In vitro primer-based RNA elongation and promoter fine mapping of the respiratory syncytial virus.

Respiratory syncytial virus (RSV) is a nonsegmented negative-sense (NNS) RNA virus and shares a similar RNA synthesis strategy with other members of NNS RNA viruses such as measles, rabies, and Ebola. RSV RNA synthesis is catalyzed by a multifunctional RNA dependent RNA Polymerase (RdRP), which comprises of a large (L) protein that catalyzes three distinct enzymatic functions and an essential co-enzyme phosphoprotein (P). Here, we successfully prepared highly pure, full-length wild-type and mutant RSV polymerase (L:P) complex. We demonstrated that the RSV polymerase could carry out both de novo and primer-based RNA synthesis. We defined the minimal length of the RNA template for in vitro de novo RNA synthesis using the purified RSV polymerase is 8 nucleotides (nts), shorter than previously reported. We showed the RSV polymerase catalyzes the primer-dependent RNA elongation with different lengths of primers on both short (10-nt) and long (25-nt) RNA templates. We compared the sequence specificity of different viral promoters and identified positions 3, 5, and 8 of the promoter sequence are essential to the in vitro RSV polymerase activity, consistent with the results previously mapped by the in vivo minigenome assay. Overall, these findings agree well with previous biochemical studies and extend our understanding of the promoter sequence and the mechanism of RSV RNA synthesis.IMPORTANCE As a major human pathogen, respiratory syncytial virus (RSV) affects 3.4 million children worldwide annually. However, no effective antivirals or vaccines are available. An in-depth mechanistic understanding of the RSV RNA synthesis machinery remains a high priority among the nonsegmented negative-sense (NNS) RNA viruses. There is a strong public health need for research on this virus due to major fundamental gaps in our understanding of NNS RNA virus replication. As the key enzyme executing transcription and replication of the virus, the RSV RNA dependent RNA polymerase (RdRP) is a logical target for novel antiviral drugs. Therefore, exploring the primer-dependent RNA elongation extends our mechanistic understanding of the RSV RNA synthesis. Further fine mapping of the promoter sequence paves the way to better understanding the function and structure of the RSV polymerase.