | ID: covidwho-1230164

ABSTRACT

Human coronavirus (HCoV)-OC43 rarely shows a cytopathic effect (CPE) after infection of various cell lines, and the indirect immunoperoxidase assay (IPA), a relatively complex procedure, has long been used as an alternative assay. Because HCoV-OC43 uses cell-surface transmembrane protease serine 2 (TMPRSS2) for cell entry, VeroE6 cells expressing TMPRSS2 may show a clear CPE after HCoV-OC43 infection. The aim of this study was to construct a 50% tissue culture infectious dose (TCID50) assay for HCoV-OC43 based on CPE evaluation using VeroE6/TMPRSS2 cells. VeroE6/TMPRSS2 cells showed clear CPEs 3 to 4 days after low-titer HCoV-OC43 infection. Evaluation of viral kinetics indicated that the viral titer in the culture supernatant of VeroE6/TMPRSS2 cells in the early stages of infection was higher than that of other cells. In comparison, between the CPE-based and the IPA-based (i.e., the reference titer) methods, the titer measured with CPE evaluation 4 to 5 days after infection using VeroE6/TMPRSS2 cells showed a much smaller difference from the reference titer than that measured using other cells. Thus, the TCID50 assay using CPE evaluation with VeroE6/TMPRSS2 cells provides the correct titer value and will greatly contribute to future research on HCoV-OC43.IMPORTANCE HCoV-OC43 rarely shows a cytopathic effect (CPE) in infected cell lines, and thus the plaque and TCID50 assays by CPE observation are not applicable for titration; the indirect immunoperoxidase assay (IPA) is used instead. However, the IPA is relatively complex, time-consuming, costly, and not suitable for simultaneous titration of many samples. We developed a TCID50 assay using CPE evaluation with TMPRSS2-expressing VeroE6/TMPRSS2 cells that provides the same accuracy as the conventional IPA-based viral titration and does not require any staining procedures using antibodies or substrates. This titration method will greatly contribute to future research on HCoV-OC43 by allowing simple, low-cost, and accurate titration of this virus.


Subject(s)
Coronavirus OC43, Human/physiology , Cytopathogenic Effect, Viral , Receptors, Virus/metabolism , Serine Endopeptidases/metabolism , Viral Load/methods , Animals , Cell Line, Tumor , Chlorocebus aethiops , Coronavirus OC43, Human/isolation & purification , Humans , Immunoenzyme Techniques , Receptors, Virus/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Serine Endopeptidases/genetics , Vero Cells/virology , Virus Cultivation , Virus Internalization , Virus Replication
11.
Proc Natl Acad Sci U S A ; 118(21)2021 05 25.
Article in English | MEDLINE | ID: covidwho-1220249

ABSTRACT

Prolonged detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and recurrence of PCR-positive tests have been widely reported in patients after recovery from COVID-19, but some of these patients do not appear to shed infectious virus. We investigated the possibility that SARS-CoV-2 RNAs can be reverse-transcribed and integrated into the DNA of human cells in culture and that transcription of the integrated sequences might account for some of the positive PCR tests seen in patients. In support of this hypothesis, we found that DNA copies of SARS-CoV-2 sequences can be integrated into the genome of infected human cells. We found target site duplications flanking the viral sequences and consensus LINE1 endonuclease recognition sequences at the integration sites, consistent with a LINE1 retrotransposon-mediated, target-primed reverse transcription and retroposition mechanism. We also found, in some patient-derived tissues, evidence suggesting that a large fraction of the viral sequences is transcribed from integrated DNA copies of viral sequences, generating viral-host chimeric transcripts. The integration and transcription of viral sequences may thus contribute to the detection of viral RNA by PCR in patients after infection and clinical recovery. Because we have detected only subgenomic sequences derived mainly from the 3' end of the viral genome integrated into the DNA of the host cell, infectious virus cannot be produced from the integrated subgenomic SARS-CoV-2 sequences.


Subject(s)
COVID-19/genetics , COVID-19/virology , SARS-CoV-2/genetics , Virus Integration/genetics , Animals , COVID-19/metabolism , Chlorocebus aethiops , Genome, Viral , HEK293 Cells , Humans , RNA, Viral/genetics , SARS-CoV-2/metabolism , Vero Cells , Virus Integration/physiology , Virus Replication/genetics , Virus Replication/physiology
12.
Phytomedicine ; 87: 153583, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1213465

ABSTRACT

BACKGROUND: A key clinical feature of COVID-19 is a deep inflammatory state known as "cytokine storm" and characterized by high expression of several cytokines, chemokines and growth factors, including IL-6 and IL-8. A direct consequence of this inflammatory state in the lungs is the Acute Respiratory Distress Syndrome (ARDS), frequently observed in severe COVID-19 patients. The "cytokine storm" is associated with severe forms of COVID-19 and poor prognosis for COVID-19 patients. Sulforaphane (SFN), one of the main components of Brassica oleraceae L. (Brassicaceae or Cruciferae), is known to possess anti-inflammatory effects in tissues from several organs, among which joints, kidneys and lungs. PURPOSE: The objective of the present study was to determine whether SFN is able to inhibit IL-6 and IL-8, two key molecules involved in the COVID-19 "cytokine storm". METHODS: The effects of SFN were studied in vitro on bronchial epithelial IB3-1 cells exposed to the SARS-CoV-2 Spike protein (S-protein). The anti-inflammatory activity of SFN on IL-6 and IL-8 expression has been evaluated by RT-qPCR and Bio-Plex analysis. RESULTS: In our study SFN inhibits, in cultured IB3-1 bronchial cells, the gene expression of IL-6 and IL-8 induced by the S-protein of SARS-CoV-2. This represents the proof-of-principle that SFN may modulate the release of some key proteins of the COVID-19 "cytokine storm". CONCLUSION: The control of the cytokine storm is one of the major issues in the management of COVID-19 patients. Our study suggests that SFN can be employed in protocols useful to control hyperinflammatory state associated with SARS-CoV-2 infection.


Subject(s)
Bronchi/virology , Interleukin-6/genetics , Interleukin-8/genetics , Isothiocyanates/pharmacology , Spike Glycoprotein, Coronavirus/toxicity , Sulfoxides/pharmacology , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Apoptosis/drug effects , Bronchi/cytology , Bronchi/drug effects , COVID-19/physiopathology , Cell Line , Chemokines/genetics , Chemokines/metabolism , Cytokine Release Syndrome/drug therapy , Cytokine Release Syndrome/metabolism , Gene Expression Regulation/drug effects , Humans , SARS-CoV-2/pathogenicity , Up-Regulation/drug effects
13.
Emerg Infect Dis ; 27(5): 1380-1392, 2021 05.
Article in English | MEDLINE | ID: covidwho-1202277

ABSTRACT

Co-infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other viruses has been reported. We evaluated cell lines commonly used to isolate viruses and diagnose related diseases for their susceptibility to SARS-CoV-2. Although multiple kidney cell lines from monkeys were susceptible to SARS-CoV-2, we found many cell types derived from humans, dogs, minks, cats, mice, and chicken were not. We analyzed MDCK cells, which are most commonly used for surveillance and study of influenza viruses, and found that they were not susceptible to SARS-CoV-2. The low expression level of the angiotensin converting enzyme 2 receptor and lower receptor affinity to SARS-CoV-2 spike, which could be overcome by overexpression of canine angiotensin converting enzyme 2 in trans, strengthened the cellular barrier to productive infection. Moreover, a D614G mutation in the spike protein did not appear to affect SARS-CoV-2 cell tropism. Our findings should help avert inadvertent propagation of SARS-CoV-2 from diagnostic cell lines.


Subject(s)
COVID-19 , Influenza, Human , Animals , Cats , Cell Line , Dogs , Humans , Mice , Peptidyl-Dipeptidase A , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics
14.
Proc Natl Acad Sci U S A ; 118(17)2021 04 27.
Article in English | MEDLINE | ID: covidwho-1172591

ABSTRACT

In order to understand the transmission and virulence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is necessary to understand the functions of each of the gene products encoded in the viral genome. One feature of the SARS-CoV-2 genome that is not present in related, common coronaviruses is ORF10, a putative 38-amino acid protein-coding gene. Proteomic studies found that ORF10 binds to an E3 ubiquitin ligase containing Cullin-2, Rbx1, Elongin B, Elongin C, and ZYG11B (CRL2ZYG11B). Since CRL2ZYG11B mediates protein degradation, one possible role for ORF10 is to "hijack" CRL2ZYG11B in order to target cellular, antiviral proteins for ubiquitylation and subsequent proteasomal degradation. Here, we investigated whether ORF10 hijacks CRL2ZYG11B or functions in other ways, for example, as an inhibitor or substrate of CRL2ZYG11B While we confirm the ORF10-ZYG11B interaction and show that the N terminus of ORF10 is critical for it, we find no evidence that ORF10 is functioning to inhibit or hijack CRL2ZYG11B Furthermore, ZYG11B and its paralog ZER1 are dispensable for SARS-CoV-2 infection in cultured cells. We conclude that the interaction between ORF10 and CRL2ZYG11B is not relevant for SARS-CoV-2 infection in vitro.


Subject(s)
COVID-19/metabolism , Cell Cycle Proteins/metabolism , Cullin Proteins/metabolism , Multiprotein Complexes/metabolism , Open Reading Frames , SARS-CoV-2/metabolism , Viral Proteins/metabolism , COVID-19/genetics , Cell Cycle Proteins/genetics , Cullin Proteins/genetics , HEK293 Cells , Humans , Multiprotein Complexes/genetics , SARS-CoV-2/genetics , Viral Proteins/genetics
15.
Front Genet ; 12: 599261, 2021.
Article in English | MEDLINE | ID: covidwho-1167316

ABSTRACT

Analyzing host cells' transcriptional response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection will help delineate biological processes underlying viral pathogenesis. First, analysis of expression profiles of lung cell lines A549 and Calu3 revealed upregulation of antiviral interferon signaling genes in response to all three SARS-CoV-2, MERS-CoV, or influenza A virus (IAV) infections. However, perturbations in expression of genes involved in inflammatory, mitochondrial, and autophagy processes were specifically observed in SARS-CoV-2-infected cells. Next, a validation study in infected human nasopharyngeal samples also revealed perturbations in autophagy and mitochondrial processes. Specifically, mTOR expression, mitochondrial ribosomal, mitochondrial complex I, lysosome acidification, and mitochondrial fission promoting genes were concurrently downregulated in both infected cell lines and human samples. SARS-CoV-2 infection impeded autophagic flux either by upregulating GSK3B in lung cell lines or by downregulating autophagy genes, SNAP29, and lysosome acidification genes in human samples, contributing to increased viral replication. Therefore, drugs targeting lysosome acidification or autophagic flux could be tested as intervention strategies. Finally, age-stratified SARS-CoV-2-positive human data revealed impaired upregulation of chemokines, interferon-stimulated genes, and tripartite motif genes that are critical for antiviral signaling. Together, this analysis has revealed specific aspects of autophagic and mitochondrial function that are uniquely perturbed in SARS-CoV-2-infected host cells.

16.
ACS Infect Dis ; 7(7): 1985-1995, 2021 07 09.
Article in English | MEDLINE | ID: covidwho-1157889

ABSTRACT

As the toll of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic continues, efforts are ongoing to identify new agents and repurpose safe drugs for its treatment. Antimalarial peroxides have reported antiviral and anticancer activities. Here, we evaluated the in vitro activities of artesunate (AS) and two ozonides (OZ418 and OZ277) against human α-coronavirus NL63 and ß-coronaviruses OC43 and SARS-CoV-2 in several cell lines. OZ418 had the best selectivity index (SI) in NL63-infected Vero cells and MK2 cells. The overall SI of the tested compounds was cell-type dependent. In OC43-infected human foreskin fibroblasts, AS had the best cell-associated SI, ≥17 µM, while the SI of OZ418 and OZ277 was ≥12 µM and ≥7 µM, respectively. AS did not inhibit SARS-CoV-2 in either Vero or Calu-3 cells. A comparison of OZ418 and OZ277 activity in SARS-CoV2-infected Calu-3 cells revealed similar EC50 (5.3 µM and 11.6 µM, respectively), higher than the EC50 of remdesivir (1.0 ± 0.1 µM), but the SI of OZ418 was higher than OZ277. A third ozonide, OZ439, inhibited SARS-CoV-2 efficiently in Vero cells, but compared to OZ418 in Calu-3 cells, it showed higher toxicity. Improved inhibition of SARS-CoV-2 was observed when OZ418 was used together with remdesivir. Although the EC50 of ozonides might be clinically achieved in plasma after intravenous administration, sustained virus suppression in tissues will require further considerations, including drug combination. Our work supports the potential repurposing of ozonides and calls for future in vivo models.


Subject(s)
Antimalarials , COVID-19 , Animals , Antimalarials/pharmacology , Chlorocebus aethiops , Humans , Peroxides/pharmacology , RNA, Viral , SARS-CoV-2 , Vero Cells
17.
Int J Mol Sci ; 22(6)2021 Mar 19.
Article in English | MEDLINE | ID: covidwho-1143519

ABSTRACT

The development of effective antiviral drugs targeting the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is urgently needed to combat the coronavirus disease 2019 (COVID-19). We have previously studied the use of semi-synthetic derivatives of oxysterols, oxidized derivatives of cholesterol as drug candidates for the inhibition of cancer, fibrosis, and bone regeneration. In this study, we screened a panel of naturally occurring and semi-synthetic oxysterols for anti-SARS-CoV-2 activity using a cell culture infection assay. We show that the natural oxysterols, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 27-hydroxycholesterol, substantially inhibited SARS-CoV-2 propagation in cultured cells. Among semi-synthetic oxysterols, Oxy210 and Oxy232 displayed more robust anti-SARS-CoV-2 activities, reducing viral replication more than 90% at 10 µM and 99% at 15 µM, respectively. When orally administered in mice, peak plasma concentrations of Oxy210 fell into a therapeutically relevant range (19 µM), based on the dose-dependent curve for antiviral activity in our cell-based assay. Mechanistic studies suggest that Oxy210 reduced replication of SARS-CoV-2 by disrupting the formation of double-membrane vesicles (DMVs); intracellular membrane compartments associated with viral replication. Our study warrants further evaluation of Oxy210 and Oxy232 as a safe and reliable oral medication, which could help protect vulnerable populations with increased risk of developing COVID-19.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Oxysterols/chemistry , Oxysterols/pharmacology , SARS-CoV-2/drug effects , Administration, Oral , Animals , Antiviral Agents/administration & dosage , Antiviral Agents/pharmacokinetics , COVID-19/drug therapy , Cell Survival/drug effects , Chlorocebus aethiops , Mice , Nucleocapsid Proteins/drug effects , Oxysterols/administration & dosage , Oxysterols/pharmacokinetics , SARS-CoV-2/genetics , Vero Cells , Viral Replication Compartments/drug effects , Virus Replication/drug effects
18.
Front Immunol ; 12: 637654, 2021.
Article in English | MEDLINE | ID: covidwho-1138709

ABSTRACT

A coronavirus SARS-CoV-2, which has caused the pandemic viral pneumonia disease COVID-19, significantly threatens global public health, highlighting the need to develop effective and safe vaccines against its infection. In this study, we developed a novel DNA vaccine candidate against SARS-CoV-2 by expressing a chimeric protein of its receptor-binding domain (RBD) fused to a 33-bp sequence (11 aa) from the hepatitis B virus (HBV) preS1 region with a W4P mutation (W4P-RBD) at the N-terminal region and evaluated its immunogenicity. In vitro transfection experiments in multiple cell lines demonstrated that W4P-RBD vs. wild-type RBD protein (W-RBD) led to enhanced production of IL-6 and TNFα at the transcription and translation levels, suggesting the adjuvant potential of N-terminal HBV preS1 sequences for DNA vaccines against SARS-CoV-2. W4P-RBD also led to enhanced production of IgG and IgA, which can neutralize and block SARS-CoV-2 infection in both blood sera and bronchoalveolar lavage (BAL) fluid from the lung in vaccinated mice. Additionally, W4P-RBD led to an enhanced T-cell-mediated cellular immune response under S1 protein stimulation. In summary, W4P-RBD led to robust humoral and cell-mediated immune responses against SARS-CoV-2 in vaccinated mice, highlighting its feasibility as a novel DNA vaccine to protect against SARS-CoV-2 infection.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/prevention & control , Hepatitis B Surface Antigens/genetics , Hepatitis B Surface Antigens/immunology , Mutation , Protein Domains/immunology , Protein Precursors/genetics , Protein Precursors/immunology , Recombinant Fusion Proteins/immunology , SARS-CoV-2/immunology , Vaccines, DNA/immunology , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19/virology , Cell Line, Tumor , Chlorocebus aethiops , HEK293 Cells , Humans , Immunogenicity, Vaccine , Male , Mice , Mice, Inbred C57BL , Vaccination/methods , Vero Cells
19.
Mol Ther Methods Clin Dev ; 21: 161-170, 2021 Jun 11.
Article in English | MEDLINE | ID: covidwho-1126997

ABSTRACT

Engineered red blood cells (RBCs) expressing viral receptors could be used therapeutically as viral traps, as RBCs lack nuclei and other organelles required for viral replication. However, expression of viral receptors on RBCs is difficult to achieve since mature erythrocytes lack the cellular machinery to synthesize proteins. Herein, we show that the combination of a powerful erythroid-specific expression system and transgene codon optimization yields high expression levels of the HIV-1 receptors CD4 and CCR5, as well as a CD4-glycophorin A (CD4-GpA) fusion protein in erythroid progenitor cells, which efficiently differentiated into enucleated RBCs. HIV-1 efficiently entered RBCs that co-expressed CD4 and CCR5, but viral entry was not required for neutralization, as CD4 or CD4-GpA expression in the absence of CCR5 was sufficient to potently neutralize HIV-1 and prevent infection of CD4+ T cells in vitro due to the formation of high-avidity interactions with trimeric HIV-1 Env spikes on virions. To facilitate continuous large-scale production of RBC viral traps, we generated erythroblast cell lines stably expressing CD4-GpA or ACE2-GpA fusion proteins, which produced potent RBC viral traps against HIV-1 and SARS-CoV-2. Our in vitro results suggest that this approach warrants further investigation as a potential treatment against acute and chronic viral infections.

20.
J Mol Cell Biol ; 13(3): 175-184, 2021 07 06.
Article in English | MEDLINE | ID: covidwho-1123315

ABSTRACT

Since chloroquine (CQ) and hydroxychloroquine (HCQ) can inhibit the invasion and proliferation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in cultured cells, the repurposing of these antimalarial drugs was considered a promising strategy for treatment and prevention of coronavirus disease (COVID-19). However, despite promising preliminary findings, many clinical trials showed neither significant therapeutic nor prophylactic benefits of CQ and HCQ against COVID-19. Here, we aim to answer the question of why these drugs are not effective against the disease by examining the cellular working mechanisms of CQ and HCQ in prevention of SARS-CoV-2 infections.


Subject(s)
COVID-19/drug therapy , Chloroquine/therapeutic use , Hydroxychloroquine/therapeutic use , Antiviral Agents/therapeutic use , COVID-19/virology , Cell Proliferation/drug effects , Chloroquine/adverse effects , Drug Repositioning , Humans , Hydroxychloroquine/adverse effects , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity
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