DISSECTION OF THE LIFE CYCLE OF HUMAN HEPATITIS B VIRUS

在这一建议中，我们希望说明 人B型肝炎病毒聚合酶基因产物。 使用网站- 定向诱变、基因转移和各种测定， 重点工程：一， 研究pol基因的策略 从3.5 kb转录本的表达：三种不同的假设， 将测试pol ene表达策略（核糖体框架， 移位、终止-反向扫描-重新启动以及直接 内部启动）。 a. 继续完成我们最初的 设计用于研究翻译控制的突变体的表征 pol基因。 B. 为了降低翻译效率， 上游核心ORF通过改变ATG周围的序列环境 起始密码子或通过引入框外的上游ATG 抑制密码子 C. 为了影响翻译效率， 下游的pol ORF通过改变c/pol重叠的长度。 D. 到 通过脊髓灰质炎病毒感染影响上游ORF的表达，或 转染 e. 为了降低翻译效率， 上游核心ORF通过杂交体捕获体外翻译。 2. 为了研究一种新发现的， 转录本（2.3kb）：a. 通过突变剪接来破坏香料 供体和受体位点; B.寻找其他未知的成绩单， 体外PCR扩增，然后进行cDNA克隆。 C. 功能 来源于剪接的cDNA克隆的表征 成绩单。 3. pol ORF的结构域定义：三个预测功能 pol结构域包括末端蛋白、逆转录酶和RNase H.大约有十几种不同的结构域突变体的特征是， 各种功能测定。

In this proposal, we wish to characterize the structure and function of the polymerase gene product of human hepatitis B virus. Using site- directed mutagenesis, gene transfer and various kinds of assays, three major projects will be pursued: 1. To study the strategy of pol gene expression from the 3.5 kb transcript: Three different hypotheses of the pol ene expression strategy will be tested (ribosomal frame- shifting, termination-backward scanning-reinitiation as well as direct internal initiation). a. To continue and complete our initial characterizations of mutants designed to study the translational control of pol gene. b. To attenuate the translational efficiency of the upstream core ORF by changing the sequence context around the ATG initiation codon or by introducing an out-of-frame, upstream ATG inhibitory codon. c. To affect the translational efficiency of the downstream pol ORF by varying the length of c/pol overlap. d. To affect the expression of the upstream ORF by polio virus infection or transfection. e. To attenuate the translational efficiency of the upstream core ORF via hybrid-arrest in vitro translation. 2. To study the functional significance of a newly discovered, spliced transcript (2.3 kb): a. To disrupt spicing by mutating the splice donor and the acceptor site; b. To look for other unknown transcript via in vitro PCR amplification, followed by cDNA cloning. c. functional characterization of the cDNA clones deriving from the spliced transcript. 3. Domain definition of the pol ORF: Three predicted functional domains of pol include terminal protein, reverse transcriptase and RNase H. About a dozen different domain mutants will be characterized by various functional assays.