Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 6 de 6
Filter
1.
PLoS Biol ; 19(12): e3001490, 2021 12.
Article in English | MEDLINE | ID: covidwho-1595018

ABSTRACT

Over the past 20 years, 3 highly pathogenic human coronaviruses (HCoVs) have emerged-Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and, most recently, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)-demonstrating that coronaviruses (CoVs) pose a serious threat to human health and highlighting the importance of developing effective therapies against them. Similar to other viruses, CoVs are dependent on host factors for their survival and replication. We hypothesized that evolutionarily distinct CoVs may exploit similar host factors and pathways to support their replication cycles. Herein, we conducted 2 independent genome-wide CRISPR/Cas-9 knockout (KO) screens to identify MERS-CoV and HCoV-229E host dependency factors (HDFs) required for HCoV replication in the human Huh7 cell line. Top scoring genes were further validated and assessed in the context of MERS-CoV and HCoV-229E infection as well as SARS-CoV and SARS-CoV-2 infection. Strikingly, we found that several autophagy-related genes, including TMEM41B, MINAR1, and the immunophilin FKBP8, were common host factors required for pan-CoV replication. Importantly, inhibition of the immunophilin protein family with the compounds cyclosporine A, and the nonimmunosuppressive derivative alisporivir, resulted in dose-dependent inhibition of CoV replication in primary human nasal epithelial cell cultures, which recapitulate the natural site of virus replication. Overall, we identified host factors that are crucial for CoV replication and demonstrated that these factors constitute potential targets for therapeutic intervention by clinically approved drugs.


Subject(s)
Autophagy/genetics , CRISPR-Cas Systems , Middle East Respiratory Syndrome Coronavirus/genetics , SARS-CoV-2/genetics , Antiviral Agents/pharmacology , Gene Knockdown Techniques , Host-Pathogen Interactions , Humans , Middle East Respiratory Syndrome Coronavirus/drug effects , Middle East Respiratory Syndrome Coronavirus/physiology , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Virus Replication
2.
Emerg Infect Dis ; 27(7): 1811-1820, 2021 07.
Article in English | MEDLINE | ID: covidwho-1278358

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally, and the number of worldwide cases continues to rise. The zoonotic origins of SARS-CoV-2 and its intermediate and potential spillback host reservoirs, besides humans, remain largely unknown. Because of ethical and experimental constraints and more important, to reduce and refine animal experimentation, we used our repository of well-differentiated airway epithelial cell (AEC) cultures from various domesticated and wildlife animal species to assess their susceptibility to SARS-CoV-2. We observed that SARS-CoV-2 replicated efficiently only in monkey and cat AEC culture models. Whole-genome sequencing of progeny viruses revealed no obvious signs of nucleotide transitions required for SARS-CoV-2 to productively infect monkey and cat AEC cultures. Our findings, together with previous reports of human-to-animal spillover events, warrant close surveillance to determine the potential role of cats, monkeys, and closely related species as spillback reservoirs for SARS-CoV-2.


Subject(s)
Animals, Wild , COVID-19 , Animals , Epithelial Cells , Humans , Respiratory System , SARS-CoV-2
3.
PLoS Biol ; 19(3): e3001158, 2021 03.
Article in English | MEDLINE | ID: covidwho-1156073

ABSTRACT

Since its emergence in December 2019, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally and become a major public health burden. Despite its close phylogenetic relationship to SARS-CoV, SARS-CoV-2 exhibits increased human-to-human transmission dynamics, likely due to efficient early replication in the upper respiratory epithelium of infected individuals. Since different temperatures encountered in the human upper and lower respiratory tract (33°C and 37°C, respectively) have been shown to affect the replication kinetics of several respiratory viruses, as well as host innate immune response dynamics, we investigated the impact of temperature on SARS-CoV-2 and SARS-CoV infection using the primary human airway epithelial cell culture model. SARS-CoV-2, in contrast to SARS-CoV, replicated to higher titers when infections were performed at 33°C rather than 37°C. Although both viruses were highly sensitive to type I and type III interferon pretreatment, a detailed time-resolved transcriptome analysis revealed temperature-dependent interferon and pro-inflammatory responses induced by SARS-CoV-2 that were inversely proportional to its replication efficiency at 33°C or 37°C. These data provide crucial insight on pivotal virus-host interaction dynamics and are in line with characteristic clinical features of SARS-CoV-2 and SARS-CoV, as well as their respective transmission efficiencies.


Subject(s)
Gene Expression Profiling/methods , Gene Expression Regulation, Viral/genetics , SARS Virus/genetics , SARS-CoV-2/genetics , Animals , Antiviral Agents/pharmacology , Cells, Cultured , Chlorocebus aethiops , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial Cells/virology , Gene Expression Regulation, Viral/drug effects , Host-Pathogen Interactions/drug effects , Host-Pathogen Interactions/genetics , Humans , Interferons/pharmacology , SARS Virus/drug effects , SARS Virus/physiology , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Species Specificity , Temperature , Vero Cells , Virus Replication/drug effects , Virus Replication/genetics
4.
Nature ; 592(7852): 122-127, 2021 04.
Article in English | MEDLINE | ID: covidwho-1104508

ABSTRACT

During the evolution of SARS-CoV-2 in humans, a D614G substitution in the spike glycoprotein (S) has emerged; virus containing this substitution has become the predominant circulating variant in the COVID-19 pandemic1. However, whether the increasing prevalence of this variant reflects a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains unknown. Here we use isogenic SARS-CoV-2 variants to demonstrate that the variant that contains S(D614G) has enhanced binding to the human cell-surface receptor angiotensin-converting enzyme 2 (ACE2), increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a human ACE2 knock-in mouse model, and markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Our data show that the D614G substitution in S results in subtle increases in binding and replication in vitro, and provides a real competitive advantage in vivo-particularly during the transmission bottleneck. Our data therefore provide an explanation for the global predominance of the variant that contains S(D614G) among the SARS-CoV-2 viruses that are currently circulating.


Subject(s)
COVID-19/transmission , COVID-19/virology , Mutation , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/genetics , Virus Replication/genetics , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , Bronchi/cytology , Bronchi/virology , COVID-19/epidemiology , Cell Line , Cells, Cultured , Cricetinae , Disease Models, Animal , Epithelial Cells/virology , Female , Ferrets/virology , Founder Effect , Gene Knock-In Techniques , Genetic Fitness , Humans , Male , Mesocricetus , Mice , Nasal Mucosa/cytology , Nasal Mucosa/virology , Protein Binding , RNA, Viral/analysis , Receptors, Coronavirus/metabolism , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity
5.
bioRxiv ; 2020 Oct 27.
Article in English | MEDLINE | ID: covidwho-915978

ABSTRACT

During the evolution of SARS-CoV-2 in humans a D614G substitution in the spike (S) protein emerged and became the predominant circulating variant (S-614G) of the COVID-19 pandemic 1 . However, whether the increasing prevalence of the S-614G variant represents a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains elusive. Here, we generated isogenic SARS-CoV-2 variants and demonstrate that the S-614G variant has (i) enhanced binding to human ACE2, (ii) increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a novel human ACE2 knock-in mouse model, and (iii) markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Collectively, our data show that while the S-614G substitution results in subtle increases in binding and replication in vitro , it provides a real competitive advantage in vivo , particularly during the transmission bottle neck, providing an explanation for the global predominance of S-614G variant among the SARS-CoV-2 viruses currently circulating.

6.
Nature ; 582(7813): 561-565, 2020 06.
Article in English | MEDLINE | ID: covidwho-164589

ABSTRACT

Reverse genetics has been an indispensable tool to gain insights into viral pathogenesis and vaccine development. The genomes of large RNA viruses, such as those from coronaviruses, are cumbersome to clone and manipulate in Escherichia coli owing to the size and occasional instability of the genome1-3. Therefore, an alternative rapid and robust reverse-genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform to genetically reconstruct diverse RNA viruses, including members of the Coronaviridae, Flaviviridae and Pneumoviridae families. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples or synthetic DNA, and these fragments were then reassembled in one step in Saccharomyces cerevisiae using transformation-associated recombination cloning to maintain the genome as a yeast artificial chromosome. T7 RNA polymerase was then used to generate infectious RNA to rescue viable virus. Using this platform, we were able to engineer and generate chemically synthesized clones of the virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)4, which has caused the recent pandemic of coronavirus disease (COVID-19), in only a week after receipt of the synthetic DNA fragments. The technical advance that we describe here facilitates rapid responses to emerging viruses as it enables the real-time generation and functional characterization of evolving RNA virus variants during an outbreak.


Subject(s)
Betacoronavirus/genetics , Cloning, Molecular/methods , Coronavirus Infections/virology , Genome, Viral/genetics , Genomics/methods , Pneumonia, Viral/virology , Reverse Genetics/methods , Synthetic Biology/methods , Animals , COVID-19 , China/epidemiology , Chlorocebus aethiops , Chromosomes, Artificial, Yeast/metabolism , Coronavirus Infections/epidemiology , DNA-Directed RNA Polymerases/metabolism , Evolution, Molecular , Humans , Mutation , Pandemics/statistics & numerical data , Pneumonia, Viral/epidemiology , Respiratory Syncytial Viruses/genetics , SARS-CoV-2 , Saccharomyces cerevisiae/genetics , Vero Cells , Viral Proteins/metabolism , Zika Virus/genetics
SELECTION OF CITATIONS
SEARCH DETAIL