Extensive Mutagenesis of the Conserved Box E Motif in Duck Hepatitis B Virus P Protein Reveals Multiple Functions in Replication and a Common Structure with the Primer Grip in HIV-1 Reverse Transcriptase
Extensive Mutagenesis of the Conserved Box E Motif in Duck Hepatitis B Virus P Protein Reveals Multiple Functions in Replication and a Common Structure with the Primer Grip in HIV-1 Reverse Transcriptase
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DOI：
10.1128/jvi.00011-12
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发表时间：
2012-04
影响因子：
5.4
通讯作者：
Yong-Xiang Wang;C. Luo;Dan Zhao;J. Beck;M. Nassal
中科院分区：
文献类型：
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作者：
Yong-Xiang Wang;C. Luo;Dan Zhao;J. Beck;M. Nassal
摘要 肝炎病毒，包括致病性乙型肝炎病毒 (HBV)，通过蛋白质引发的逆转录复制其小 DNA 基因组，该逆转录由其 P 蛋白中的末端蛋白 (TP) 结构域和前基因组 RNA (pgRNA) 上的 RNA 茎环 ϵ 介导。 P 蛋白没有直接的结构数据，但它们的逆转录酶 (RT) 结构域包含在所有 RT 中保守的基序（A 框到 G 框），这意味着具有相似的结构；然而，对这一概念的实验支持是有限的。利用适用于鸭 HBV (DHBV) 而不是 HBV P 蛋白的检测方法，我们评估了 E 框内众多突变的功能后果，E 框形成了人类免疫缺陷病毒 1 型 (HIV-1) RT 中的 DNA 引物夹。该子结构在聚合循环过程中协调引物 3' 端定位和 RT 子结构域移动，是 HIV-1 RT 的非核苷 RT 抑制剂 (NNRTI) 的主要靶点。 E框确实对于DHBV复制至关重要，其突变以类似于HIV-1 RT的位置和氨基酸侧链依赖性方式影响P蛋白的折叠、εRNA相互作用和聚合酶活性。分子模型强调了与 HIV-1 RT 的结构相似性，并通过携带来自 HIV-1 RT 的框 E、甚至框 C 到框 E 的嵌合 P 蛋白的复制活性得到证实。因此，DHBV P 蛋白中的框 E 和可能的 HBV P 蛋白形成引物夹状结构，可能为抗 HBV NNRTI 提供新靶点。
ABSTRACT Hepadnaviruses, including the pathogenic hepatitis B virus (HBV), replicate their small DNA genomes through protein-primed reverse transcription, mediated by the terminal protein (TP) domain in their P proteins and an RNA stem-loop, ϵ, on the pregenomic RNA (pgRNA). No direct structural data are available for P proteins, but their reverse transcriptase (RT) domains contain motifs that are conserved in all RTs (box A to box G), implying a similar architecture; however, experimental support for this notion is limited. Exploiting assays available for duck HBV (DHBV) but not the HBV P protein, we assessed the functional consequences of numerous mutations in box E, which forms the DNA primer grip in human immunodeficiency virus type 1 (HIV-1) RT. This substructure coordinates primer 3′-end positioning and RT subdomain movements during the polymerization cycle and is a prime target for nonnucleosidic RT inhibitors (NNRTIs) of HIV-1 RT. Box E was indeed critical for DHBV replication, with the mutations affecting the folding, ϵ RNA interactions, and polymerase activity of the P protein in a position- and amino acid side chain-dependent fashion similar to that of HIV-1 RT. Structural similarity to HIV-1 RT was underlined by molecular modeling and was confirmed by the replication activity of chimeric P proteins carrying box E, or even box C to box E, from HIV-1 RT. Hence, box E in the DHBV P protein and likely the HBV P protein forms a primer grip-like structure that may provide a new target for anti-HBV NNRTIs.