Researchers reveal novel mechanisms of viral RdRP nucleotide addition cycle

RNA viruses include a variety of pathogenic agents and pose a threat
to human health.
The genome replication and transcription process of RNA viruses requires RNA-dependent RNA polymerase (RdRP) to be led by its own encoding
.
The process goes through initiation, elongation, and termination phases and consists
of thousands of nucleotide addition cycles (NACs).
The mechanism of this cycle is not only an important content to understand the essential characteristics of RNA viruses, but also an important basis
for the development of antiviral strategies against RdRP.
Existing studies on viral RdRP have shown that each NAC undergoes four steps: reaction substrate NTP enters the RdRP activity center; RdRP active site closure; The nucleotide transfer reaction occurs (a nucleotide is added to the 3'-end of the product RNA); RdRP translocates the next template nucleotide translocation, thus initiating the next cycle
.
In the past ten years, structural biology and enzymology studies have basically completely outlined the mechanism of this NAC, but the RdRP structure information when the catalytic reaction (step 3) is about to occur has not been accurately obtained, so there is controversy
about which amino acid residues play a key role in the catalytic reaction.
The team of Gong Peng, a researcher at the Wuhan Institute of Virology, Chinese Academy of Sciences, has long been engaged in the study
of the catalytic mechanism of virus RdRP.
Recently, the team made multiple attempts in the crystal immersion experiment of enterovirus 71 (EV71) RdRP-RNA complex, and resolved the RdRP crystal structure (Figure A, PDB number: 7W9S, resolution 2.
5 angstroms) that is highly close to the state in which the catalytic reaction occurred, and found that a lysine residue K360 located near RdRP motif D has a close interaction (3.
3 angstroms) with the g-phosphate of the substrate CTP in the structure
。 In previous studies of poliovirus, which is an enterovirus with EV71, equivalent K359 residues were thought to be directly involved in catalyzing the reaction and the proton transfer process therein, and the close interaction observed in the crystal structure above further suggests this possibility
。 Accordingly, the team selected the K360 residue of EV71 RdRP and another motif F arginine residue R174 that interacts with the phosphate group of CTP as mutation sites to design a series of mutants, and used enzymatic methods to use enzymatic methods to use the single-step extension rate constant and CTP Michael's constant (Figure B) of wild-type RdRP and mutants.
Various parameters and properties such as the pH-dependent catalysis under the elongation state and pre-elongation state of RdRP were characterized
.
The results showed that the mutation of K360 site reduced the catalytic efficiency and affected the pH-dependent characteristics of RdRP in both the extension and pre-extension states, while the mutation of the R174 site caused a similar effect to K360 and could additionally reduce the affinity
of substrate CTP.
These data suggest that both K360 and R174 are directly involved in catalytic reactions, but whether these two residues are directly involved in proton transfer needs further evidence
.
The above results are another achievement of the team's NAC series of work (corresponding to NAC step 3) such as analyzing the partially closed structure of the active center of RdRP (corresponding to NAC step 2) and revealing the asymmetric transposition phenomenon (corresponding to NAC step 4) (PNAS 2016; Nature Communications 2020), and clarifying the whole process mechanism of this cycle (Figure C, GIF).

The results were published online in
Nucleic Acids Research.
The research work has been supported
by the National Key R&D Program, the National Natural Science Foundation of China, and the major biosafety science and technology projects of Jiangxia Laboratory of Hubei Province.