MOLECULAR CHARACTERIZATION OF HAV

HAV在细胞培养中通常不致细胞病变。几个高细胞 培养适应的菌株已显示出对 一些细胞基质。其中一个是从我们的原型菌株中提取的， HM175。我们已经克隆并测序了这种HM175的致细胞病变变体， 将这个序列与亲本病毒进行了比对有44个变化， 与非致细胞病变细胞培养适应亲本相比，致细胞病变病毒 病毒这些变化目前正在研究个别网站指导 诱变和通过替换cDNA片段分组，以确定 导致表型改变的突变III与 博士柠檬在U在北卡罗来纳州，已经证明这些细胞病变 变异似乎是由病毒变异的基因重组引起的， 在细胞培养中。甲型肝炎病毒基因组5'非编码区的作用 翻译正在通过突变分析进行研究。相比 脊髓灰质炎病毒，似乎前50个碱基是至关重要的， 体外病毒蛋白的翻译。HAV似乎有一个独特的VP4 蛋白大多数小核糖核酸病毒具有75至80个氨基酸的VP4。HAV VP4只有23个氨基酸。我们将VP4替换为 脊髓灰质炎病毒用于HAV的VP 4，以确定非常小的VP 4是否赋予 HAV的任何生物学特性。使用一种新技术， 聚合酶链反应和定点突变 产生了嵌合病毒，用精确的脊髓灰质炎VP4代替了 HAV VP4.研究的其他小核糖核酸病毒的VP4具有其氨基末端， 甲硫氨酸裂解并加入肉豆蔻酸酯。HAV具有潜在的肉豆蔻酰化 VP4的氨基末端内的位点4氨基酸。突变分析 该位点表明肉豆蔻酰化在HAV中不发生。我们 表达与病毒蛋白酶结合的HAV的整个衣壳区， 一种杆状病毒来生产合成的空衣壳。

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.