KANAZAWA, Japan, July 29, 2024 /PRNewswire/ -- Researchers at Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, IMDEA Nanoscience (Madrid, Spain) and CNB-CSIC (Madrid, Spain) report in ACS Nano experiments that reveal a cycle of conformational stages that recombinant Influenza A genomes pass through during RNA synthesis.

Influenza A is a global health risk responsible for local epidemics and deadly pandemics. As such, the mechanism by which the virus replicates itself has attracted significant interest. Researchers led by Shingo Fukuda at Nano Life Science Institute (WPI-NanoLSI) at Kanazawa University in Japan, Jaime Martin-Benito at CNB-CSIC and Borja Ibarra  at IMDEA Nanociencia in Spain, used high-speed atomic force microscopy and electron microscopy to pin down the conformational dynamics of recombinant viral genomes (or rRNPs) during RNA synthesis.

Previous attempts to understand what possible conformational changes occur during Influenza A viral multiplication cycle had been hindered by what the researchers describe as the "bulky double-helical structure" of the viral RNPs (vRNP, Fig.1 A), which made it hard to see what was going on. As a result the researchers produced a circular recombinant ribonucleoprotein complex (rRNP) (Fig.1 B), which allowed them to overcome the 'bulky issue'. The authors used HS-AFM to follow conformational changes of individual rRNP complexes in real-time during active RNA synthesis (Fig.1 C).

Their work provides first direct experimental evidence showing that individual rRNPs can be recycled for multiple transcription and replication cycles. This is a key feature for viral multiplication. In addition, their study highlights how factors that affect the stability of the secondary structures of the nascent RNA affect the rate of RNA synthesis.

The authors concluded that the approach is useful for investigating viral transcription and replication mechanisms. "Transcriptional pausing is an intrinsic property of most RNA polymerases, and its regulation constitutes one of the central mechanisms of control of gene expression," they add. "Future single-molecule experiments of real-time RNA synthesis kinetics by the IAV RdRp [influenza A virus RNA dependent RNA polymerase] within the context of the RNP will help to elucidate the nature of putative pause states and their roles in viral transcription and replication."

Figure

https://nanolsi.kanazawa-u.ac.jp/wp/wp-content/uploads/Figure-1-2.jpg

Caption : Fig.1 (A, B) HS-AFM images of vRNP (A) and rRNP (B). (C) Successive HS-AFM images of rRNP during RNA synthesis. © 2024 Carlero, et al. Published by American Chemical Society

Glossary

RNA synthesis

Ribonucleic acid (RNA) is a polymer and a nucleic acid. Its replication is catalyzed by an enzyme known as RNA polymerase. The synthesis only proceeds in the presence of the nucleotide and proteins required to build up the RNA molecule.

Although RNA synthesis can use DNA as the synthesis template, a number of viruses replicate with RNA as the template. The enzyme that catalyzes this type of synthesis is known as RNA dependent RNA polymerase.

Recombinant ribonucleic proteins

Recombinant ribonucleoproteins are useful for ways of studying RNA processes. In this instance the authors used a recombinant ribonucleoprotein made up of the same protein components but just 352 nucleotides long, where the RNA segment used had been shown to avoid supercoiling.

High-speed atomic force microscopy

This imaging technique uses a nanosized tip at the end of a cantilever that is scanned over a sample. It can be used to determine the topography of a sample surface from the change in the strength of forces between the tip and the sample with distance, and the resulting deflection of the cantilever. It was first developed in the 1980s but a number of modifications have augmented the functionality of the technique since. It is better suited to imaging biological samples than the scanning tunneling microscope that had been developed because it does not require a conducting sample.

In the 2000s Toshio Ando at Kanazawa University was able to improve the scanning speed to such an extent that moving images could be captured. This allowed people to use the technique to visualize molecular processes for the first time.

Reference

Diego Carlero, Shingo Fukuda, Rebeca Bocanegra, Toshio Ando, Jaime Martin-Benito, and Borja Ibarra Conformational Dynamics of Influenza A Virus Ribonucleoprotein Complexes during RNA Synthesis ACS Nano 2024.

DOI：10.1021/acsnano.4c01362

URL: https://pubs.acs.org/doi/10.1021/acsnano.4c01362

Funding acknowledgements

This work was supported by the NanoLSI Visiting Fellows Program 2019 (to B.I.), the World Premier International Research Center Initiative (WPI), MEXT (Japan), and grants PGC2018-099341-B-I00 (to B.I.), PID2021-126755NB-I00 (to B.I.), and PID2020-117752RB-I00 (to J.M.B.) financed by MCIU/AEI/10.13039/501100011033 and FEDER, UE,  Grant TED2021-132748B-I00 financed by the European Union "NextGeneration EU"/PRTR (to J.M.B.), and Grant No. 20K15140 financed by JSPS KAKENHI (to S.F.). IMDEA Nanociencia acknowledges support from the Severo Ochoa Program for Centers of Excellence in R&D (CEX2020-001039-S).

Contact

Hiroe Yoneda (Ms)
Senior Specialist in Project Planning and Outreach
NanoLSI Administration Office, Nano Life Science Institute (WPI-NanoLSI)
Kanazawa University
Kakuma-machi, Kanazawa 920-1192, Japan
Email: nanolsi-office@adm.kanazawa-u.ac.jp
Tel: +81 (76) 234-4555

About Nano Life Science Institute (WPI-NanoLSI), Kanazawa University

Understanding nanoscale mechanisms of life phenomena by exploring "uncharted nano-realms"

Cells are the basic units of almost all life forms. We are developing nanoprobe technologies that allow direct imaging, analysis, and manipulation of the behavior and dynamics of important macromolecules in living organisms, such as proteins and nucleic acids, at the surface and interior of cells. We aim at acquiring a fundamental understanding of the various life phenomena at the nanoscale.

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About Kanazawa University

As the leading comprehensive university on the Sea of Japan coast, Kanazawa University has contributed greatly to higher education and academic research in Japan since it was founded in 1949. The University has three colleges and 17 schools offering courses in subjects that include medicine, computer engineering, and humanities.

The University is located on the coast of the Sea of Japan in Kanazawa – a city rich in history and culture. The city of Kanazawa has a highly respected intellectual profile since the time of the fiefdom (1598-1867). Kanazawa University is divided into two main campuses: Kakuma and Takaramachi for its approximately 10,200 students including 600 from overseas.

http://www.kanazawa-u.ac.jp/en/

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