RNA synthesis initiation by the nonsegmented negative-sense RNA virus polymerases
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Nonsegmented negative-sense RNA viruses (nsNSVs) include the human pathogens respiratory syncytial virus (RSV) and Ebola virus (EBOV). The genomes of these viruses are replicated and transcribed by a virus-encoded RNA dependent RNA polymerase (RdRp). The RdRps of nsNSVs initiate RNA synthesis in a primer-independent (de novo) manner. RSV RdRp initiates replication opposite the uridine at template position 1 (T1U) and transcription opposite the cytidine at template position 3 (T3C). The EBOV RdRp initiation site(s) was not known. We sought to better understand nsNSV RNA synthesis initiation by investigating the factors affecting initiation including promoter sequence, initiating NTPs, and RdRp structural determinants. We investigated the importance of promoter sequence for ebolavirus RNA synthesis initiation. Most nsNSVs have complementary genomic ends, however published ebolavirus sequences lack complementarity. We sequenced ebolavirus genomic termini and discovered the 3′ end has sequence variability and an overhang compared to the 5′ end of the replication product. Characterization of promoter sequence requirements for initiation revealed ebolavirus RdRp initiates internally (T2C) in contrast to most nsNSV RdRps, including RSV RdRp which initiates replication from T1U . We investigated the role of promoter sequence along with initiating NTPs for RSV RNA synthesis initiation. We showed that initiation by the RSV RdRp at T1U and T3C occurred independently of each other with the same RdRp selecting the two sites. Site selection could be modulated by the relative concentrations of ATP versus GTP. RSV template mutation analysis indicated the RdRp could bind ATP and CTP, or GTP, independently of template nucleotides. The data suggest a model in which the RdRps innate affinity for particular NTPs, coupled with a repeating sequence element within the promoter, allows precise initiation of replication at T1U or transcription at T3C. Other RNA virus polymerases utilize a priming residue to form base stacking interactions with the initiating NTP and stabilize the initiation complex. The RSV RdRp priming residue has not been described. Our data suggest RSV RdRp large subunit L proline 1261 is involved in initiation from T1U and T3C, but not RNA elongation. Alanine substitution analysis revealed the initiation defect at T3C, but not T1U, was partially rescued by increasing initiating NTP levels. These data suggest proline 1261 is the RSV priming residue and is more strictly required for terminal initiation (T1U) than internal initiation (T3C). The nsNSV have divergent initiation mechanisms and better understanding will aid in rational antiviral and vaccine design.