De novo synthetic virus RdRP initiation-extension conversion mechanism

Nucleic acid polymerase is a key protein machine for nucleic acid biosynthesis and plays a central role
in important processes such as replication, transcription, and reverse transcription.
The process by which polymerases initiate nucleic acid synthesis is also known as "initiation" and can be achieved through two basic modes: the first is called "primer-dependent", which requires the use of nucleic acids or proteins as primers, represented by the process of DNA replication; The other is called "denovo synthesis", which is initiated by the reaction of two nucleoside triphosphate (NTP) molecules to produce dinucleotide products, represented by the transcription process
.
The initiation process is usually sequence-specific, less stable and slow to synthesize, and polymerases need to further complete the transition from the initiation stage to the less sequence-specific elongation stage to achieve stable and rapid synthesis
of long-chain nucleic acids 。 RNA-dependent RNA polymerase (RdRP) encoded by RNA viruses is the necessary and only common gene for RNA viruses, and with the continuous impact of RNA viruses such as Ebola virus and the 2019 novel coronavirus in recent years, RdRP has increasingly become a hot spot
in basic and antiviral research of RNA viruses.
Similar to other nucleic acid polymerases, the initiation mode of RdRP is also divided into two types: primer-dependent and de novo synthesis, represented by poliovirus 3D^pol and dengue virus NS5, respectively, since the first three-dimensional structure of RdRP was resolved in 1997, although the structure and function of RdRP have been progressed, but how these two types of RdRP achieve the transition from initiation to extension has not been elucidated
.

Since 2011, the team of Gong Peng, a researcher from the Wuhan Institute of Virology, Chinese Academy of Sciences, has been committed to the study of the initiation-extension conversion mechanism of de novo synthetic RdRP, and has recently successfully elucidated the initiation-extension conversion mechanism
of dengue virus NS5 RdRP through more than ten years of "predecessor-succession" exploration 。 The team analyzed the crystal structure of the elongation complex (EC) formed by dengue virus NS5 RdRP with a resolution of 2.
6-2.
9 angstroms and template-product double-stranded RNA (Figure A) (PDB numbers 7XD8 and 7XD9, data collected at Shanghai Light Source BL19U1 and BL17U1 line stations, respectively), and found that the "priming element" (PE, A circular structure located in the thumb domain of RdRP) exits the active center of RdRP and refolds into a new morphology, while the initiator that undergoes the allosteric process establishes a new interaction with the double-stranded RNA in the extension complex (Figure B).

The research team further discovered that the allosteric initiator improved the stability of the elongation complex through a variety of enzymatic characterization methods, thereby confirming that the initiator element can play a completely different function through allosteric in the two important stages of initiation and extension of RdRP synthesis (Figure C).

Due to the high sequence and structural diversity of initiation elements of de novo synthetic RdRP, the research team proposes that the details of the initiation element of different RdRPs may be quite different, but the mode of their dual functions in the initiation and extension stages may be the same (Figure D), that is, to achieve "similarity"
.

This work is an important breakthrough made by the team in the field of RdRP on the basis of the early analysis of the full-length three-dimensional structure of flavivirus NS5 (PLoS Pathogens 2013) and the successful assembly of the flavivirus NS5 extension complex (PLoS Pathogens 2020), and also contributed to the poliovirus 3D^pol, The initiation-extension conversion mechanism of primer-dependent RdRP such as the 2019 novel coronavirus nsp12 provides an important reference
.
The related paper was recently published online in Proc Natl Acad Sci U S A (Proceedings of the National Academy of Sciences), which was supported by the National Key Research and Development Program of China (2018YFA0507200, the project leader is Professor Chen Xinwen), the National Natural Science Foundation of China (32070185; 32000136), supported
by the project of the Youth Innovation Promotion Association of the Chinese Academy of Sciences.
Wu Jiqin, a young researcher at Wuhan Institute of Virology (who realized the assembly of the extended complex, mainly completed structural biology research) and Wang Xinyu, a doctoral student (solved the problem of the solubility of the extended complex and screened the crystallization conditions of the complex, mainly completed the enzymatic research) were the co-first authors of the paper, experimentalist Liu Qiaojie (participated in enzymatic research) and doctoral student/assistant researcher Lu Guoliang (participated in structural biology research, now an associate researcher of Fudan University) as co-authors, and researcher Gong Peng was the corresponding author of the paper
。

Links to the papers and previous series are:

style="font-family:;line-height: 150%;;font-size:16px" _msthash="101743" _msttexthash="6416423">https://journals.
plos.
org/plospathogens/article/file?id=10.
1371/journal.
ppat.
1008484&type=printable

https://journals.
plos.
org/plospathogens/article/file?id=10.
1371/journal.
ppat.
1003549&type=printable

Figure: De novo synthetic virus RdRP initiation-extension conversion mechanism
.
A) Dengue virus NS5 RdRP extended complex crystal structure
.
B) initiating element allosteric (left to right) and establishing a new interaction
with double-stranded RNA.
C) Initiation elements improve the stability of
the extension compound.
D) Initiating component decomposition diagrams (analogies with GIFs).

GIF: The RdRP initiator element is dual-function through allosteric transformation, similar to the low-speed mode switching achieved by deformation of bicycle transmissions