MOLECULAR CHARACTERIZATION OF HAV

HAV 的分子表征

• 批准号: 3811273
• 负责人: S M FEINSTONE
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3811273
• 关键词: Hepatovirus; chemical structure function; fusion gene; genetic recombination; genetic translation; genome; insect virus; molecular cloning; mutant; nucleic acid sequence; peptidases; poliovirus; polymerase chain reaction; site directed mutagenesis; tissue /cell culture; virus DNA; virus cytopathogenic effect; virus envelope; virus genetics; virus protein

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.

HAV 在细胞培养物中通常不致细胞病变。几个高度细胞 培养适应菌株已被证明可产生细胞病变效应 一些细胞基质。其中之一来自我们的原型菌株， HM175。我们已经克隆并测序了 HM175 的这种细胞病变变体 将此序列与亲本病毒进行比较。共有44处变化 细胞病变病毒与非细胞病变细胞培养适应亲本相比 病毒。这些变化现在正在由站点定向单独研究 诱变并通过替换 cDNA 片段进行分组以确定 导致表型改变的突变。 Ⅲ 与以下机构的合作 北卡罗来纳州的 Lemon 博士表示，这些细胞病变 变异似乎是由病毒变异的基因重组产生的 在细胞培养中。 HAV 基因组 5' 非编码区的作用 iii 正在通过突变分析来研究翻译。相比之下 脊髓灰质炎病毒，看来前 50 个碱基对于 病毒蛋白的体外翻译。 HAV似乎有一个独特的VP4 蛋白质。大多数小核糖核酸病毒的 VP4 含有 75 至 80 个氨基酸。甲型肝炎病毒 VP4 中似乎只有 23 个氨基酸。我们将 VP4 替换为 用于 HAV VP4 的脊髓灰质炎病毒，以确定非常小的 VP4 是否赋予 HAV 的任何生物学特性。使用一种新颖的技术结合 我们拥有聚合酶链式反应和定点诱变 产生的嵌合病毒具有精确的脊髓灰质炎 VP4 代替 HAV VP4。研究的其他小核糖核酸病毒的 VP4 具有氨基末端 裂解蛋氨酸并添加肉豆蔻酸酯。 HAV 具有潜在的肉豆蔻酰化作用 VP4 氨基末端内的第 4 位氨基酸。突变分析 该网站表明，HAV 中不会发生肉豆蔻酰化。我们是 表达 HAV 的整个衣壳区域以及病毒蛋白酶 产生合成空衣壳的杆状病毒。