Understanding respiratory syncytial virus genome synthesis

Factors affecting de novo RNA synthesis and back-priming by the respiratory syncytial virus polymerase

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Respiratory syncytial virus (RSV) is a paramyxovirus, one of the non-segmented negative strand RNA viruses. The long-term goal of our research is to understand how RSV mRNA, antigenome and genome synthesis are regulated. We previously showed that the RSV trailer-complement (trc) promoter, which lies at the 3´ end of the antigenome, is modified by back-priming, whereby the RNA folds into a panhandle structure and 1-3 nucleotides are added to the 3´ end by the viral polymerase. Here we used an in vitro assay to examine which factors influence this process. The key findings are that more than 20 nucleotides of trc sequence are necessary for back-priming to be efficient and accurate. The leader (le) promoter, which lies at the 3´ end of the genome, is predicted to have a more stable secondary structure than trc sequence. We found the le can also be modified by back-priming in vitro, but with an apparently lower efficiency than the trc promoter.

We embarked on this study to follow up our finding that the trc promoter, is modified by back-priming in vitro and in RSV infected cells. This was a surprising finding because back-priming can only occur if trc RNA folds into a secondary structure, but it is widely believed that paramyxovirus replicative RNA is completely encapsidated and devoid of secondary structure. We had also found that in contrast to the trc promoter, the le promoter is not modified during infection. This prompted us to dissect which sequences in the trc region are necessary for back-priming and to determine if sequence differences between trc and le account for why le is not modified in infected cells.

The findings that the efficiency of back-priming is not directly correlated to predicted RNA secondary structure stability, and that the number of nucleotides that are added is affected by sequence downstream of the back-priming site were surprising. These findings suggest that this process involves an intimate relationship between the polymerase and an extended region of trc RNA sequence. The finding that the le RNA sequence can support back-priming in vitro, even though this does not occur in infected cells, supports the idea that back-priming is regulated during infection. We have hypothesized that back-priming might be a mechanism for sensing if there is sufficient viral nucleoprotein for newly synthesized genome RNA to become encapsidated; in future work we hope to determine if this is the case.

In RSV infected cells and in vitro, the trc promoter sequence (left) can be modified by back-priming. The RNA folds into a secondary structure, with nucleotides 1 and 2 base-pairing with nucleotides 14 and 13, and one to three nucleotides (red) are added to the RNA 3´ end by the viral polymerase (orange oval). Back-priming can also occur on the le promoter sequence in vitro, but does not occur in infected cells.
In RSV infected cells and in vitro, the trc promoter sequence (left) can be modified by back-priming. The RNA folds into a secondary structure, with nucleotides 1 and 2 base-pairing with nucleotides 14 and 13, and one to three nucleotides (red) are added to the RNA 3´ end by the viral polymerase (orange oval). Back-priming can also occur on the le promoter sequence in vitro, but does not occur in infected cells.

Introducing the authors
Noton

Rachel Fearns (left) and Sarah Noton (right) from Boston University School of Medicine.

About the research

Factors affecting de novo RNA synthesis and back-priming by the respiratory syncytial virus polymerase
Virology, Volumes 462–463, August 2014, Pages 318–327
Sarah L. Noton, Waleed Aljabr, Julian A. Hiscox, David A. Matthews, Rachel Fearns

Read the full article on ScienceDirect.

 

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