Virion instability of human immunodeficiency virus type 1 reverse transcriptase (RT) mutated in the protease cleavage site between RT p51 and the RT RNase H domain
Virion instability of human immunodeficiency virus type 1 reverse transcriptase (RT) mutated in the protease cleavage site between RT p51 and the RT RNase H domain
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DOI：
10.1128/jvi.79.18.11952-11961.2005
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发表时间：
2005-09-01
影响因子：
5.4
通讯作者：
Parniak, MA
中科院分区：
文献类型：
--
作者：
Abram, ME;Parniak, MA
人类免疫缺陷病毒1型（HIV-1）pol编码的酶、蛋白酶（PR）、逆转录酶（RT）和整合酶（IN）中的每一种仅作为二聚体（或在IN的情况下更高阶的寡聚体）具有活性，但仅RT包含不同质量的亚基。RT是66-kDa和51-kDa亚基的异二聚体。后者是通过在病毒体成熟过程中HIV PR催化的p66裂解形成的，导致p66亚基的RNA酶H（RNH）结构域的去除。为了研究在病毒体的情况下对RT异二聚体的明显需要，我们在感染性HIV-1分子克隆的RT p51-RNH蛋白酶切割位点中引入了各种突变。令人惊讶的是，不是导致具有增加的RT p66含量的病毒体，大多数突变导致显著减毒的病毒，其含有大大降低的RT水平，在许多情况下主要是p51 RT。然而，大多数突变体表现出正常水平的Pr160（gag-pol）前体多蛋白，这表明减少病毒体RT产生的蛋白水解不稳定性，而不是减少掺入。突变病毒粒子p24 Gag水平与野生型相当，表明Gag掺入和加工不受影响。暴露于突变病毒的MT-2细胞的重复传代导致病毒的出现，其复制能力提高;这些病毒体含有接近野生型水平的正常加工的RT。这些结果表明，额外的蛋白水解处理RT的p66/p51异源二聚体是必不可少的，以提供蛋白水解稳定性的RT在HIV-1成熟。
Each of the human immunodeficiency virus type 1 (HIV-1) pol-encoded enzymes, protease (PR), reverse transcriptase (RT), and integrase (IN), is active only as a dimer (or higher-order oligomer in the case of IN), but only RT comprises subunits of different masses. RT is a heterodimer of 66-kDa and 51-kDa subunits. The latter is formed by HIV PR-catalyzed cleavage of p66 during virion maturation, resulting in the removal of the RNase H (RNH) domain of a p66 subunit. In order to study the apparent need for RT heterodimers in the context of the virion, we introduced a variety of mutations in the RT p51-RNH protease cleavage site of an infectious HIV-1 molecular clone. Surprisingly, rather than leading to virions with increased RT p66 content, most of the mutations resulted in significantly attenuated virus that contained greatly decreased levels of RT that in many cases was primarily p5l RT. IN levels were also reduced in several mutants. However, most mutants showed normal levels of the Pr160(gag-pol) precursor polyprotein, suggesting that reduced virion RT arose from proteolytic instability rather than decreased incorporation. Mutant virion p24 Gag levels were equivalent to wild type, indicating that Gag incorporation and processing were not affected. Repeated passage of MT-2 cells exposed to mutant viruses led to the appearance of virus with improved replication capacity; these virions contained normally processed RT at near-wild-type levels. These results imply that additional proteolytic processing of RT to the p66/p51 heterodimer is essential to provide proteolytic stability of RT during HIV-1 maturation.