Researchers continue to achieve a better understanding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus behind the ongoing coronavirus disease 2019 (COVID-19) pandemic. One such effort has been reported in a new preprint on the bioRxiv* server, dealing with the phenomenon of backtracking.
Backtracking in viral replication
Backtracking refers to the retrograde movement of the RNA-dependent RNA polymerase (RdRp), which is the crucial enzyme within the SARS-CoV-2 replication-transcription complex. This enzyme comprises three viral non-structural proteins sp7/nsp82/nsp12, and requires many other cofactors to fulfill its function. These include nsp13 helicase and the nsp10/nsp14 proofreading assembly.
Backtracking is important in regulating transcription via the DdRp molecules. The DdRp and the transcription machinery are associated with a move backward on the DNA, but the RNA transcript picks its way backward through the complex. This results in a single-stranded 3’ RNA transcript that is pushed out through a secondary exit called the nucleoside-triphosphate (NTP)-entry tunnel.
The current study attempts to solve a puzzle created by the putative structural arrangement of nsp13 helicase bound to the replication-transcription complex (RTC) of the virus. This involves both nsp13 and RdRp undergoing translocation on the RNA strand in opposite directions, the former in the 5'->3' and the latter in the 3'->5' direction on the same template RNA strand.
If the nsp 13 helicase succeeds, it will push back the RdRp, in a reversible backward sliding motion. This backtracking has been earlier reported in the cellular DNA-dependent RNA polymerases.
The researchers postulated that helicase translocation mediates RdRp backtracking in SARS-CoV-2 replication, thus making it energetically favorable. This process would allow RNA proofreading and template switching to occur during the transcription of subgenomic RNA.
Nsp 13 binds template RNA strand
They constructed RNA scaffolds based on the original SARS-CoV-2 RTC-scaffold to test RdRp backtracked complexes (BTCs). They found that the holo-RdRp bound to the RTC scaffold, but could not bind efficiently to the BTC scaffold without nsp13.
In the presence of nsp13, stable complexes were formed, however. Using cryo-EM, they examined the nsp 13-RdRp-BTC assemble, and found two major divergences from the nsp 13-RTC structures. Firstly, a template-RNA-nsp 13 complex was seen, while secondly, a single-stranded p-RNA 3’ segment was extruded into the secondary NTP-entry tunnel.
The engagement of the single-stranded 5'-segment of the t-RNA with the nsp 13 occurred in a seven-nucleotide stretch between +14 to +8. A stretch of five nucleotides connected the t-RNA between the engaged nsp 13 and the RdRp in a disorderly manner.
Backtracked RNA extruded into NTP-entry tunnel
The finding of the extruded segment into the RdRp NTP-entry tunnel confirmed the presence of a BTC that is strongly analogous to the DdRp BTCs.
The researchers had previously resolved the architecture of the DdRp active site cleft, which was split into a channel for the DNA template strand and the NTP-entry tunnel. Of these, one went above and the other below the DdRp bridge helix.
In a similar manner to the bridge helix, the F motif of the viral RdRp separates the two strands for the RNA BTC. The presence of this entry tunnel allows an energetically favorable environment without steric hindrances. This permits the backtracked RNA to leave the active site while avoiding obstructive polar interactions between the RNA and the protein.
This tunnel has an electrostatic surface containing positively charged arginine and lysine residues on the motif F, completed by the conserved residues of the RdRp motifs C and E.
Nsp 13 promotes backtracking
The SARS-CoV-2 wild-type holo-RdRp requires the nsp13 helicase for efficient binding to the BTC-scaffolds. This is not the case with the holo-RdRp containing nsp12 substituted with D760A.
Nsp12-D760 is a residue on the RdRp motif C that removes an essential magnesium ion from the catalytic complex. Magnesium is absent, however, from all substrate-empty RdRp structures such as the BTC in this case.
The removal of D760 stabilizes BTC-binding to the scaffolds. This is because the original amino acid prevents tracking of the phosphate backbone of the backtracked RNA without magnesium ions, according to the BTC structures suggested in the current study.
Alternatively, the nsp 13 helicase is required to overcome this energy barrier. They found that the highest probability was of a post-translocation state for the RTC, with a 4-thio-U residue being sequestered in the RNA-RNA hybrid. While this makes it unavailable for crosslinking with the nsp12, the addition of nsp 13 increases crosslinking markedly.
Misincorporation promotes backtracking
Moreover, the study also shows that if a mismatched nucleotide is added to the growing strand of p-RNA at the 3’ end, it undergoes spontaneous fraying to enter the RdRp NTP-entry tunnel about 60% of the time, whereas a matched nucleotide spent 100% of the time in the active catalytic site pocket.
These findings indicate the existence of a secondary tunnel to incorporate backtracked RNA, which is important for proofreading during RNA synthesis to ensure fidelity of transcription. This is analogous to the cellular DdRps, though not related to them evolutionarily, indicating that this feature is essential for transcription enzymes.
Mismatched nucleotides spent more than 50% of their time frayed from the template strand, either within or towards the NTP-entry tunnel, which probably prevents further translocation and elongation of the strand. This is further promoted by the electrostatic and steric conditions of the NTP-entry tunnel that encourage backtracking.
Since translocation is prevented, the nsp 13 helicase may bind tightly to the single-stranded t-RNA, which facilitates helicase-mediated backtracking of the complex more easily via its 5'->3' translocation activity.
SARS-CoV-2 backtrack complex. A. RNA scaffolds: (top) replication-transcription complex (RTC) scaffold (14); (bottom) backtrack complex scaffolds (BTC3 and BTC5). B. A native gel electrophoretic mobility shift assay reveals that holo-RdRp requires nsp13(ADP-AlF3) to bind the BTC scaffolds efficiently. C. Cryo-EM structures of SARS-CoV-2 BTCs.
What are the implications?
The study thus validates an important prediction of the existing model of subgenomic transcription and proofreading, centered on template switching. The ability of SARS-CoV-2 to backtrack proves that template switching can occur via this process.
Secondly, the incorporation of antivirals like remdesivir, that is, nucleotide analogs, that are incorporated into the product RNAs by RdRps, could lead to backtracking. The misincorporation stops RdRp activity, so that nsp13 engages with the template strand downstream and triggers backtracking.
The extrusion of the p-RNA 3'-end from the NTP-entry tunnel would then allow the viral proofreading components to break it down, and so remove the mismatched nucleotide. This activity is key to the resistance of coronaviruses against multiple nucleotide analog antivirals.
Understanding RdRp backtracking and its potential role in CoV proofreading can facilitate the development of therapeutics.”
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.