Counting errors sheds new light on viral activity

11 February 2015 by Communication TNW

TU Delft researchers have, in collaboration with their colleagues at the University of Helsinki, revealed the pausing behaviour of a viral RNA-dependent RNA polymerase, a key protein that is found in all RNA viruses. They also related this pausing behaviour to errors made by this protein.

These findings are of great importance for our understanding of the mutation rate by viruses and thus impact our ability to inhibit viral activity. The research results were published on February 12 in the open access scientific journal Cell Reports.

Viral RNA-dependent RNA polymerase
Many viruses store their genetic information in the form of single-stranded RNA. Even others encode their genome into double-stranded RNA. For such viruses, a single-stranded RNA copy is synthesized (transcribed) by an RNA-dependent RNA polymerase in order to infect their hosts. When new viral progeny are formed, RNA-dependent RNA polymerases also copy (replicate) this single-stranded RNA back into double-stranded RNA.

The errors in synthesis made by the RNA-dependent RNA polymerase play a crucial role in the viability of the virus: if the errors are too few, then the virus cannot synthesize slightly modified proteins that could enhance survival probability under changing conditions; conversely, if the errors are too numerous, the protein function may be impaired, threatening virus survival.

Thus, knowledge of the manner in which RNA-dependent RNA polymerase incorporates errors is not only important for understanding the mechanics of this particular case, but impacts our ability to inhibit viral activity.

Measuring transcription
In the course of their research, the scientists used a magnetic tweezer capable of measuring on individually tethered RNA molecules to follow the process of transcription by an RNA-dependent RNA polymerase in real time, with a resolution of a few RNA bases. A special feature of this magnetic tweezer configuration was that its read-out process was highly parallelized: the activity of hundreds of RNA-dependent RNA polymerases could be processed simultaneously, allowing the researchers to gather very large datasets. These datasets, which reported on the time spent by RNA-dependent RNA polymerase in transcribing many different short segments of RNA, could then be fitted to stochastic models of polymerase pausing.

Measurement of error rate
By repeating their measurements under differing conditions, the researchers could determine that pauses of a certain duration could be associated with the incorporation of an incorrect nucleotide (an ‘error’) by RNA-dependent RNA polymerase. They were able to measure the error rate under different conditions and also proposed a model that predicts under which conditions the polymerase should be particularly error prone. These findings are of great importance for our understanding of the mutation rate by viruses. This study was performed on the RNA-dependent RNA polymerase from the bacteriophage Φ6, but future studies will determine whether the same mechanism underlies error incorporation by RNA-dependent RNA polymerase from other viruses.

Figure 1. Schematic of the magnetic tweezers setup used to monitor the activity of an RNA-dependent RNA polymerase (denoted P2 RdRP). As the polymerase synthesizes a complementary strand to the existing RNA, the .omolecule tethered between the bead and the surface is converted from double-stranded to single-stranded RNA. During its synthesis process, the polymerase occasionally pauses. A particular class of these pauses could be linked to the incorporation of errors into the newly transcribed RNA strand by the polymerase.

For more information, please contact:

David Dulin, Igor D. Vilfan, Bojk A. Berghuis, Susanne Hage, Dennis H. Bamford, Minna M. Poranen, Martin Depken, and Nynke H. Dekker
"Elongation-Competent Pauses Govern the Fidelity of a Viral RNA-Dependent RNA Polymerase”
Cell Reports
(2015)
www.cell.com/cell-reports/fulltext/S2211-1247%2815%2900056-X

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