Comparison of RNA synthesis initiation properties of non-segmented negative strand RNA virus polymerases

It is generally thought that the promoters of non-segmented, negative strand RNA viruses (nsNSVs) direct the polymerase to initiate RNA synthesis exclusively opposite the 3 ' terminal nucleotide of the genome RNA by a de novo (primer independent) initiation mechanism. However, recent studies have revealed that there is diversity between different nsNSVs with pneumovirus promoters directing the polymerase to initiate at positions 1 and 3 of the genome, and ebolavirus polymerases being able to initiate at position 2 on the template. Studies with other RNA viruses have shown that polymerases that engage in de novo initiation opposite position 1 typically have structural features to stabilize the initiation complex and ensure efficient and accurate initiation. This raised the question of whether different nsNSV polymerases have evolved fundamentally different structural properties to facilitate initiation at different sites on their promoters. Here we examined the functional properties of polymerases of respiratory syncytial virus (RSV), a pneumovirus, human parainfluenza virus type 3 (PIV-3), a paramyxovirus, and Marburg virus (MARV), a filovirus, both on their cognate promoters and on promoters of other viruses. We found that in contrast to the RSV polymerase, which initiated at positions 1 and 3 of its promoter, the PIV-3 and MARV polymerases initiated exclusively at position 1 on their cognate promoters. However, all three polymerases could recognize and initiate from heterologous promoters, with the promoter sequence playing a key role in determining initiation site selection. In addition to examining de novo initiation, we also compared the ability of the RSV and PIV-3 polymerases to engage in back-priming, an activity in which the promoter template is folded into a secondary structure and nucleotides are added to the template 3 ' end. This analysis showed that whereas the RSV polymerase was promiscuous in back-priming activity, the PIV-3 polymerase generated barely detectable levels of back-primed product, irrespective of promoter template sequence. Overall, this study shows that the polymerases from these three nsNSV families are fundamentally similar in their initiation properties, but have differences in their abilities to engage in back-priming. Author summaryThe non-segmented negative strand RNA viruses are a large group of viruses that includes a number of significant human pathogens. Their viral genome is transcribed and replicated by a virally-encoded polymerase, which is considered to be a good target for intervention with antiviral drugs. Defining the similarities and differences between the polymerases of different viruses could be helpful for developing inhibitors with potency against a broad spectrum of related viruses. In this study, we examined the RNA synthesis initiation properties of polymerases of viruses from three different non-segmented negative strand RNA virus families and showed that they are fundamentally similar in the ways that they can initiate RNA synthesis. However, we also found that polymerases from two different virus families differed significantly in their ability to perform another activity, referred to as back-priming, which suggests that there might be structural differences between them. Thus, this study identifies similarities and differences between the polymerases in this group of viruses.

Automated summary

The study explores the RNA synthesis initiation properties of polymerases from three different non-segmented negative strand RNA virus families, revealing that they are fundamentally similar in their initiation properties but differ in their ability to engage in back-priming activity. This suggests potential structural differences between polymerases from different virus families and highlights the importance of understanding the similarities and differences in developing antiviral drugs with broad efficacy.