Functional analysis of flavivirus methyltransferase using an improved infectiouscDNA clone of West Nile virus

Many flaviviruses are globally important human pathogens, including yellow fever virus, dengue viruses, and West Nile virus (WNV). Development of a reliable reverse genetic system is critical to study replication and pathogenesis of flaviviruses. Here I report the improvement of an infectious cDNA clone of an epidemic WNV strain (lineage I strain 3356). The full-length WNV cDNA was cloned into a low-copy-number plasmid pACYC177. The new cDNA clone exhibited superior stability compared with the previously developed pBR322-based clone, as evidenced by multiple rounds of transformation-isolation of the cDNA plasmid in E. coli followed by sequencing of the viral cDNA inserts. RNA transcribed from this clone was highly infectious. The cDNA clone was further improved by engineering a modified T7 promoter and a hepatitis delta virus self-cleaving ribozyme (HDVr) at the 5'- and 3'-end of the viral cDNA, respectively; in vitro transcription using this cDNA template results in RNA with authentic viral ends. The RNA with authentic ends yielded a specific infectivity over 110-fold higher than the RNA with extra terminal nucleotides. The robustness of the new infectious clone was further demonstrated by the ease of its utilization to prepare six recombinant WNV, each of which contained mutations derived from viruses continuously passaged in mosquitoes or mosquito C6/36 cells. The results demonstrate that the improved infectious cDNA clone will greatly facilitate WNV study.; The 5'-end capping is critical for the stability and efficient translation of most eukaryotic and viral mRNA. The plus-strand RNA genome of flavivirus contains a 5'-terminal cap 1 structure (m7GpppAmG). The flaviviruses encode one methyltransferase (MTase), located at the N-terminal portion of the NS5 protein, to sequentially catalyze guanine N-7 and ribose 2'-OH methylations during viral cap formation. Using the infectious WNV cDNA clone, I investigated the functions of residues within the MTase active sites in the context of complete virus. Mutant viruses depleted solely in 2'-O methylation (K61A, K182A, or E218A) are attenuated in cell culture and in mice and can be used to protect mice from challenge with wild-type WNV. However, a mutation (D146A) defective in both the N-7 and 2'-O methylations is lethal for virus replication; interestingly, partial rescue of N-7 MTase activity by a D146E mutation supported viral replication, but at a reduced level. The results demonstrate that the N-7 methylation activity is essential for the WNV life cycle, whereas 2'-O methylation activity is important, but not essential, for the WNV reproduction. Using a luciferase-reporting replicon of WNV, I found that N-7 methylation, but not 2'-O methylation, of viral RNA cap increases viral translation. In combination with biochemical analyses, the results suggest that WNV MTase methylates the N-7 and 2'-O positions of RNA cap using two distinct active sites and mechanisms. In addition, specific residues within a GTP-binding pocket (K13 and F24), and SAM-binding pocket (S56) are also important, but not essential, for cap methylation and viral replication. Overall, our data indicate that flavivirus MTase represents a novel target for antiviral therapy.