scholarly journals SARS-CoV-2 RdRp is a versatile enzyme with proofreading activity and ability to incorporate NHC into RNA by using diphosphate form molnupiravir as a substrate

2021 ◽  
Author(s):  
Maofeng Wang ◽  
Cancan Wu ◽  
Nan Liu ◽  
Fengyu Zhang ◽  
Hongjie Dong ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Drug Development ◽  
Rna Polymerase ◽  
Human Health ◽  
Antiviral Drug ◽  
Polymerase Activity ◽  
Genome Replication ◽  
Rna Dependent Rna Polymerase ◽  
The World ◽  
Rna Polymerase Activity

The coronavirus disease 2019 (COVID-19) has been ravaging throughout the world for almost two years and has severely impaired both human health and the economy. The causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) employs the viral RNA-dependent RNA polymerase (RdRp) complex for genome replication and transcription, making RdRp an appealing target for antiviral drug development. Although the structure of the RdRp complex has been determined, the function of RdRp has not been fully characterized. Here we reveal that in addition to RNA dependent RNA polymerase activity, RdRp also shows exoribonuclease activity and consequently proofreading activity. We observed that RdRp and nsp14-ExoN, when combined, exhibit higher proofreading activity compared to RdRp alone. Moreover, RdRp can recognize and utilize nucleoside diphosphate (NDP) as substrate to synthesize RNA and can also incorporate β-d-N4-hydroxycytidine (NHC) into RNA while using diphosphate form molnupiravir as substrate.

scholarly journals A dynamic regulatory interface on SARS-CoV-2 RNA polymerase

2020 ◽  
Author(s):  
Wei Shi ◽  
Ming Chen ◽  
Yang Yang ◽  
Wei Zhou ◽  
Shiyun Chen ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Active Center ◽  
Antiviral Drug ◽  
Polymerase Activity ◽  
Genome Replication ◽  
Specific Interactions ◽  
Global Public Health ◽  
Rna Dependent Rna Polymerase ◽  
Viral Genome Replication

AbstractThe RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 is the core machinery responsible for the viral genome replication and transcription and also a major antiviral target. Here we report the cryo-electron microscopy structure of a post-translocated SARS-CoV-2 RdRp core complex, comprising one nsp12, one separate nsp8(I) monomer, one nsp7-nsp8(II) subcomplex and a replicating RNA substrate. Compared with the recently reported SARS-CoV-2 RdRp complexes, the nsp8(I)/nsp7 interface in this RdRp complex shifts away from the nsp12 polymerase. Further functional characterizations suggest that specific interactions between the nsp8(I) and nsp7, together with the rearrangement of nsp8(I)/nsp7 interface, ensure the efficient and processive RNA synthesis by the RdRp complex. Our findings provide a mechanistic insight into how nsp7 and nsp8 cofactors regulate the polymerase activity of nsp12 and suggest a potential new intervention interface, in addition to the canonical polymerase active center, in RdRp for antiviral design.Author summarySince it was first discovered and reported in late 2019, the coronavirus disease 2019 (COVID-19) pandemic caused by highly contagious SARS-CoV-2 virus is wreaking havoc around the world. Currently, no highly effective and specific antiviral drug is available for clinical treatment. Therefore, the threat of COVID-19 transmission necessitates the discovery of more effective antiviral strategies. Viral RNA-dependent RNA polymerase (RdRp) is an important antiviral drug target. Here, our cryo-EM structure of a SARS-CoV-2 RdRp/RNA replicating complex reveals a previously uncharacterized overall shift of the cofactor nsp8(I)/nsp7 interface, leading to its rearrangement. Through in vitro functional test, we found that the specific interactions on the interface are important to the efficient RNA polymerase activity of SARS-CoV-2 RdRp. These observations let us to suggest this interface as a potential new drug intervention site, outside of the canonical polymerase active center, in RdRp for antiviral design. Our findings would provide new insights into regulatory mechanism of this novel SARS-CoV-2 RdRp, contribute to the design of antiviral drugs against SARS-CoV-2, and benefit the global public health.

scholarly journals Thermolabile L-A virus-like particles from pet18 mutants of Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (2) ◽  
pp. 404-410 ◽  
Author(s):  
T Fujimura ◽  
R B Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Polymerase Activity ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Rna Dependent Rna Polymerase ◽  
Virus Like Particles ◽  
Rna Polymerase Activity ◽  
Lines Of Evidence

pet18 mutations in Saccharomyces cerevisiae confer on the cell the inability to maintain either L-A or M double-stranded RNAs (dsRNAs) at the nonpermissive temperature. In in vitro experiments, we examined the effects of pet18 mutations on the RNA-dependent RNA polymerase activity associated with virus-like particles (VLPs). pet18 mutations caused thermolabile RNA polymerase activity of L-A VLPs, and this thermolability was found to be due to the instability of the L-A VLP structure. The pet18 mutations did not affect RNA polymerase activity of M VLPs. Furthermore, the temperature sensitivity of wild-type L-A RNA polymerase differed substantially from that of M RNA polymerase. From these results, and from other genetic and biochemical lines of evidence which suggest that replication of M dsRNA requires the presence of L-A dsRNA, we propose that the primary effect of the pet18 mutation is on the L-A VLP structure and that the inability of pet18 mutants to maintain M dsRNA comes from the loss of L-A dsRNA.

An Assay for RNA-Dependent RNA Polymerase Activity

2006 ◽  
Vol 2006 (1) ◽  
pp. pdb.prot4322
Author(s):  
Luyun Huang ◽  
John Gledhill ◽  
Craig E. Cameron
Keyword(s):  
Rna Polymerase ◽  
Polymerase Activity ◽  
Rna Dependent Rna Polymerase ◽  
Rna Polymerase Activity

scholarly journals High-throughput identification of compounds targeting influenza RNA-dependent RNA polymerase activity

2010 ◽  
Vol 107 (45) ◽  
pp. 19151-19156 ◽  
Author(s):  
C.-Y. Su ◽  
T.-J. R. Cheng ◽  
M.-I. Lin ◽  
S.-Y. Wang ◽  
W.-I. Huang ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
High Throughput ◽  
Polymerase Activity ◽  
Rna Dependent Rna Polymerase ◽  
Rna Polymerase Activity
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