Mapping of the Hepatitis B Virus Reverse Transcriptase TP and RT Domains by Transcomplementation for Nucleotide Priming and by Protein-Protein Interaction

Hepadnavirus polymerases initiate reverse transcription in a protein-primed reaction. We previously described a complementation assay for analysis of the roles of the TP and RT domains of HBV reverse transcriptase (pol) in the priming reaction. Independently expressed TP and RT domains form a complex functional for in vitro priming reactions. To map the minimal functional TP and RT domains, we prepared baculoviruses expressing amino- and carboxyl-terminal deletions of both the TP and RT domains and analyzed the proteins for the ability to participate in transcomplementation for the priming reaction. The minimal TP domain spanned amino acids 20 to 175; however, very little activity was observed without a TP domain spanning amino acids 1 to 199. The minimal RT domain spanned amino acids 300 to 775; however, little activity was observed unless the carboxyl end of the RT domain extended to amino acid 800. Thus, most of the RNase H domain was required. In previous studies, we observed a TP inhibitory domain between amino acids 199 and 344. The current analysis narrowed this domain to residues 300 to 334, which is a portion of the minimal RT domain. In addition, the ability of TP and RT deletion mutants to form stable TP-RT complexes was examined in coimmunoprecipitation assays. The minimal TP and RT domains capable of protein-protein interaction were considerably smaller than the domains required for functional interaction in the transcomplementation assays, and unlike priming activity, TP-RT interaction did not require the epsilon RNA stem-loop. These studies help to further define the complex protein-protein interactions required in HBV genome replication.

嗜肝DNA病毒（Hepadnavirus）聚合酶可通过蛋白引发反应启动逆转录过程。此前我们曾报道一套互补试验方法，用于分析乙型肝炎病毒（HBV）逆转录酶（Reverse Transcriptase, RT，又称聚合酶pol）的末端蛋白（Terminal Protein, TP）结构域与RT结构域在引发反应中的作用。单独表达的TP与RT结构域可形成具备活性的复合物，可用于体外引发反应实验。为定位最小功能TP与RT结构域，我们构建了分别携带TP、RT结构域氨基端与羧基端缺失突变的重组杆状病毒，并检测各突变蛋白参与引发反应反式互补的能力。结果显示，最小功能TP结构域的氨基酸跨度为20至175位，但仅当TP结构域保留至1至199位时才可检测到显著活性，截短至该范围外的TP结构域几乎无活性。最小功能RT结构域的氨基酸跨度为300至775位，但仅当RT结构域的羧基端延伸至800位氨基酸时才可观测到显著活性，这表明绝大多数核糖核酸酶H（RNase H）结构域均为该反应所必需。既往研究中，我们曾发现位于氨基酸199至344位区间的TP抑制结构域，本次分析将该抑制结构域的范围缩小至300至334位残基，该区域恰好属于最小RT结构域的一部分。此外，我们通过免疫共沉淀（coimmunoprecipitation）试验检测了TP与RT缺失突变体形成稳定TP-RT复合物的能力。可介导蛋白质相互作用的最小TP与RT结构域，其尺寸远小于反式互补功能试验中所需的功能结构域；且与引发活性不同，TP-RT的相互作用并不需要ε RNA茎环结构（epsilon RNA stem-loop）的参与。本研究进一步明确了HBV基因组复制过程中所需的复杂蛋白质相互作用机制。