Anti-Frameshifting Ligand Active against SARS Coronavirus-2 is Resistant to Natural Mutations of the Frameshift-Stimulatory Pseudoknot

Neupane, Krishna; Munshi, Sneha; Zhao, Meng; Ritchie, Dustin B.; Ileperuma, Sandaru M.; Woodside, Michael T.

doi:10.1016/j.bpj.2020.11.1838

Cited by 6 publications

(28 citation statements)

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“…We first measured the basal efficiency of −1 PRF stimulated by each of the 6 frameshift signals from the testing panel using cell-free translation of a dual-luciferase reporter system (26,37), to confirm that they did indeed induce −1 PRF and assess the efficiency with which they did so. The reporter system consisted of the Renilla luciferase gene in the 0 frame upstream of the firefly luciferase gene in the −1 frame, with the two separated by the CoV frameshift signal in the 0 frame.…”

Section: Resultsmentioning

confidence: 99%

“…Preparation of mRNA constructs: (1) Constructs for testing inhibitors. For measuring −1 PRF efficiency in the panel of CoV frameshift signals used to test inhibitors, we used a dual-luciferase reporting system based on a plasmid containing the sequence for Renilla luciferase and the multiple cloning site (MCS) from the plasmid pMLuc-1 (Novagen) upstream of the firefly luciferase sequence in the plasmid pISO (addgene), as described previously (19,26). The frameshift signals for all CoVs in the testing panel as well as the two viruses used as negative controls (HIV-1 and PEMV1) were cloned into the MCS between the restriction sites PstI and SpeI.…”

Section: Clustering Of Bat Coronavirus Genomesmentioning

confidence: 99%

“…Sequences of frameshift signals for all constructs are listed in Table S1. We amplified transcription templates from these plasmids by PCR, using a forward primer that included the T7 polymerase sequence as a 5′ extension to the primer sequence (19,26). We then produced the mRNA for dual-luciferase measurements from the transcription templates by in-vitro transcription (MEGAclear).…”

Section: Clustering Of Bat Coronavirus Genomesmentioning

confidence: 99%

“…Compounds were dissolved in DMSO, leading to a final DMSO concentration in the assays of 1% by volume. The results were averaged from 3-9 replicates, as described previously (19,26). The Renilla and firefly luciferase luminescence levels were similar for the different frameshift signals.…”

Section: Clustering Of Bat Coronavirus Genomesmentioning

confidence: 99%

“…Several studies have identified small-molecule ligands that modulate −1 PRF in human CoVs. Computational docking against the SARS-CoV pseudoknot found a compound, 2-[{4-(2-methylthiazol-4ylmethyl)- [1,4]diazepane-1-carbonyl]-amino}-benzoic acid ethyl ester (hereafter denoted MTDB) that inhibited −1 PRF in SARS-CoV (24,25), SARS-CoV-2 (19), and mutant variants of SARS-CoV-2 (26), and also suppressed viral replication (10). More recently, empirical screens for modulation of −1 PRF in SARS-CoV-2 have found a number of compounds that either enhance or suppress −1 PRF (11,27,28), including merafloxacin, a fluoroquinolone antibacterial that was also effective at suppressing viral replication and showed resistance to natural mutants of the pseudoknot (11).…”

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confidence: 99%

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Small-molecule ligands can inhibit −1 programmed ribosomal frameshifting in a broad spectrum of coronaviruses

Munshi

Neupane

Ileperuma

et al. 2021

Preprint

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Recurrent outbreaks of novel zoonotic coronavirus (CoV) diseases since 2000 have high-lighted the importance of developing therapeutics with broad-spectrum activity against CoVs. Because all CoVs use −1 programmed ribosomal frameshifting (−1 PRF) to control expression of key viral proteins, the frameshift signal in viral mRNA that stimulates −1 PRF provides a promising potential target for such therapeutics. To test the viability of this strategy, we explored a group of 6 small-molecule ligands, evaluating their activity against the frameshift signals from a panel of representative bat CoVs—the most likely source of future zoonoses—as well as SARS-CoV-2 and MERS-CoV. We found that whereas some ligands had notable activity against only a few of the frameshift signals, the serine protease inhibitor nafamostat suppressed −1 PRF significantly in several of them, while having limited to no effect on −1 PRF caused by frameshift signals from other viruses used as negative controls. These results suggest it is possible to find small-molecule ligands that inhibit −1 PRF specifically in a broad spectrum of CoVs, establishing the frameshift signal as a viable target for developing pan-coronaviral therapeutics.

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Section: Resultsmentioning

confidence: 99%

Section: Clustering Of Bat Coronavirus Genomesmentioning

confidence: 99%

Section: Clustering Of Bat Coronavirus Genomesmentioning

confidence: 99%

Section: Clustering Of Bat Coronavirus Genomesmentioning

confidence: 99%

mentioning

confidence: 99%

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Small-molecule ligands can inhibit −1 programmed ribosomal frameshifting in a broad spectrum of coronaviruses

Munshi

Neupane

Ileperuma

et al. 2021

Preprint

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The short isoform of the host antiviral protein ZAP acts as an inhibitor of SARS-CoV-2 programmed ribosomal frameshifting

et al. 2021

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Programmed ribosomal frameshifting (PRF) is a fundamental gene expression event in many viruses, including SARS-CoV-2. It allows production of essential viral, structural and replicative enzymes that are encoded in an alternative reading frame. Despite the importance of PRF for the viral life cycle, it is still largely unknown how and to what extent cellular factors alter mechanical properties of frameshift elements and thereby impact virulence. This prompted us to comprehensively dissect the interplay between the SARS-CoV-2 frameshift element and the host proteome. We reveal that the short isoform of the zinc-finger antiviral protein (ZAP-S) is a direct regulator of PRF in SARS-CoV-2 infected cells. ZAP-S overexpression strongly impairs frameshifting and inhibits viral replication. Using in vitro ensemble and single-molecule techniques, we further demonstrate that ZAP-S directly interacts with the SARS-CoV-2 RNA and interferes with the folding of the frameshift RNA element. Together, these data identify ZAP-S as a host-encoded inhibitor of SARS-CoV-2 frameshifting and expand our understanding of RNA-based gene regulation.

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SARS-CoV-2 Ribosomal Frameshifting Pseudoknot: Detection of Inter-viral Structural Similarity

Trinity

Lansing

Jabbari

et al. 2021

2021 IEEE 9th International Conference on Healthcare Informatics (ICHI)

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the COVID-19 pandemic. After over 160 million cases and 3.3 million deaths worldwide as of May 2021, it is still unclear when this crisis will end. Thus, there is an urgent need for treatments that improve the prognosis of seriously affected patients. Multiple viruses including HIV, MERS-CoV (coronavirus responsible for Middle East Respiratory Syndrome, MERS), SARS-CoV (coronavirus responsible for SARS) and SARS-CoV-2 use a mechanism known as -1 programmed ribosomal frameshifting (-1 PRF) to successfully replicate. SARS-CoV and SARS-CoV-2 possess a unique RNA pseudoknotted structure that stimulates -1 PRF. Recent experiments identified small molecules as antiviral agents that can bind to the pseudoknot and disrupt its stimulation of -1 PRF. Targeting -1 PRF in SARS-CoV-2 can be an excellent strategy to impair viral replication and improve patients' prognoses. Crucial to developing these successful therapies is modeling the structure of the SARS-CoV-2 -1 PRF pseudoknot. Following a structural alignment approach, we identify similarities in -1 PRF pseudoknots of SARS-CoV-2, SARS-CoV, and MERS-CoV. In addition, we provide a better understanding of the SARS-CoV-2 -1 PRF pseudoknot by investigating the structural landscape using a hierarchical folding approach. We provide in-depth analysis on alternative structure prediction methods based on SARS-CoV-2 structural reactivity (SHAPE) data to contextualize and motivate future RNA structure-function research. Since understanding the impact of mutations is vital to long-term success of treatments based on predicted RNA functional structures, we provide insight on SARS-CoV-2 -1 PRF pseudoknot sequence mutations and their effect on the resulting structure.

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Anti-Frameshifting Ligand Active against SARS Coronavirus-2 is Resistant to Natural Mutations of the Frameshift-Stimulatory Pseudoknot

Abstract: Riboswitches are structured non-coding RNA elements typically found in the 5' untranslated regions of bacterial mRNAs. They regulate gene expression, most

Cited by 6 publications

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Small-molecule ligands can inhibit −1 programmed ribosomal frameshifting in a broad spectrum of coronaviruses

Small-molecule ligands can inhibit −1 programmed ribosomal frameshifting in a broad spectrum of coronaviruses

The short isoform of the host antiviral protein ZAP acts as an inhibitor of SARS-CoV-2 programmed ribosomal frameshifting

SARS-CoV-2 Ribosomal Frameshifting Pseudoknot: Detection of Inter-viral Structural Similarity

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