A Genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor

Geethakumari, Anupriya M.; Ahmed, Wesam S.; Rasool, Saad; Fatima, Asma; Uddin, Shahab; Aouida, Mustapha; Biswas, Kabir H.

doi:10.1101/2022.01.31.478460

Cited by 5 publications

(9 citation statements)

References 109 publications

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“…Nanoscale Molecular Dynamics (NAMD) V2.13 software ( 67 ) and CHARMM36 force field ( 68 ) were utilized to perform the MD simulations as described previously ( 69 – 71 ). The simulation systems consisting of the biomolecular complexes of HC+β2m and HC+ORF7a described above were generated using the QwikMD Toolkit ( 72 ) available as a plugin in Visual Molecular Dynamics (VMD) software V1.9.3 ( 73 ).…”

Section: Methodsmentioning

confidence: 99%

SARS-CoV-2 accessory proteins ORF7a and ORF3a use distinct mechanisms to down-regulate MHC-I surface expression

Arshad

Laurent-Rolle

Ahmed

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Major histocompatibility complex class I (MHC-I) molecules, which are dimers of a glycosylated polymorphic transmembrane heavy chain and the small-protein β 2 -microglobulin (β 2 m), bind peptides in the endoplasmic reticulum that are generated by the cytosolic turnover of cellular proteins. In virus-infected cells, these peptides may include those derived from viral proteins. Peptide-MHC-I complexes then traffic through the secretory pathway and are displayed at the cell surface where those containing viral peptides can be detected by CD8 + T lymphocytes that kill infected cells. Many viruses enhance their in vivo survival by encoding genes that down-regulate MHC-I expression to avoid CD8 + T cell recognition. Here, we report that two accessory proteins encoded by SARS-CoV-2, the causative agent of the ongoing COVID-19 pandemic, down-regulate MHC-I expression using distinct mechanisms. First, ORF3a, a viroporin, reduces the global trafficking of proteins, including MHC-I, through the secretory pathway. The second, ORF7a, interacts specifically with the MHC-I heavy chain, acting as a molecular mimic of β 2 m to inhibit its association. This slows the exit of properly assembled MHC-I molecules from the endoplasmic reticulum. We demonstrate that ORF7a reduces antigen presentation by the human MHC-I allele HLA-A*02:01. Thus, both ORF3a and ORF7a act post-translationally in the secretory pathway to lower surface MHC-I expression, with ORF7a exhibiting a specific mechanism that allows immune evasion by SARS-CoV-2.

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Section: Methodsmentioning

confidence: 99%

SARS-CoV-2 accessory proteins ORF7a and ORF3a use distinct mechanisms to down-regulate MHC-I surface expression

Arshad

Laurent-Rolle

Ahmed

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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“…Nanoscale Molecular Dynamics (NAMD) V2.13 software (62) and CHARMM36 force field (63) were utilized to perform the MD simulations as described previously (64–66). The simulation systems consisting of the biomolecular complexes of HC+β 2 m and HC+ORF7a described above were generated using the QwikMD Toolkit (67) available as a plugin in Visual Molecular Dynamics (VMD) software V1.9.3 (68).…”

Section: Methodsmentioning

confidence: 99%

SARS-CoV-2 accessory proteins ORF7a and ORF3a use distinct mechanisms to downregulate MHC-I surface expression

Arshad

Laurent-Rolle

Ahmed

et al. 2022

Preprint

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Major histocompatibility complex class I (MHC-I) molecules, which are dimers of a glycosylated polymorphic transmembrane heavy chain and the small protein β2-microglobulin (β2m), bind peptides in the endoplasmic reticulum that are generated by the cytosolic turnover of cellular proteins. In virus-infected cells these peptides may include those derived from viral proteins. Peptide-MHC-I complexes then traffic through the secretory pathway and are displayed at the cell surface where those containing viral peptides can be detected by CD8+ T lymphocytes that kill infected cells. Many viruses enhance their in vivo survival by encoding genes that downregulate MHC-I expression to avoid CD8+ T cell recognition. Here we report that two accessory proteins encoded by SARS-CoV-2, the causative agent of the ongoing COVID-19 pandemic, downregulate MHC-I expression using distinct mechanisms. One, ORF3a, a viroporin, reduces global trafficking of proteins, including MHC-I, through the secretory pathway. The second, ORF7a, interacts specifically with the MHC-I heavy chain, acting as a molecular mimic of β2m to inhibit its association. This slows the exit of properly assembled MHC-I molecules from the endoplasmic reticulum. We demonstrate that ORF7a reduces antigen presentation by the human MHC-I allele HLA-A*02:01. Thus, both ORF3a and ORF7a act post-translationally in the secretory pathway to lower surface MHC-I expression, with ORF7a exhibiting a novel and specific mechanism that allows immune evasion by SARS-CoV-2.Significance StatementViruses may down-regulate MHC class I expression on infected cells to avoid elimination by cytotoxic T cells. We report that the accessory proteins ORF7a and ORF3a of SARS-CoV-2 mediate this function and delineate the two distinct mechanisms involved. While ORF3a inhibits global protein trafficking to the cell surface, ORF7a acts specifically on MHC-I by competing with β2m for binding to the MHC-I heavy chain. This is the first account of molecular mimicry of β2m as a viral mechanism of MHC-I down-regulation to facilitate immune evasion.

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confidence: 99%

“…Meanwhile, based on the versatility of nanomaterials, − we developed sensitive colorimetric sensors for the detection of M pro . ,, However, these colorimetric sensors cannot be used to accurately visualize M pro activity in infected cells. As another approach, genetically encoded biosensors have generated several excellent sensors for M pro . , Although these genetically encoded biosensors provide the selective and noninvasive detection of M pro , they can only be used in gene-edited cells.…”

mentioning

confidence: 99%

“…As another approach, genetically encoded biosensors have generated several excellent sensors for M pro . 28,29 Although these genetically encoded biosensors provide the selective and noninvasive detection of M pro , they can only be used in gene-edited cells. Fluorescence as a nondestructive energy form can be used to achieve the sensitive, measurable, and contrastive detection of all kinds of biological molecules.…”

mentioning

confidence: 99%

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A Self-Immolative Fluorescent Probe for Selective Detection of SARS-CoV-2 Main Protease

Zhou

Cheng

et al. 2022

Anal. Chem.

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Existing tools to detect and visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suffer from low selectivity, poor cell permeability, and high cytotoxicity. Here we report a novel self-immolative fluorescent probe (MP590) for the highly selective and sensitive detection of the SARS-CoV-2 main protease (M pro ). This fluorescent probe was prepared by connecting a M pro -cleavable peptide ( N -acetyl-Abu-Tle-Leu-Gln) with a fluorophore ( i . e ., resorufin) via a self-immolative aromatic linker. Fluorescent titration results show that MP590 can detect M pro with a limit of detection (LoD) of 35 nM and is selective over interferents such as hemoglobin, bovine serum albumin (BSA), thrombin, amylase, SARS-CoV-2 papain-like protease (PL pro ), and trypsin. The cell imaging data indicate that this probe can report M pro in HEK 293T cells transfected with a M pro expression plasmid as well as in TMPRSS2-VeroE6 cells infected with SARS-CoV-2. Our results suggest that MP590 can both measure and monitor M pro activity and quantitatively evaluate M pro inhibition in infected cells, making it an important tool for diagnostic and therapeutic research on SARS-CoV-2.

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A Genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor

Cited by 5 publications

References 109 publications

SARS-CoV-2 accessory proteins ORF7a and ORF3a use distinct mechanisms to down-regulate MHC-I surface expression

SARS-CoV-2 accessory proteins ORF7a and ORF3a use distinct mechanisms to down-regulate MHC-I surface expression

SARS-CoV-2 accessory proteins ORF7a and ORF3a use distinct mechanisms to downregulate MHC-I surface expression

A Self-Immolative Fluorescent Probe for Selective Detection of SARS-CoV-2 Main Protease

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