MOLECULAR CHARACTERIZATION OF HAV

点击翻译按钮获取中文摘要

HAV is generally not cytopathic in cell culture. Several highly cell culture adapted strains have been shown to produce a cytopathic effect on some cell substrates. One of these was derived from our prototype strain, HM175. We have cloned and sequenced this cytopathic variant of HM175 and compared this sequence with the parental virus. There are 44 changes in the cytopathic virus compared to a non-cytopathic cell culture adapted parent virus. These changes are now being studied individually by site directed mutagenesis and in groups by substitution of cDNA fragments to determine the mutations responsible for the altered phenotype. Iii collaboration with Dr. Lemon at U. North Carolina, it has been shown that these cytopathic variants seem to arise by genetic recombination of viral variants arising in cell culture. The role of the 5' non-coding region of the HAV genome iii translation is being investigated by mutational analysis. In contrast to poliovirus, it appears that the first 50 bases are vitally important for translation of viral proteins in vitro. HAV seems to have a unique VP4 protein. Most picornaviruses have a VP4 of 75 to 80 amino acids. HAV appears to have only 23 amino acids in it VP4. We substituted the VP4 of poliovirus for the VP4 of HAV to determine if the very small VP4 confers any of the biologic properties of HAV. Using a novel technique combining the polymerase chain reaction and site directed mutagenesis we have produced chimeric viruses that have the precise polio VP4 in place of the HAV VP4. The VP4 of other picornaviruses studied have their amino terminal methionine cleaved and a myristate added. HAV has a potential myristylation site 4 amino acids inside the amino terminus of VP4. Mutational analysis of this site suggests that myristylation does not occur in HAV. We are expressing the entire capsid region of HAV combined with viral protease in a baculovirus to produce synthetic empty capsids.