Biology subjects

Krafcikova, P.

Publications and source records attributed to Krafcikova, P..

3 recordsLinked to original sources

bioRxiv2021

Structural analysis of the OC43 coronavirus 2'-O-RNA methyltransferase

The OC43 coronavirus is a human pathogen that usually causes only the common cold. One of its key enzymes, similar to other coronaviruses, is the 2'-O-RNA methyltransferase (MTase) that is essential for viral RNA stability and expression. Here, we report the crystal structure of the 2'-O-RNA MTase in a complex with the pan-methyltransferase inhibitor sinefungin solved at 2.2 [A] resolution. The structure revealed an overall fold consistent with the fold observed in other coronaviral MTases. The major differences are in the conformation of the C-terminus of the nsp16 subunit and an additional helix in the N-terminus of the nsp10 subunits. The structural analysis also revealed very high conservation of the SAM binding pocket suggesting that the SAM pocket is a suitable spot for the design of antivirals effective against all human coronaviruses. ImportanceSome coronaviruses are dangerous pathogens while some cause only common colds. The reasons are not understood although the spike proteins probably play an important role. However, to understand the coronaviral biology in sufficient detail we need to compare the key enzymes from different coronaviruses. We solved the crystal structure of 2'-O-RNA methyltransferase of the OC43 coronavirus, a virus that usually causes mild colds. The structure revealed some differences in the overall fold but also revealed that the SAM binding site is conserved suggesting that development of antivirals against multiple coronaviruses is feasible.

biophysics↗

bioRxiv2020

Substrate specificity of SARS-CoV-2 nsp10-nsp16 methyltransferase

The ongoing COVID-19 pandemic exemplifies the general need to better understand viral infections. The positive single strand RNA genome of its causative agent, the SARS coronavirus 2 (SARS-CoV-2) encodes all viral enzymes. In this work, we focus on one particular methyltransferase (MTase), nsp16, which in complex with nsp10 is capable of methylating the first nucleotide of a capped RNA strand at the 2'-O position. This process is part of a viral capping system and is crucial for viral evasion of the innate immune reaction. In light of recently discovered non-canonical RNA caps, we tested various dinucleoside polyphosphate-capped RNAs as substrates for nsp10-nsp16 MTase. We developed an LC-MS-based method and discovered five types of capped RNA (m7Gp3A(G)-, Gp3A(G)- and Gp4A-RNA) that are substrates of the nsp10-nsp16 MTase. Our technique is an alternative to the classical isotope labelling approach for measurement of 2'-O-MTase activity. Further, we determined the IC50 value of sinefungin (286 {+/-} 66 nM) to illustrate the value of our approach for inhibitor screening. In the future, this approach can be used for screening inhibitors of any type of 2'-O-MTase.

molecular biology↗

bioRxiv2020

Structural analysis of the SARS-CoV-2 methyltransferase complex involved in coronaviral RNA cap creation

COVID-19 pandemic is caused by the SARS-CoV-2 virus that has several enzymes that could be targeted by antivirals including a 2-O RNA methyltransferase (MTase) that is involved in the viral RNA cap formation; an essential process for RNA stability. This MTase is composed of two nonstructural proteins, the nsp16 catalytic subunit and the activating nsp10 protein. We have solved the crystal structure of the nsp10-nsp16 complex bound to the pan-MTase inhibitor sinefungin in the active site. Based on the structural data we built a model of the MTase in complex with RNA that illustrates the catalytic reaction. A structural comparison to the Zika MTase revealed low conservation of the catalytic site between these two RNA viruses suggesting preparation of inhibitors targeting both these viruses will be very difficult. Together, our data will provide the information needed for structure-based drug design.

molecular biology↗