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1.
ACS Infect Dis ; 8(7): 1265-1279, 2022 Jul 08.
Article in English | MEDLINE | ID: covidwho-1908084

ABSTRACT

There is a pressing need for host-directed therapeutics that elicit broad-spectrum antiviral activities to potentially address current and future viral pandemics. Apratoxin S4 (Apra S4) is a potent Sec61 inhibitor that prevents cotranslational translocation of secretory proteins into the endoplasmic reticulum (ER), leading to anticancer and antiangiogenic activity both in vitro and in vivo. Since Sec61 has been shown to be an essential host factor for viral proteostasis, we tested Apra S4 in cellular models of viral infection, including SARS-CoV-2, influenza A virus, and flaviviruses (Zika, West Nile, and Dengue virus). Apra S4 inhibited viral replication in a concentration-dependent manner and had high potency particularly against SARS-CoV-2 and influenza A virus, with subnanomolar activity in human cells. Characterization studies focused on SARS-CoV-2 revealed that Apra S4 impacted a post-entry stage of the viral life-cycle. Transmission electron microscopy revealed that Apra S4 blocked formation of stacked double-membrane vesicles, the sites of viral replication. Apra S4 reduced dsRNA formation and prevented viral protein production and trafficking of secretory proteins, especially the spike protein. Given the potent and broad-spectrum activity of Apra S4, further preclinical evaluation of Apra S4 and other Sec61 inhibitors as antivirals is warranted.


Subject(s)
COVID-19 , Influenza A virus , Zika Virus Infection , Zika Virus , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Depsipeptides , Humans , Pandemics , SARS-CoV-2 , Zika Virus Infection/drug therapy
2.
PLoS Pathog ; 17(9): e1009929, 2021 09.
Article in English | MEDLINE | ID: covidwho-1430555

ABSTRACT

Remdesivir (RDV), a broadly acting nucleoside analogue, is the only FDA approved small molecule antiviral for the treatment of COVID-19 patients. To date, there are no reports identifying SARS-CoV-2 RDV resistance in patients, animal models or in vitro. Here, we selected drug-resistant viral populations by serially passaging SARS-CoV-2 in vitro in the presence of RDV. Using high throughput sequencing, we identified a single mutation in RNA-dependent RNA polymerase (NSP12) at a residue conserved among all coronaviruses in two independently evolved populations displaying decreased RDV sensitivity. Introduction of the NSP12 E802D mutation into our SARS-CoV-2 reverse genetics backbone confirmed its role in decreasing RDV sensitivity in vitro. Substitution of E802 did not affect viral replication or activity of an alternate nucleoside analogue (EIDD2801) but did affect virus fitness in a competition assay. Analysis of the globally circulating SARS-CoV-2 variants (>800,000 sequences) showed no evidence of widespread transmission of RDV-resistant mutants. Surprisingly, we observed an excess of substitutions in spike at corresponding sites identified in the emerging SARS-CoV-2 variants of concern (i.e., H69, E484, N501, H655) indicating that they can arise in vitro in the absence of immune selection. The identification and characterisation of a drug resistant signature within the SARS-CoV-2 genome has implications for clinical management and virus surveillance.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/pharmacology , COVID-19 , Coronavirus RNA-Dependent RNA Polymerase/genetics , Drug Resistance, Microbial/genetics , SARS-CoV-2/drug effects , Adenosine Monophosphate/pharmacology , Alanine/pharmacology , Animals , Biological Evolution , COVID-19/drug therapy , Chlorocebus aethiops , Humans , Mutation , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
3.
PLoS Biol ; 19(2): e3001091, 2021 02.
Article in English | MEDLINE | ID: covidwho-1102372

ABSTRACT

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.


Subject(s)
COVID-19 Vaccines , COVID-19/diagnosis , COVID-19/virology , Reverse Genetics , SARS-CoV-2/genetics , A549 Cells , Angiotensin-Converting Enzyme 2/metabolism , Animals , Chlorocebus aethiops , Codon , Humans , Hydrazones/pharmacology , Mice , Morpholines/pharmacology , Open Reading Frames , Plasmids/genetics , Pyrimidines/pharmacology , Serine Endopeptidases/metabolism , Vero Cells , Viral Proteins/metabolism
4.
Pept Sci (Hoboken) ; : e24217, 2021 Jan 08.
Article in English | MEDLINE | ID: covidwho-1014097

ABSTRACT

COVID-19 is caused by a novel coronavirus called severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). Virus cell entry is mediated through a protein-protein interaction (PPI) between the SARS-CoV-2 spike protein and angiotensin-converting enzyme 2 (ACE2). A series of stapled peptide ACE2 peptidomimetics based on the ACE2 interaction motif were designed to bind the coronavirus S-protein RBD and inhibit binding to the human ACE2 receptor. The peptidomimetics were assessed for antiviral activity in an array of assays including a neutralization pseudovirus assay, immunofluorescence (IF) assay and in-vitro fluorescence polarization (FP) assay. However, none of the peptidomimetics showed activity in these assays, suggesting that an enhanced binding interface is required to outcompete ACE2 for S-protein RBD binding and prevent virus internalization.

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