Perturbation in the conserved methyltransferase-polymerase interface of flavivirus NS5 differentially affects polymerase initiation and elongation.J Virol.

　　Wu J, Lu G, Zhang B, Gong P.

      J Virol. 2014 Oct 15. pii: JVI.02085-14.

      Abstract

　　The flavivirus NS5 is a natural fusion of a methyltransferase (MTase) and an RNA-dependent RNA polymerase (RdRP). Analogous to DNA-dependent RNA polymerases, the NS5 polymerase initiates RNA synthesis through a de novo mechanism and then makes a transition to a processive elongation phase. However, whether and how the MTase affects polymerase activities through intra-molecular interactions remains elusive. By solving the crystal structure of Japanese encephalitis virus (JEV) NS5, we recently identified an MTase-RdRP interface containing a set of six hydrophobic residues highly conserved among flaviviruses. To dissect the functional relevance of this interface, we made a series of JEV NS5 constructs with mutations of these hydrophobic residues and/or with the N-terminal 261-303 residues deleted. Comparing to the wild type (WT) NS5, full-length NS5 variants exhibited consistent up- or down-regulation of the initiation activities in two types of polymerase assays. Five representative full-length NS5 constructs were then tested in an elongation assay from which the apparent single-nucleotide incorporation rate constant was estimated. Interestingly, two constructs exhibited different elongation kinetics from the WT NS5, with an effect rather opposite to what was observed at initiation. Moreover, constructs with MTase and/or the linker region (residues 266-275) removed still retained polymerase activities, albeit at overall lower levels. However, further removal of the N-terminal extension (residues 276-303) abolished regular template-directed synthesis. Together, our data showed that the MTase-RdRP interface was relevant in both polymerase initiation and elongation, likely with different regulation mechanisms in these two major phases of RNA synthesis.

　　IMPORTANCE:

　　The flavivirus NS5 is very unique in placing a methyltransferase (MTase) to the immediate N-terminus of its RNA-dependent RNA polymerase (RdRP). We recently solved the crystal structure of the full-length NS5 that reveals a conserved interface between MTase and RdRP. Building on this discovery, here we carried out in vitro polymerase assays to address the functional relevance of the interface interactions. By explicitly probing polymerase initiation and elongation activities, we found that perturbation in the MTase-RdRP interface had different impacts on different phases of synthesis, suggesting that the roles and contribution of the interface interactions may change upon phase transitioning. By comparing the N-terminal truncated enzymes with the full-length NS5, we collected data to indicate the indispensability to regular polymerase activities of a region that was functionally un-clarified previously. Taken together, we have provided biochemical evidences and mechanistic insights for the crosstalk between the two enzyme modules of flavivirus NS5.

　　http://www.ncbi.nlm.nih.gov/pubmed/25320292