key: cord-290218-dvyeg5fk
authors: Jiang, Yi; Yin, Wanchao; Xu, H. Eric
title: RNA-dependent RNA polymerase: Structure, mechanism, and drug discovery for COVID-19
date: 2020-09-04
journal: Biochem Biophys Res Commun
DOI: 10.1016/j.bbrc.2020.08.116
sha: 
doc_id: 290218
cord_uid: dvyeg5fk

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has rapidly become a global pandemic. Although great efforts have been made to develop effective therapeutic interventions, only the nucleotide analog remdesivir was approved for emergency use against COVID-19. Remdesivir targets the RNA-dependent RNA polymerase (RdRp), an essential enzyme for viral RNA replication and a promising drug target for COVID-19. Recently, several structures of RdRp in complex with substrate RNA and remdesivir were reported, providing insights into the mechanisms of RNA recognition by RdRp. These structures also reveal the mechanism of RdRp inhibition by nucleotide inhibitors and offer a molecular template for the development of RdRp-targeting drugs. This review discusses the recognition mechanism of RNA and nucleotide inhibitor by RdRp, and its implication in drug discovery.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was initially emerged in December 2019 [1, 2] . The exponential growth of infections and death cases makes COVID-19 a worldwide health threat, and the World Health Organization (WHO) declares it a global pandemic in March 2020. There have been over 20 million infections and 800,000 deaths in 188 countries as of August 2020 (GISAID database). Genome sequence analysis has revealed that SARS-CoV-2 belongs to the coronaviridae family. Its sequence is closely related to the severe acute respiratory syndrome activity. Motif F (residues L544 to V557) interacts with the phosphate group of incoming NTP.

Structurally, the side chains of K545 and R555 contact with the +1 base to direct the incoming NTP to the correct position for catalysis. Motif G (residues D499 to L514) interacts with the template strand and may direct the RNA template to the active catalytic site (Fig. 2E ).

The recent structures of SARS-CoV-2 RdRp in complex with double-stranded RNA helix reveal the recognition mechanism of substrate RNA by SARS-CoV-2 RdRp [17, 18] . Like other RNA polymerases, the primer-template entry, NTP entry, and the nascent strand exit routes are positively charged and solvent accessible, providing a preferable environment for RNA binding (Fig. 3A) . The primer-template RNA is embraced by the finger-palm-thumb subdomains (Fig. 3B) . A total of 41 residues from nsp12 direct contact with helical RNA, of which 26 residues to the template strand and 15 to the primer strand. No interactions are observed between base pairs of RNA and residues from nsp12, providing a structural basis for RNA sequence-independent binding of RdRp. Additionally, most of these interactions are mediated by the phosphate-ribose backbone, especially the 2'-OH groups, which offers a structural explanation for RdRp to distinguish RNA from DNA [17] .

Interestingly, the structure of complexed nsp12 is almost identical to nsp12 in apo RdRp, with an RMSD of 0.5 Å [17] , coinciding with the high processivity of the viral RNA polymerase, which does not need to consume extra energy for conformation changes in the active site during the replication cycle (Fig. 3B ). Although nearly identical conformation between the apo and RNA bound nsp12 structures, several subtle structural rearrangements can still be observed (Fig.3C ). Compared to the structure of apo RdRp complex, the loop in motif G (residues D449 to L514) moves outward by 2.2-3.0 Å (as measured by nsp12 residue S501), and loop (residues S682 to T686) in motif B by 1.7-2.0 Å (as measured by nsp12 residue A685) [17, 18] . The loop connecting the first and second helices of the thumb subdomain also shifts outward by 6.0 Å (as measured by nsp residue I847) [17] . These conformational A recent structure of RdRp-RNA complex represents a long template-product helical RNA strands exiting from the active core of RdRp, which is not observed in other RdRp-RNA complex [38] . The two copies of nsp8 exhibit long helical extensions at their N-terminus fragments, and serve as platforms for coordinating the exiting RNA backbones, forming positively charged "sliding poles" (Fig. 3D) [38] . These "sliding poles" are stabilized by interactions formed between the positively charged residues at the extended N-terminal of nsp8 and bases in RNA backbones ( Fig. 3E ) and reported to account for the known processivity of the RdRp, which is required for replicating the long coronavirus genomes [39] .

In this structure of RdRp in complex with the protruding RNA, the prominent nsp8-2 extends up to 28 base pairs away from the active site, which is a unique conformation not observed in structures of other RNA viruses [33, 34] and SARS-CoV-2 RdRp-RNA complexes [17, 19] .

The N-terminal ends of two copies of nsp8 direct to different orientations, which synergistically prevent premature dissociation of helical RNA from RdRp ( Fig. 3D and E).

Similarly, a pre-released structure of helicase-RdRp-RNA complex also exhibits a similar nsp8 conformation [40] , while structure of pre-translocated SARS-CoV-2 RdRp-RNA complex exhibits two conformations of nsp8-2 with a ~45º rotation at its N-terminus, one of which structurally overlaps with the "sliding poles"-like conformation of nsp8-2 [18] . The alanine mutation of K58, which located in the nsp8 extension, is lethal to the virus, supporting the model of "sliding poles" [26] . Collectively, these findings indicate that the N-terminal conformation of nsp8 is flexible in solution and dominates the "sliding pole"-like state during RNA elongation.

Although the sequence identity of nsp12 across the RNA viruses is low, the polymerase active site is structurally highly conserved, suggesting that RdRp inhibitors may serve as a potential J o u r n a l P r e -p r o o f broad-spectrum antiviral drug against RNA viruses. Remdesivir (RDV, GS-5734) is a 1'-cyano-substituted adenosine nucleotide analog prodrug, which is originally developed as a treatment for the Ebola virus by inhibiting RNA synthesis. It is metabolized into its active form (RDV-MP, GS-441524-MP), which has a monophosphate moiety to enhance intracellular metabolism into its active triphosphate metabolite (RDV-TP) (Fig. 4A ) [41] .

Like many nucleotide analog inhibitors, RDV-TP competes with the incorporation of nucleotide counterparts and inhibits transcription of viral RNA. The structure of the RdRp-RNA-RDV complex reveals a molecular mechanism for transcription inhibition by remdesivir [17] . Only one RDV-MP molecule incorporates into the primer strand at +1 position and forms base-stacking interaction with bases in primer strand and two hydrogen bonds with the uridine base from the template strand. RDV-MP also interacts with residues K545 and R555 in motif F. Near RDV-MP are two magnesium ions and pyrophosphate, whose densities are missing in other structures of SARS-CoV-2 RdRp-RNA complexes. The pyrophosphate may block the entry of NTP to the active site by occupying the entrance of nucleotides. Two magnesium ions contact with phosphate diester backbone and form polar interactions with two conserved aspartic acids in motif C (Fig. 4B) .

In contrast to other classic chain terminators, the featured inhibition mechanism of remdesivir is a delayed chain termination of nascent viral RNA at i+3 position [42] . When the RNA synthesis process to the i+3 position, the incorporated RTP will be at -3 position. The molecular simulation analysis shows that the 1'-cyano substituent of incorporated RDV sterically clashed with the side chain of S861, a residue faces with -3 position, and probably causes significant distortion of the position of RNA, hampering the translocation of RNA to the -4 position [42] . The S861A mutant abates the chain termination reaction, supporting this steric clash hypothesis for the delayed chain termination [18] .

The RdRp is critical for the replication of viral RNA, and also a promising drug target for COVID-19 treatment. Firstly, like other proteins of SARS-CoV-2, RdRp lacks the closed-related host cell counterparts. Thus, targeting RdRp may circumvent the off-target side effects. Secondly, compared to the spike protein and other virus surface proteins, the active catalytic motifs of the RdRp are highly conserved among RNA viruses, making RdRp an attractive antiviral drug target for a broad-spectrum of viruses. Several nucleoside analog inhibitors have shown inhibitory activities against a broad spectrum of RNA viruses [43] . therapeutics.

The replication and transcription of SARS-CoV-2 is a spatiotemporally regulated multi-step process, which is mediated by the replication-transcription complex composed by primarily the virus-encoded non-structural proteins. The field of structural studies on the RTC is rapidly developed, as witnessed by the copious amount of the recent RdRp complex structures J o u r n a l P r e -p r o o f [16] [17] [18] [19] 38] . Recently, a structure of SARS-CoV-2 holo-RdRp-RNA in complex with two molecules of the nsp13 helicase was pre-released, providing a structural basis for putative nsp13 helicase functions during viral genome replication and transcription [40] . The structures of SARS-CoV-2 RdRp and lessons learned from other coronaviruses have shed light on the mechanisms of RNA binding and inhibitor recognition of RdRp. However, the structural organization and the regulation mechanism of the replication-transcription complex have not been thoroughly studied. More high-resolution structures with diverse nsps organizations are essential to understand how these cofactors conduct the orchestra of RNA processing enzyme to achieve the replication and transcription of virus RNA. Moreover, the complete picture of intricate RNA processing will also provide new candidate targets for designing drugs against COVID-19. 

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