Abstract
Nucleotide addition cycle (NAC) is a fundamental process utilized by nucleic acid polymerases when carrying out nucleic acid biosynthesis. An induced-fit mechanism is usually taken by these polymerases upon NTP/dNTP substrate binding, leading to active site closure and formation of a phosphodiester bond. In viral RNA-dependent RNA polymerases, the post-chemistry translocation is stringently controlled by a structurally conserved motif, resulting in asymmetric movement of the template-product duplex. This perspective focuses on viral RdRP NAC and related mechanisms that have not been structurally clarified to date. Firstly, RdRP movement along the template strand in the absence of catalytic events may be relevant to catalytic complex dissociation or proofreading. Secondly, pyrophosphate or non-cognate NTP-mediated cleavage of the product strand 3′-nucleotide can also play a role in reactivating paused or arrested catalytic complexes. Furthermore, non-cognate NTP substrates, including NTP analog inhibitors, can not only alter NAC when being misincorporated, but also impact on subsequent NACs. Complications and challenges related to these topics are also discussed.
Highlights
RNA viruses are a large collection of diverse, rapidly evolving viruses with a wide host range covering bacteria and eukaryotes (Krupovic et al, 2018)
Numerous Nucleotide addition cycle (NAC)-state-related structures from various viral RNA-dependent RNA polymerase (RdRP) have provided a relatively complete structural view of the cognate NTP-driven NAC, several aspects directly or indirectly related to NAC have rarely been addressed structurally in RdRPs including nucleotide additionfree translocation, intrinsic product cleavage activities, and perturbation of NAC by non-cognate NTPs and NTP analogs. These events are possibly related to important events including but not limited to RdRP catalytic complex dissociation, proofreading, reactivation, fidelity control, and effective intervention
Pyrophosphorolysis did not occur in the crystal, solution trials mimicking the crystal soaking condition led to observation of PPi-mediated cleavage (Wang et al, 2020a) (Figure 1Ad–k), suggesting reverse translocation as a prerequisite of the cleavage of a post-translocation complex
Summary
RNA viruses are a large collection of diverse, rapidly evolving viruses with a wide host range covering bacteria and eukaryotes (Krupovic et al, 2018). Understanding the fundamental features of RNA viruses has become an attractive and rapid growing research area ever since the emergence of severe and acute syndrome coronavirus 2 (SARS-CoV-2) causing the coronavirus disease 2019 (COVID-19) (Zhou et al, 2020). One unique feature of RNA viruses is that their genome replication and transcription processes are DNA-independent, requiring a virally-encoded RNA-dependent RNA polymerase (RdRP) to carry out these essential processes of the virus life cycle (Wolf et al, 2018). Due to their essentialness and highest conservation level, RdRPs have become attractive targets to develop antivirals with high potency and/or broad-spectrum potential. Being considered the most conserved protein of RNA viruses, RdRPs are still quite diverse with respect to their global structure organization (Lesburg et al, 1999; Thompson and Peersen, 2004; Lu and Gong, 2013; Pflug et al, 2014; Liang et al, 2015; Jia and Gong, 2019; Kirchdoerfer and Ward, 2019), initiation mechanisms
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