SARS-CoV-2 enzyme RdRp

Show:Asymmetric Unit Biological Assembly RNA-dependent RNA polymerase/ RNA-directed RNA polymerase (RdRp)/ nsp12 Function Responsible for replication and transcription of the viral RNA genome. Together with the proteins nsp7 and two different conformations of nsp8, nsp12 forms the minimal core polymerase complex, as without its co-factors, nsp12 has a low efficiency in polymerase activity. This complex mediates the RNA synthesis of the virus and is therefore an important part in the replication procedure. Disease The global COVID-19 pandemic, which started in 2019, is caused by the SARS-CoV-2. Structure Nsp12 consists of three domains, the nidoviruses specific N-terminal nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain (D60-R249), the C-terminal RdRp domain (S367-F920) as well as an interface domain (A250-R365) connecting both. At the N-terminus a beta-hairpin motif binds at the interface between the palm subdomain and the NiRAN domain. The catalytic domain is a right hand RdRp domain, consisting of three subdomains: the finger, palm and thumb. Together with the thumb, the long extended finger forms a closed-ring structure. It contains the motifs A-F that are highly conserved among viral RdRps and motif G . Motifs A-E compose the active site at the palm subdomain, with motif C binding to the 3’ end of the RNA template. The motifs F and G are located in the fingers and are responsible for the positioning of the RNA template, while the first turn of the RNA is bound between the fingers and the thumb. The fingertip consists of the motif F, interacting with the thumb subdomain and the finger extension loop. The finger extension loops, which themselves are supported by interactions with the nsp7-nsp8.1 heterodimer, stabilize the fingertip loop. The main part of the interaction of the nsp7-nsp8 complex with nsp12 has the nsp7 above the thumb subdomain, while it stabilizes the conformation of the finger extension loops. The second nsp8 (nsp8.2) binds to the top of the finger subdomain and the interface domain and forms a significantly different conformation than nsp8.1. For entering the catalytic chamber, the RNA gets guided through the template entrance, stabilized by the fingertip and finger extension loops. At the back side of the palm subdomain is a channel for the nucleotide triphosphate to enter the active site. The RNA duplex, made from template and product, can exit at the front of the polymerase, and has later to be separated in further steps by other nsps in order to function. Studies suggest an interaction between the nsp12 and the helicase nsp13 (PDB: 6XEZ), as stable complexes can be formed of these two proteins. The helicase in complex with the replication-transcription complex might play a role in possible backtracking. The to date published structures of complexes with nsp12 include 6M71, 6NUR, 6BTF, 7C2K, 7BZF, 7BV2, 6XEZ, 6XQB, 7CTT, 7BV1, 7BW4 and 6YYT. Variations The sequences of the nsp12 in SARS-CoV and SARS-CoV-2 have a high degree of identity (95.8%) as well as a high similarity in their structure. The amino acid substitutions occur in all of the three subunits, but are not part of the active site or the interfaces between subunits of the polymerase. A study on their enzymatic behavior showed a much lower efficiency of the SARS-CoV-2 nsp12-nsp7-nsp8 complex of around 35% compared to SARS-CoV. A cross-combination analysis of the different proteins of both viruses showed that the variations in nsp12 and nsp8 are the cause of the negative effect on the polymerase activity. Further a comparison on the thermostability of the proteins showed a lower melting temperature for nsp12 and nsp8 compared to those of SARS-CoV. Relevance Through its essential role in the virus’s life cycle, the polymerase complex, especially nsp12, is the primary target for studies on nucleotide analog antiviral inhibitors. An example is remdesivir, an adenosine nucleoside triphosphate analog, acting with a delayed chain termination mechanism. It has been authorized as a drug for emergency use by the US Food and Drug Administration. See also Coronavirus_Disease 2019 (COVID-19)