Functional analysis of the L Protein of Nipah Virus Using

Nipah virus (NiV) was identified in Malaysia in late September 1998 as the etiological agent of an outbreak of acute encephalitis with the high mortality rate in human. NiV polymerase gene (L) encodes RNA-dependent RNA polymerase (RdRp) which is an enzyme required for viral replication. The aim of this study is to investigate the role of the N terminal of polymerase protein in replication of NiV. Based on the extensive deduced amino acid sequence analyses of a number of L proteins of non-segmented negative-strand (NNS) RNA viruses, a cluster of high-homology sequence segments have been identified within the body of the L proteins. The functional characterization of the NiV L polymerase was addressed in this research by generating a series of progressive N- deletions in the cloned gene and testing them in a minigenome assay. Five mutations that delete increasing amounts of the amino terminus of the L protein (20 amino acids) were generated at the amino-terminal. The ability of new recombinants plasmids in viral replication were tested by using minigenome system which is based on an intracellular and plasmid-based replication assay. This system contains an inserted chloramphenicol acetyltransferase (CAT) gene, for readout of the replication of the mini genome directed by wild type or mutant L protein. The results demonstrated that the first 100 amino acids of the NH2-terminal domain spanning a highly conserved motif are directly involved in transcription of the genome RNA. The possible functional significance of the NH2-terminal domain of paramyxovirus L protein is discussed