Researchers propose a de novo synthetic virus RdRP triggers RNA synthesis and induces a fit mechanism

RNA virus transcription and genome replication processes do not involve the form of DNA, and require RNA-dependent RNA polymerase (RNA-dependent RNA polymerase, RdRP) encoded by the virus itself to complete it
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The precise and efficient initiation of the polymerization reaction by RdRP at a specific site is essential for maintaining the integrity of the viral genome and synthesizing the correct transcription product
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According to different priming methods, RdRP can be divided into a primer-dependent type and a de novo type.
The latter is usually assisted by a “priming element” (PE) located in the thumb domain to achieve precision.
Efficient initiation
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However, the initiating elements of RdRP of viruses of different families and genus show high diversity in amino acid sequence and three-dimensional structure, and existing studies have failed to use a universal mechanism to explain the initiation process of this type of RdRP
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Gong Peng's team, a researcher at the Wuhan Institute of Virology, Chinese Academy of Sciences, is dedicated to the study of the catalysis and regulation mechanism of viral RdRP
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Recently, the scientific research team analyzed the crystal structure of a classic swine fever virus (classical swine fever virus, CSFV) RdRP with a resolution of 3.
1 angstroms (Figure 1, PDB number: 7EKJ).
Complete structural information, and structural analysis shows that the initiating element is far away from the initiation site (up to about 9 angstroms), and the initiation can be achieved after undergoing transformation
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The team used enzymatic methods to further discover that a conserved glycine residue G671 in the priming element has an important contribution to the priming reaction
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Compared with wild-type (WT) RdRP, the initiation efficiency of the mutant protein at this site was significantly reduced, and the initiation rate constant kcat value of the most influential G671P mutant was reduced by about 7 times (Figure 2)
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At the same time, through the characterization of truncated RdRP with different lengths at the C-terminal, it was found that the 665-680 region of the initiating element tail played an important role in the initiation of RdRP
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Combined with a comparative study in CSFV's hepatitis C virus (HCV) and dengue virus (DENV), the team proposed that the initiating element of RdRP is in RdRP and template RNA and NTP.
When the substrate is bound, the initiation can be achieved by allosteric in the “induced fit” method, and then this mechanism may be generalized by investigating the structure and sequence characteristics of the initiating element of representative de novo RdRP (Figure 3)
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This research provides an important basis for a comprehensive and in-depth understanding of the initiation mechanism of viral RdRP
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Related papers were recently published online in Nucleic Acids Research (Nucleic Acids Research)
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The research work was funded by the National Key Research and Development Program "Study on the Co-infection and Co-pathogenic Mechanism of Important Poultry and Livestock Pathogens", the National Natural Science Foundation of China "Functional Research on the N-terminal Domain of the Swine Fever Virus NS5B Protein", and the Special Research Assistant of the Chinese Academy of Sciences support the project
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Figure 1.
The crystal structure of CSFV RdRP shows a relatively complete initiating element
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A.
The crystal structure of the HCV RdRP initiator complex; B.
The overall structure of the CSFV RdRP, in which the initiator element is purple-red; C.
The comparison of the structure of the Plastivirus RdRP initiator element; D.
The stereo pair of the CSFV RdRP initiator element and its surrounding area Image diagram; E.
Conservation analysis of the amino acid sequence of the initiating element of Plastivirus RdRP
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Figure 2.
Enzymatic characterization of CSFV RdRP and its mutants
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AB: Single-step initiation reaction kinetic analysis of WT (A) and G671P mutant (B)
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CD: Kinetic analysis of the initiation reaction of other mutants (C) and C-terminal truncation (D) at G671
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Among them, the Mie equation fitting curves of WT and G671P are taken from A and B respectively
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Figure 3.
The comparison of the structure of the initiating element of RdRP suggests that the de novo synthesis of RdRP may have a universal mechanism for inducing fit
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Comparison of initiating elements between AI and representative virus RdRP
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J.
Induced fit model when induced by de novo synthesis of RdRP
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When RdRP is in a non-substrate-bound state, the priming element can have a variety of conformations (upper left and Figure 1-4 below).
After RdRP binds to the template RNA and NTP, the priming element can undergo a conformational change and reach the end of the RNA template.
At the same time, NTP, which participates in the initiation reaction, is stabilized to achieve accurate and efficient initiation
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The arrow in the figure below indicates the direction of the conformational change required for the initiating element to reach its initiating state
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Source: Wuhan Institute of Virology, Chinese Academy of Sciences