Analysis of RNA Splicing in Woodchuck Hepatitis Virus by Splice Junction Mutagenesis

Abstract

Previous studies have identified two spliced transcripts of Woodchuck Hepatitis Virus (WHV). The larger spliced RNA transcript lacks an intron of 1300 nucleotides, and the smaller transcript lacks an additional downstream intron of 1104 nucleotides. Oligonucleotide directed site specific mutagenesis was used to create an alteration to the consensus splice acceptor sequence of the second intron. Mutant plasmid constructs containing WHY deficient in splicing capabilities were transfected into a HepG2 cell line and RNA was purified from these cells. Polymerase Chain Reaction- analysis of cDNA showed that the smaller RNA transcript was not produced. The cDNA analysis also revealed a cryptic splice donor which appeared to replace the mutated splice donor and produce a transcript with a second intron of approximately 450 nucleotides. This construct may be used in future studies to further our understanding of the role spliced RNA transcripts have in virus production.