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.