The Respiratory Syncytial Virus Polymerase Has Multiple RNA Synthesis Activities at the Promoter

Abstract

Respiratory syncytial virus (RSV) is an RNA virus in the Family Paramyxoviridae. Here, the activities performed by the RSV polymerase when it encounters the viral antigenomic promoter were examined. RSV RNA synthesis was reconstituted in vitro using recombinant, isolated polymerase and an RNA oligonucleotide template representing nucleotides 1–25 of the trailer complement (TrC) promoter. The RSV polymerase was found to have two RNA synthesis activities, initiating RNA synthesis from the +3 site on the promoter, and adding a specific sequence of nucleotides to the 3′ end of the TrC RNA using a back-priming mechanism. Examination of viral RNA isolated from RSV infected cells identified RNAs initiated at the +3 site on the TrC promoter, in addition to the expected +1 site, and showed that a significant proportion of antigenome RNAs contained specific nucleotide additions at the 3′ end, demonstrating that the observations made in vitro reflected events that occur during RSV infection. Analysis of the impact of the 3′ terminal extension on promoter activity indicated that it can inhibit RNA synthesis initiation. These findings indicate that RSV polymerase-promoter interactions are more complex than previously thought and suggest that there might be sophisticated mechanisms for regulating promoter activity during infection.

Sensitivity of the Polymerase of Vesicular Stomatitis Virus to 2′ Substitutions in the Template and Nucleotide Triphosphate during Initiation and Elongation

“…Such a discrimination could be manifested by alterations to the nucleotide entrance tunnel, the template tunnels, the catalytic site itself, or a combination of all three. The ability to catalyze RNA synthesis on naked RNA has been established for negativesense RNA viruses that are pathogenic for humans, including respiratory syncytial virus (13). Such assays represent a significant advance in our ability to interrogate whether nucleotide analogs can block polymerase activity, but it will be important to consider how the template-associated N protein influences such results.…”

“…This latter assay has been replicated to study the biology of other negative-strand RNA viruses, including the significant human pathogens Machupo and respiratory syncytial virus. This is an achievement that holds promise in studies to define the activities of polymerase inhibitors that could serve as important start points for antiviral development (12,13).…”

An active site mutation increases the polymerase activity of the guinea pig-lethal Marburg virus

“…L, together with the MARV protein VP35, forms the RNAdependent RNA polymerase complex which is essential for transcription and replication of the viral genome . Based on the sequence alignment of MARV L and polymerases of other NNS RNA viruses, six conserved sequence regions have been identified, designated domains I-VI (Mühlberger et al, 1992), which are believed to constitute various enzymatic activities of L protein (Liang et al, 2015;Poch et al, 1990;Sidhu et al, 1993;Stec et al, 1991). Domain III of MARV L is anticipated to represent the RNA polymerase active site.…”

“…This site contains a GDNQ/E motif, which is highly conserved in polymerases of NNS RNA viruses (Magoffin et al, 2007;Mühlberger et al, 1992;Poch et al, 1990). The insertion of mutations within or in close proximity to the GDNQ/E motif has been shown to abolish polymerase activity of rhabdo-and paramyxoviruses (Magoffin et al, 2007;Malur et al, 2002;Noton et al, 2012;Schnell & Conzelmann, 1995;Sleat & Banerjee, 1993;Smallwood et al, 2002). Remarkably, three mutations in the MARV polymerase L (S741C, D758A and A759D)-located N-and C-terminal of the GDNQ/E motif (residues 744-747) have been detected in a MARV which became lethal for guinea pigs after sequential passages (Lofts et al, 2007).…”

“…Rhabdoviridae (Kim et al, 2014), Filoviridae (Ebihara et al, 2006) and Bornaviridae (Ackermann et al, 2007). This Cterminal component of L proteins of NNS RNA viruses, especially the region between domains V and VI, is highly variable, and considered as a domain of L which interacts with unique host cell transacting transcriptional cofactors (Dortmans et al, 2011;Poch et al, 1990;Sidhu et al, 1993). The mutations in guinea pig-lethal MARV L protein, however, affect the domain III close to the GDNQ/E motif, presumably directly modulating the processivity of viral RNA-dependent RNA polymerase (Liang et al, 2015).…”