Fidelity rotator virus

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You will be subject to the destination website's privacy policy when you follow the link. The y -axis is on a log scale. Error bars represent deviations in nonlinear regression analysis. However, K d,app for wobble base pair in KR Based on the specificity for UTP, we computed the discrimination factor for both wild-type and KR, which serves as a quantitative measurement for the transcription fidelity by computing the ratio of specificities for cognate CTP:G template and wobble UTP:G template.

To further explore the molecular mechanisms by which KR enhanced the incorporation rate k pol in comparison with wild-type Figure 5A , we performed MD simulations to survey the active site of wild-type and KR mutant RdRps five ns simulations for each system, see Supplementary Sections 1.

Overall, the KR mutant not only preserves distances that are critical to catalysis but also forms extra interactions between KR and CTP to further stabilize the catalytically active conformation. This provides the mechanistic interpretation for the higher k pol in KR Figure 5A.

KR mutation accelerates the incorporation of the cognate CTP. Overall, the distance analysis from MD simulations explains the closer distance between KR and substrate NTP can lead to a faster incorporation rate k pol. It is noted that the sufficient space in the active site of IAV RdRp is important to ensure the KR mutant with a longer side chain while maintaining the positive charge could work together with R to approach the triphosphate moiety of CTP without imposing any steric hindrance, thereby exerting a higher propensity for withdrawing electron density from the phosphate and facilitating the phosphodiester bond formation.

This mutant showed increased fidelity and resistance to nucleoside reverse transcriptase inhibitors NRTIs but resulted in a significant reduction in retroviral transcription activity We noticed that the specificity and fidelity are also contributed by K d,app in addition to k pol.

For NTP loading, it is challenging to directly quantify its contribution to K d,app. This observed conformational flexibility together with the results of the free energy calculations explains how the KR mutation deters the UTP binding i. Altogether, the single-turnover NTP incorporation kinetics assays allow us to quantify the transcription fidelity of KR mutant against the wild-type RdRp from the derived incorporation constant k pol and apparent substrate dissociation constant K d,app for the cognate CTP and the noncognate UTP.

The longer side chain imposed by the KR mutant renders the residue closer to the triphosphate moiety of CTP and thereby considerably facilitates the incorporation of the cognate CTP faster k pol , leading to higher transcription efficiency than the wild-type. Meanwhile, the mutant shows lower transcription efficiency to incorporate the noncognate UTP mainly due to the higher K d,app for the KR mutant.

More importantly, our findings suggest that the higher fidelity of KR mutant is originated from higher transcription efficiency for incorporating cognate NTP over noncognate NTP. This discovery is distinct from previous studies where a more accurate NTP incorporation is always accompanied by a slower transcription activity 64— Hence, in our single-turnover NTP incorporation assay, other than the primary extension products initiated from the third base G , the mismatched products extended from the second base C may also contribute to the single-cycle extension signal for CTP or UTP incorporation, which could probably interfere with the measurement.

Our discovery of the KR mutant IAV may have significant implications to vaccine development by overcoming two main challenges. First, most high-fidelity mutant RNA viruses were identified through blind passages of the virus in the presence of antiviral drugs. Second, to harness fidelity variants as stable attenuated virus strains for the vaccine design, the fidelity variant mutant viruses should possess high fidelity and high activity.

However, thus far, the accurate transcription of identified high-fidelity viruses always exhibits reduced polymerase activity in various degrees. To address these challenges, we here adopted a mechanistic-insight driven approach to discover a novel variant in IAV RdRp. To achieve this, we employed the single-turnover NTP incorporation assay, which can selectively measure the specificity of cognate versus noncognate NTP tightly to quantify fidelity, and coupled with all-atom MD simulations to explore the molecular explanation on the mechanism of altered incorporation rate.

We showed here, as the first exception, that a single Lysine-to-Arginine mutation on the IAV RdRp can enhance both the incorporation rate and fidelity. In the future, we anticipate that this mutation holds great promise for the development of vaccines owing to its high fidelity for fast attenuation in infection while maintaining a high profile in viral replication for mass production.

After removing the adapter, the sequencing reads were mapped to the reference IAV genome. With the high coverage of both systems, we were confident to further compute SNVs by following a previously published method The NGS results indicated that the KR mutant live virus had a significantly lower mutation rate than that of the wild-type live virus, which are also consistent with our findings based on biochemical assays and confirm that KR is indeed a high-fidelity mutant at the live virus level.

NGS sequencing on wild-type and KR mutant virus genome. The plaques were visualized by crystal violet stain. B The genome-wide depth distribution of sequencing reads from P3 wild-type and KR viral genome.

D The number of SNV was screened and reported in the bar chart. On the contrary, the wild-type K is preserved because it exhibits high polymerase activity and optimum fidelity without impairing viral fitness. Indeed, the high-fidelity phenotype KR is only present in a small number of swine and avian strains e. In this study, we utilized a multiple-level approach by integrating enzymatic, virological assays, MD simulations to elucidate that the highly conserved R and K are essential for transcription activity and fidelity in IAV.

More intriguingly, we found that K is critical to maintaining transcription activity in IAV due to its positively charged side chain. These remarkable properties obtained from a single lysine-to-arginine mutation have not been characterized before, which renders this mutant a promising target for the structural-base design of attenuated vaccine development and a model to study the transcription rate and fidelity control of viral polymerases.

The authors would like to thank Prof. Aartjan te Velthuis, Prof. George Brownlee, and Prof. Chris Ka Pun Mok for sharing the plasmids. The authors would also like to thank Prof. Peng Gong, Dr. Jinping Lei, Mr.

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Picelli S. Full-length RNA-seq from single cells using Smart-seq2. Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. Fast and accurate short read alignment with Burrows-Wheeler transform. The U. Ministers believe the move is logical now that omicron cases in the U.

Johnson said omicron would account for the majority of cases in London by Tuesday. There are now proven cases of the more infectious type of the virus to date in the country, up from 33 cases on Dec. The city of Tianjin reported the first case of omicron in mainland China Monday, the Global Times reported , citing the city. The infection came from an overseas arrival and the patient is receiving treatment at a designated hospital, the Chinese news outlet reported.

Prime Minister Boris Johnson repeatedly declined to rule out imposing further coronavirus restrictions before Christmas to tackle the spread of omicron, as he confirmed the first U.

Johnson also said omicron would account for the majority of cases in London by Tuesday. A plan to replace self-isolation for contacts of positive Covid cases was hampered when the government website ran out of free at-home rapid lateral flow tests. Biden to nominate Raskin, 2 others to Fed board. Load Error. Microsoft and partners may be compensated if you purchase something through recommended links in this article.

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