Coding sequences upstream of the human immunodeficiency virus type 1 reverse transcriptase domain in gag-pol are not essential for incorporation of the Pr160gag-pol into virus particles
Coding sequences upstream of the human immunodeficiency virus type 1 reverse transcriptase domain in gag-pol are not essential for incorporation of the Pr160gag-pol into virus particles
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DOI：
10.1128/jvi.76.7.3221-3231.2002
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发表时间：
2002-04-01
影响因子：
5.4
通讯作者：
Wang, CT
中科院分区：
文献类型：
--
作者：
Chiu, HC;Yao, SY;Wang, CT
人类免疫缺陷病毒1型（HIV-1）Gag-Pol掺入病毒体被认为是由N-末端Gag结构域通过与Gag前体相互作用介导的。然而，最近的一项研究表明，鼠白血病病毒Pol可以独立于Gag-Pol表达被掺入病毒体中，这意味着Pol和Gag前体之间可能存在相互作用。为了测试HIV-1 Pol是否可以在去除N-末端Gag结构域后掺入病毒体中，并更详细地定义Gag-Pol掺入病毒体所需的序列，构建了一系列在逆转录酶（RT）结构域上游区域具有各种广泛缺失的HIV Gag-Pol表达质粒，并通过用表达Pr 55（gag）的质粒共转染293 T细胞来检查Gag-Pol缺失突变体的病毒掺入。分析表明，gag编码区的N-末端三分之二的缺失并不显著影响Gag-Pol掺入病毒粒子。相比之下，Gag-Pol蛋白与覆盖衣壳（CA）主要同源区域和相邻的C-末端CA区域的缺失相对于组装成病毒体受损。然而，Gag-Pol与删除上游的蛋白酶的序列，或RT结构域，但保留15个N-末端gag密码子，仍然可以拯救到病毒体的水平约20%的野生型水平。当在nonmyristylated Gag-Pol背景下测定时，所有的Gag-Pol缺失突变体都以与其myristylated对应物相当的水平掺入病毒体中，这表明Gag-Pol缺失突变体掺入病毒体中不依赖于N-末端myristylation信号。
Incorporation of the human immunodeficiency virus type 1 (HIV-1) Gag-Pol into virions is thought to be mediated by the N-terminal Gag domain via interaction with the Gag precursor. However, one recent study has demonstrated that the murine leukemia virus Pol can be incorporated into virions independently of Gag-Pol expression, implying a possible interaction between the Pol and Gag precursor. To test whether the HIV-1 Pol can be incorporated into virions on removal of the N-terminal Gag domain and to define sequences required for the incorporation of Gag-Pol into virions in more detail, a series of HIV Gag-Pol expression plasmids with various extensive deletions in the region upstream of the reverse transcriptase (RT) domain was constructed, and viral incorporation of the Gag-Pol deletion mutants was examined by cotransfecting 293T cells with a plasmid expressing Pr55(gag). Analysis indicated that deletion of the N-terminal two-thirds of the gag coding region did not significantly affect the incorporation of Gag-Pol into virions. In contrast, Gag-Pol proteins with deletions covering the capsid (CA) major homology regions and the adjacent C-terminal CA regions were impaired with respect to assembly into virions. However, Gag-Pol with sequences deleted upstream of the protease, or of the RT domain but retaining 15 N-terminaI gag codons, could still be rescued into virions at a level about 20% of the wild-type level. When assayed in a nonmyristylated Gag-Pol context, all of the Gag-Pol deletion mutants were incorporated into virions at a level comparable to their myristylated counterparts, suggesting that the incorporation of the Gag-Pol deletion mutants into virions is independent of the N-terminal myristylation signal.