A Genetic Investigation of Pregenomic RNA Expression in the Woodchuck Hepatitis Virus
McFalone, Mairi C.
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Woodchuck hepatitis virus (WHV) belongs to the hepadnavirus family along with its close relative human hepatitis B virus (HBV). HBV infection is a public health problem of worldwide importance. Because WHV is genetically homologous to HBV, it is an appropriate experimental model for studying disease pathogenesis. Two RN A transcripts have been detected from the small 3.3 kb genome. The longer of these two, termed pregenomic RNA, serves as the template for virus DNA replication. In previous work, it was discovered that two splicing events occur in the pregenomic RNA transcript. Mutating the donor site of intron 1 abolished expression of pregenome RNA. It is hypothesized that the donor 1 site may be a cis-acting sequence. A cis-acting sequence is one that affects the function its own DNA. The donor 1 site may interact with other sequences of DNA in the genome, and therefore be required for expression of pregenomic RN A by the virus. In a study of duck hepatitis B virus, a sequence termed pet was found to be required for pregenomic RNA expression, like the donor 1 site. However, pet was only necessary for 'pregenomic RN A expression if other downstream sequences were also present. The objective of this study is to look for downstream sequences that interact with the donor 1 splice site. First, WHY expression vectors were constructed with downstream deletions. Th,ese new vectors contained either a wildtype or mutant donor 1 site. These viruses were then assayed for pregenomic RN A expression. It was found that the genome deletions have no effect 0 n viral RN A expression in wild type virus. It was also discovered that genome deletions do not restore the expression of RNA in donor 1 mutant virus. Therefore, these downstream sequences appears not to interact with the donor 1 mutant site in mediating RN A expression. The role of the splice sites remains a mystery that needs to be investigated further.