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

Abstract

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.

Distinctive Features of the Respiratory Syncytial Virus Priming Loop Compared to Other Non-Segmented Negative Strand Rna Viruses

“…In a previous study, we showed that although the PIV-3 polymerase initiates only at position 1 of its own promoter, it initiates at positions 1 and 3 of the RSV promoter (like the RSV polymerase). Likewise, we showed that although MARV polymerase only initiates at position 1 of its own promoter, it too can initiate efficiently at an internal site at position 2 of the Ebola virus promoter (39). These findings indicate that while nsNSVs have differences in their promoters and sites of initiation, their polymerases have similar properties.…”

“…As shown in the alignment presented in Fig 1B, the filovirus MARV L protein does not have an aromatic residue corresponding to the rabies virus L Trp1180, but it does have a proline residue (Pro1217) that aligns with RSV L Pro1261 and an aromatic tyrosine residue (Y1218) that aligns with RSV L Trp1262. Since we have recently established an in vitro RNA synthesis initiation assay for the MARV polymerase (39), we used this approach to test the effect of substituting MARV L Pro1217 and Tyr1218 with alanine. The mutant L-VP35 complexes were isolated and tested in an on-bead RNA synthesis assay using an RNA template consisting of nucleotides 1-30 of the MARV le promoter sequence (Fig 7A and 7B).…”

“…However, at a detailed level, there are differences between them. For example, whereas rhabdoviruses, paramyxoviruses and filoviruses have a single initiation site in their promoters, the pneumoviruses, RSV and HMPV, have two initiation sites (9, 14, 30, 39, 40) and whereas as most nsNSVs investigated so far initiate RNA replication at position 1 of the promoter, ebolaviruses initiate at position 2 (41, 42). It is of interest to understand to what extent these different mechanisms reflect differences in the polymerase protein.…”

“…Under these limiting NTP concentrations, reactions that contained Mg 2+ did not yield detectable pppApC dinucleotide from position 1 of the tr promoter, with either wt L-P or the P1261A mutant (Fig 6C, lanes 4 and 5). However, in reactions containing Mn 2+ , a significant amount of product was generated by both polymerases (Fig 6C, MARV polymerase (39), we used this approach to test the effect of substituting MARV L Pro1217 and Tyr1218 with alanine. The mutant L-VP35 complexes were isolated and tested in an on-bead RNA synthesis assay using an RNA template consisting of nucleotides 1-30 of the MARV le promoter sequence (Fig 7A and 7B).…”

A comprehensive list of the replication promoters of Bunyavirales reveals a unique promoter structure in Nairoviridae differing from other virus families

“…The 5'-pppA is generated by viral RdRp that recognizes the opposite U as the template. As seen in several segmented and non-segmented RNA viral polymerases (18)(19)(20), bunyaviral RdRp synthesizes RNA from an internal nt, and not from the terminus of the template. In LACV, RNA synthesis is initiated with A using the U at the +4 position of the antigenome 10 (3'-UCAUCA) as the template during genome replication (9).…”