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1.
Signal Transduct Target Ther ; 7(1): 23, 2022 01 25.
Article in English | MEDLINE | ID: covidwho-1655541
2.
Proc Natl Acad Sci U S A ; 119(5)2022 02 01.
Article in English | MEDLINE | ID: covidwho-1642084

ABSTRACT

Novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants pose a challenge to controlling the COVID-19 pandemic. Previous studies indicate that clinical samples collected from individuals infected with the Delta variant may contain higher levels of RNA than previous variants, but the relationship between levels of viral RNA and infectious virus for individual variants is unknown. We measured infectious viral titer (using a microfocus-forming assay) and total and subgenomic viral RNA levels (using RT-PCR) in a set of 162 clinical samples containing SARS-CoV-2 Alpha, Delta, and Epsilon variants that were collected in identical swab kits from outpatient test sites and processed soon after collection. We observed a high degree of variation in the relationship between viral titers and RNA levels. Despite this, the overall infectivity differed among the three variants. Both Delta and Epsilon had significantly higher infectivity than Alpha, as measured by the number of infectious units per quantity of viral E gene RNA (5.9- and 3.0-fold increase; P < 0.0001, P = 0.014, respectively) or subgenomic E RNA (14.3- and 6.9-fold increase; P < 0.0001, P = 0.004, respectively). In addition to higher viral RNA levels reported for the Delta variant, the infectivity (amount of replication competent virus per viral genome copy) may be increased compared to Alpha. Measuring the relationship between live virus and viral RNA is an important step in assessing the infectivity of novel SARS-CoV-2 variants. An increase in the infectivity for Delta may further explain increased spread, suggesting a need for increased measures to prevent viral transmission.


Subject(s)
COVID-19/epidemiology , Gene Expression Regulation, Viral , Genome, Viral , RNA, Viral/genetics , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Animals , COVID-19/pathology , COVID-19/transmission , COVID-19/virology , Cell Line, Tumor , Chlorocebus aethiops , Coronavirus Envelope Proteins/genetics , Coronavirus Envelope Proteins/metabolism , Hepatocytes/metabolism , Hepatocytes/virology , Humans , RNA, Viral/metabolism , SARS-CoV-2/classification , SARS-CoV-2/metabolism , Vero Cells , Viral Load , Virulence
3.
PLoS Pathog ; 17(9): e1009840, 2021 09.
Article in English | MEDLINE | ID: covidwho-1403328

ABSTRACT

COVID-19 vaccines based on the Spike protein of SARS-CoV-2 have been developed that appear to be largely successful in stopping infection. However, therapeutics that can help manage the disease are still required until immunity has been achieved globally. The identification of repurposed drugs that stop SARS-CoV-2 replication could have enormous utility in stemming the disease. Here, using a nano-luciferase tagged version of the virus (SARS-CoV-2-ΔOrf7a-NLuc) to quantitate viral load, we evaluated a range of human cell types for their ability to be infected and support replication of the virus, and performed a screen of 1971 FDA-approved drugs. Hepatocytes, kidney glomerulus, and proximal tubule cells were particularly effective in supporting SARS-CoV-2 replication, which is in-line with reported proteinuria and liver damage in patients with COVID-19. Using the nano-luciferase as a measure of virus replication we identified 35 drugs that reduced replication in Vero cells and human hepatocytes when treated prior to SARS-CoV-2 infection and found amodiaquine, atovaquone, bedaquiline, ebastine, LY2835219, manidipine, panobinostat, and vitamin D3 to be effective in slowing SARS-CoV-2 replication in human cells when used to treat infected cells. In conclusion, our study has identified strong candidates for drug repurposing, which could prove powerful additions to the treatment of COVID.


Subject(s)
COVID-19/drug therapy , Drug Discovery/methods , Drug Repositioning , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Animals , Biomarkers , Cell Line , Chlorocebus aethiops , Hepatocytes/virology , Humans , Luciferases/pharmacology , Nanostructures , SARS-CoV-2/genetics , Vero Cells , Virus Replication/drug effects
4.
Signal Transduct Target Ther ; 5(1): 221, 2020 10 06.
Article in English | MEDLINE | ID: covidwho-1387195
6.
J Biol Chem ; 296: 100111, 2021.
Article in English | MEDLINE | ID: covidwho-1066049

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a ß-coronavirus, is the causative agent of the COVID-19 pandemic. Like for other coronaviruses, its particles are composed of four structural proteins: spike (S), envelope (E), membrane (M), and nucleoprotein (N) proteins. The involvement of each of these proteins and their interactions are critical for assembly and production of ß-coronavirus particles. Here, we sought to characterize the interplay of SARS-CoV-2 structural proteins during the viral assembly process. By combining biochemical and imaging assays in infected versus transfected cells, we show that E and M regulate intracellular trafficking of S as well as its intracellular processing. Indeed, the imaging data reveal that S is relocalized at endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) or Golgi compartments upon coexpression of E or M, as observed in SARS-CoV-2-infected cells, which prevents syncytia formation. We show that a C-terminal retrieval motif in the cytoplasmic tail of S is required for its M-mediated retention in the ERGIC, whereas E induces S retention by modulating the cell secretory pathway. We also highlight that E and M induce a specific maturation of N-glycosylation of S, independently of the regulation of its localization, with a profile that is observed both in infected cells and in purified viral particles. Finally, we show that E, M, and N are required for optimal production of virus-like-particles. Altogether, these results highlight how E and M proteins may influence the properties of S proteins and promote the assembly of SARS-CoV-2 viral particles.


Subject(s)
Coronavirus Envelope Proteins/genetics , Nucleocapsid Proteins/genetics , SARS-CoV-2/growth & development , Spike Glycoprotein, Coronavirus/genetics , Viral Matrix Proteins/genetics , Virion/growth & development , Virus Assembly/physiology , Animals , Biomimetic Materials/chemistry , Biomimetic Materials/metabolism , Cell Line, Tumor , Chlorocebus aethiops , Coronavirus Envelope Proteins/metabolism , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/ultrastructure , Endoplasmic Reticulum/virology , Gene Expression , Golgi Apparatus/metabolism , Golgi Apparatus/ultrastructure , Golgi Apparatus/virology , HEK293 Cells , Hepatocytes/metabolism , Hepatocytes/ultrastructure , Hepatocytes/virology , Host-Pathogen Interactions/genetics , Humans , Nucleocapsid Proteins/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Viral Matrix Proteins/metabolism , Virion/genetics , Virion/metabolism , Virus Internalization , Virus Release/physiology
7.
Arch Toxicol ; 94(12): 4037-4041, 2020 12.
Article in English | MEDLINE | ID: covidwho-716276

ABSTRACT

Besides lung drastic involvement, SARS-CoV-2 severely affected other systems including liver. Emerging epidemiological studies brought the attentions towards liver injury and impairment as a potential outcome of COVID19. Angiotensin-converting enzyme 2 (ACE2) and Transmembrane serine protease (TMPRSS2) are the main cell entry receptors of SARS-CoV-2. We have tested the ability of medications to regulate expression of SARS-CoV-2 receptors. Understanding that may reflect how such medications may affect the level of infectivity and permissibility of the liver following COVID-19. Using transcriptomic datasets, Toxicogenomic Project-Genomics Assisted Toxicity Evaluation System (Open TG-GATEs) and GSE30351, we have tested the ability of ninety common medications to regulate COVID-19 receptors expression in human primary hepatocytes. Most medications displayed a dose-dependent change in expression of receptors which could hint at a potentially more pronounced change with chronic use. The expression level of TMPRSS2 was increased noticeably with a number of medications such as metformin. Within the analgesics, acetaminophen revealed a dose-dependent reduction in expression of ACE2, while non-steroidal anti-inflammatory drugs had mixed effect on receptors expression. To confirm the observed effects on primary human hepatocytes, rat hepatocyte treatments data was obtained from DrugMatrix toxicogenomic database (GSE57805), which showed a similar ACE2 and TMPRSS2 expression pattern. Treatment of common co-morbidities often require chronic use of multiple medications, which may result in an additive increase in the expression of ACE2 and TMPRSS2. More research is needed to determine the effect of different medications on COVID-19 receptors.


Subject(s)
Betacoronavirus/pathogenicity , Hepatocytes/drug effects , Hepatocytes/virology , Peptidyl-Dipeptidase A/genetics , Serine Endopeptidases/genetics , Virus Internalization/drug effects , Acetaminophen/administration & dosage , Acetaminophen/pharmacology , Angiotensin-Converting Enzyme 2 , Animals , COVID-19 , Cells, Cultured , Coronavirus Infections/therapy , Dose-Response Relationship, Drug , Griseofulvin/pharmacology , Host-Pathogen Interactions/drug effects , Humans , Hypoglycemic Agents/pharmacology , Liver/cytology , Liver/virology , Pandemics , Pneumonia, Viral/therapy , Rats , SARS-CoV-2
8.
Cell Stem Cell ; 27(1): 125-136.e7, 2020 07 02.
Article in English | MEDLINE | ID: covidwho-610467

ABSTRACT

SARS-CoV-2 has caused the COVID-19 pandemic. There is an urgent need for physiological models to study SARS-CoV-2 infection using human disease-relevant cells. COVID-19 pathophysiology includes respiratory failure but involves other organ systems including gut, liver, heart, and pancreas. We present an experimental platform comprised of cell and organoid derivatives from human pluripotent stem cells (hPSCs). A Spike-enabled pseudo-entry virus infects pancreatic endocrine cells, liver organoids, cardiomyocytes, and dopaminergic neurons. Recent clinical studies show a strong association with COVID-19 and diabetes. We find that human pancreatic beta cells and liver organoids are highly permissive to SARS-CoV-2 infection, further validated using adult primary human islets and adult hepatocyte and cholangiocyte organoids. SARS-CoV-2 infection caused striking expression of chemokines, as also seen in primary human COVID-19 pulmonary autopsy samples. hPSC-derived cells/organoids provide valuable models for understanding the cellular responses of human tissues to SARS-CoV-2 infection and for disease modeling of COVID-19.


Subject(s)
Betacoronavirus/physiology , Coronavirus Infections/virology , Induced Pluripotent Stem Cells/metabolism , Models, Biological , Organoids/virology , Pneumonia, Viral/virology , Tropism , Angiotensin-Converting Enzyme 2 , Animals , Autopsy , COVID-19 , Cell Line , Coronavirus Infections/pathology , Hepatocytes/pathology , Hepatocytes/virology , Humans , Induced Pluripotent Stem Cells/virology , Liver/pathology , Mice , Pancreas/pathology , Pancreas/virology , Pandemics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/pathology , SARS-CoV-2 , Virus Internalization
9.
J Med Virol ; 92(10): 2096-2104, 2020 10.
Article in English | MEDLINE | ID: covidwho-379986

ABSTRACT

The micro-neutralization assay is a fundamental test in virology, immunology, vaccine assessment, and epidemiology studies. Since the SARS-CoV-2 outbreak at the end of December 2019 in China, it has become extremely important to have well-established and validated diagnostic and serological assays for this new emerging virus. Here, we present a micro-neutralization assay with the use of SARS-CoV-2 wild type virus with two different methods of read-out. We evaluated the performance of this assay using human serum samples taken from an Italian seroepidemiological study being performed at the University of Siena, along with the human monoclonal antibody CR3022 and some iper-immune animal serum samples against Influenza and Adenovirus strains. The same panel of human samples have been previously tested in enzyme-linked immunosorbent assay (ELISA) as a pre-screening. Positive, borderline, and negative ELISA samples were evaluated in neutralization assay using two different methods of read-out: subjective (by means of an inverted optical microscope) and objective (by means of a spectrophotometer). Our findings suggest that at least 50% of positive ELISA samples are positive in neutralization as well, and that method is able to quantify different antibody concentrations in a specific manner. Taken together, our results confirm that the colorimetric cytopathic effect-based microneutralization assay could be used as a valid clinical test method for epidemiological and vaccine studies.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19/diagnosis , Colorimetry/standards , Microscopy/standards , Neutralization Tests/standards , SARS-CoV-2/immunology , Animals , Antibodies, Monoclonal/analysis , COVID-19/immunology , COVID-19/virology , Cell Line, Tumor , Chlorocebus aethiops , Colorimetry/methods , Enzyme-Linked Immunosorbent Assay , Hepatocytes/immunology , Hepatocytes/virology , Humans , Immune Sera/chemistry , Microscopy/methods , Spectrophotometry , Vero Cells , Viral Load/immunology
10.
Antiviral Res ; 175: 104706, 2020 03.
Article in English | MEDLINE | ID: covidwho-2162

ABSTRACT

Rocaglates, a class of natural compounds isolated from plants of the genus Aglaia, are potent inhibitors of translation initiation. They are proposed to form stacking interactions with polypurine sequences in the 5'-untranslated region (UTR) of selected mRNAs, thereby clamping the RNA substrate onto eIF4A and causing inhibition of the translation initiation complex. Since virus replication relies on the host translation machinery, it is not surprising that the rocaglate Silvestrol has broad-spectrum antiviral activity. Unfortunately, synthesis of Silvestrol is sophisticated and time-consuming, thus hampering the prospects for further antiviral drug development. Here, we present the less complex structured synthetic rocaglate CR-31-B (-) as a novel compound with potent broad-spectrum antiviral activity in primary cells and in an ex vivo bronchial epithelial cell system. CR-31-B (-) inhibited the replication of corona-, Zika-, Lassa-, Crimean Congo hemorrhagic fever viruses and, to a lesser extent, hepatitis E virus (HEV) at non-cytotoxic low nanomolar concentrations. Since HEV has a polypurine-free 5'-UTR that folds into a stable hairpin structure, we hypothesized that RNA clamping by Silvestrol and its derivatives may also occur in a polypurine-independent but structure-dependent manner. Interestingly, the HEV 5'-UTR conferred sensitivity towards Silvestrol but not to CR-31-B (-). However, if an exposed polypurine stretch was introduced into the HEV 5'-UTR, CR-31-B (-) became an active inhibitor comparable to Silvestrol. Moreover, thermodynamic destabilization of the HEV 5'-UTR led to reduced translational inhibition by Silvestrol, suggesting differences between rocaglates in their mode of action, most probably by engaging Silvestrol's additional dioxane moiety.


Subject(s)
Antiviral Agents/pharmacology , Benzofurans/pharmacology , Triterpenes/pharmacology , Virus Replication/drug effects , Viruses/drug effects , A549 Cells , Animals , Antiviral Agents/chemical synthesis , Benzofurans/chemical synthesis , Bronchi/cytology , Cell Culture Techniques , Cells, Cultured , Epithelial Cells/virology , Eukaryotic Initiation Factor-4A/antagonists & inhibitors , Hepatocytes/virology , Humans , Mice , Viruses/classification
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