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Interface-based design of the favipiravir-binding site in SARS-CoV-2 RNA-dependent RNA polymerase reveals mutations conferring resistance to chain termination.

ABSTRACT: Favipiravir is a broad-spectrum inhibitor of viral RNA-dependent RNA polymerase (RdRp) currently being used to manage COVID-19. Accumulation of mutations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RdRp may facilitate antigenic drift, generating favipiravir resistance. Focussing on the chain-termination mechanism utilized by favipiravir, we used high-throughput interface-based protein design to generate > 100 000 designs of the favipiravir-binding site of RdRp and identify mutational hotspots. We identified several single-point mutants and designs having a sequence identity of 97%-98% with wild-type RdRp, suggesting that SARS-CoV-2 can develop favipiravir resistance with few mutations. Out of 134 mutations documented in the CoV-GLUE database, 63 specific mutations were already predicted as resistant in our calculations, thus attaining ˜ 47% correlation with the sequencing data. These findings improve our understanding of the potential signatures of adaptation in SARS-CoV-2 against favipiravir.

SUBMITTER: Padhi AK

PROVIDER: S-EPMC8426738 | biostudies-literature |

REPOSITORIES: biostudies-literature

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RNA polymerase II mutations conferring defects in poly(A) site cleavage and termination in Saccharomyces cerevisiae.

Project description:Transcription termination by RNA polymerase (Pol) II is an essential but poorly understood process. In eukaryotic nuclei, the 3' ends of mRNAs are generated by cleavage and polyadenylation, and the same sequence elements that specify that process are required for downstream release of the polymerase from the DNA. Although Pol II is known to bind proteins required for both events, few studies have focused on Pol II mutations as a means to uncover the mechanisms that couple polyadenylation and termination. We performed a genetic screen in the yeast Saccharomyces cerevisiae to isolate mutations in the N-terminal half of Rpb2, the second largest Pol II subunit, that conferred either a decreased or increased response to a well-characterized poly(A) site. Most of the mutant alleles encoded substitutions affecting either surface residues or conserved active site amino acids at positions important for termination by other RNA polymerases. Reverse transcription polymerase chain reaction experiments revealed that transcript cleavage at the poly(A) site was impaired in both classes of increased readthrough mutants. Transcription into downstream sequences beyond where termination normally occurs was also probed. Although most of the tested readthrough mutants showed a reduction in termination concomitant with the reduced poly(A) usage, these processes were uncoupled in at least one mutant strain. Several rpb2 alleles were found to be similar or identical to published mutants associated with defective TFIIF function. Tests of these and additional mutations known to impair Rpb2-TFIIF interactions revealed similar decreased readthrough phenotypes, suggesting that TFIIF may have a role in 3' end formation and termination.

| S-EPMC3564978 | biostudies-literature

Yhh1p/Cft1p directly links poly(A) site recognition and RNA polymerase II transcription termination.

Project description:RNA polymerase II (pol II) transcription termination requires co-transcriptional recognition of a functional polyadenylation signal, but the molecular mechanisms that transduce this signal to pol II remain unclear. We show that Yhh1p/Cft1p, the yeast homologue of the mammalian AAUAAA interacting protein CPSF 160, is an RNA-binding protein and provide evidence that it participates in poly(A) site recognition. Interestingly, RNA binding is mediated by a central domain composed of predicted beta-propeller-forming repeats, which occurs in proteins of diverse cellular functions. We also found that Yhh1p/Cft1p bound specifically to the phosphorylated C-terminal domain (CTD) of pol II in vitro and in a two-hybrid test in vivo. Furthermore, transcriptional run-on analysis demonstrated that yhh1 mutants were defective in transcription termination, suggesting that Yhh1p/Cft1p functions in the coupling of transcription and 3'-end formation. We propose that direct interactions of Yhh1p/Cft1p with both the RNA transcript and the CTD are required to communicate poly(A) site recognition to elongating pol II to initiate transcription termination.

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Transcription termination by the eukaryotic RNA polymerase III.

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DNA-directed termination of RNA polymerase II transcription.

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Tuning promoter strength through RNA polymerase binding site design in Escherichia coli.

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Cidofovir Diphosphate Inhibits Adenovirus 5 DNA Polymerase via both Nonobligate Chain Termination and Direct Inhibition, and Polymerase Mutations Confer Cidofovir Resistance on Intact Virus.

Project description:Human adenovirus (AdV) can cause fatal disease in immune-suppressed individuals, but treatment options are limited, in part because the antiviral cytidine analog cidofovir (CDV) is nephrotoxic. The investigational agent brincidofovir (BCV) is orally bioavailable, nonnephrotoxic, and generates the same active metabolite, cidofovir diphosphate (CDVpp). However, its mechanism of action against AdV is poorly understood. Therefore, we have examined the effect of CDVpp on DNA synthesis by a purified adenovirus 5 (AdV5) DNA polymerase (Pol). CDVpp was incorporated into nascent DNA strands and promoted a nonobligate form of chain termination (i.e., AdV5 Pol can extend, albeit inefficiently, a DNA chain even after the incorporation of a first CDVpp molecule). Moreover, unlike a conventional mismatched base pair, misincorporated CDVpp was not readily excised by the AdV5 Pol. At elevated concentrations, CDVpp inhibited AdV5 Pol in a manner consistent with both chain termination and direct inhibition of Pol activity. Finally, a recombinant AdV5 was constructed, containing Pol mutations (V303I and T87I) that were selected following an extended passage of wild-type AdV5 in the presence of BCV. This virus had a 2.1-fold elevated 50% effective concentration (EC50) for BCV and a 1.9-fold increased EC50 for CDV; thus, these results confirmed that viral resistance to BCV and CDV can be attributed to mutations in the viral Pol. These findings show that the anti-AdV5 activity of CDV and BCV is mediated through the viral DNA Pol and that their antiviral activity may occur via both (nonobligate) chain termination and (at high concentration) direct inhibition of AdV5 Pol activity.

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Point mutations in the Rpb9-homologous domain of Rpc11 that impair transcription termination by RNA polymerase III.

Project description:RNA polymerase III recognizes and pauses at its terminator, an oligo(dT) tract in non-template DNA, terminates 3' oligo(rU) synthesis within this sequence, and releases the RNA. The pol III subunit Rpc11p (C11) mediates RNA 3'-5' cleavage in the catalytic center of pol III during pausing. The amino and carboxyl regions of C11 are homologous to domains of the pol II subunit Rpb9p, and the pol II elongation and RNA cleavage factor, TFIIS, respectively. We isolated C11 mutants from Schizosaccharomyces pombe that cause pol III to readthrough terminators in vivo. Mutant RNA confirmed the presence of terminator readthrough transcripts. A predominant mutation site, F32, resides in the C11 Rpb9-like domain. Another mutagenic approach confirmed the F32 mutation and also isolated I34 and Y30 mutants. Modeling Y30, F32 and I34 of C11 in available cryoEM pol III structures predicts a hydrophobic patch that may interface with C53/37. Another termination mutant, Rpc2-T455I, appears to reside internally, near the RNA-DNA hybrid. We show that the Rpb9 and TFIIS homologous mutants of C11 reflect distinct activities, that differentially affect terminator recognition and RNA 3' cleavage. We propose that these C11 domains integrate action at the upper jaw and center of pol III during termination.

| S-EPMC3152337 | biostudies-literature

Molecular dissection of mammalian RNA polymerase II transcriptional termination.

Project description:Transcriptional termination of mammalian RNA polymerase II (Pol II) is an essential but little-understood step in protein-coding gene expression. Mechanistically, termination by all DNA-dependent RNA polymerases can be reduced to two steps, namely release of the RNA transcript and release of the DNA template. Using a simple nuclear fractionation procedure, we have monitored transcript and template release in the context of both natural and artificial Pol II terminator sequences. We describe the timing and relationship between these events and in so doing establish the roles of the poly(A) signal, cotranscriptional RNA cleavage events, and 5'-3' exonucleolytic RNA degradation in the mammalian Pol II termination process.

| S-EPMC2288634 | biostudies-literature

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