Effect of human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein on HIV-1 reverse transcriptase activity in vitro
Effect of human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein on HIV-1 reverse transcriptase activity in vitro
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DOI：
10.1021/bi951707e
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发表时间：
1996-01-09
期刊：
影响因子：
2.9
通讯作者：
Preston, BD
中科院分区：
文献类型：
--
作者：
Ji, XD;Klarmann, GJ;Preston, BD
人类免疫缺陷病毒1型(HIV-1)基因组RNA向病毒DNA的转化是病毒生命周期中必不可少的步骤。这种转化是由逆转录酶(RT)催化的，逆转录酶与包括核衣壳(NC)在内的几种病毒蛋白组成的大型核蛋白复合体相关。为了更好地表征病毒DNA合成的生化机制，我们对重组HIV-1NC进行了高效表达和纯化，并研究了其在体外HIV-1模板序列聚合过程中对HIV-1逆转录酶活性和加工能力的影响。NC对稳态RT活性的影响依赖于反应组分的加入顺序。在RT-引物形成前加入NC。模板-dNTP三元复合体通过减缓稳态RT翻转，抑制了引物延伸并降低了总产物产量。相反，将NC加入到预制的三元复合体中，可以有效地延伸引物，并增加特定DNA模板位点的RT处理能力。NC通过HIV-1 env的cRRE区的13个位点中的8个促进聚合(2-4倍)，并通过HIV-1 Poll的D3/CTS区提高聚合速度10倍。数据表明，NC通过模板二级结构区域促进聚合，从而影响RT加工能力。因此，NC在体外可以作为单链结合(SSB)样的辅助复制因子用于体外RT，并且可能是多组分逆转录病毒复制复合体的一部分。
Conversion of human immunodeficiency virus type 1 (HIV-1) genomic RNA to viral DNA is a requisite step in the virus life cycle. This conversion is catalyzed by reverse transcriptase (RT) associated with a large nucleoprotein complex composed of several viral proteins including nucleocapsid (NC). To better characterize the biochemical mechanisms of viral DNA synthesis, we overexpressed and purified recombinant HIV-1 NC and studied its effect on the activity and processivity of HIV-1 RT during polymerization of HIV-1 template sequences in vitro. The effect of NC on steady-state RT activity was dependent on the order of addition of reaction components. Addition of NC prior to formation of RT-primer . template-dNTP ternary complexes inhibited primer extension and reduced total product yields by slowing steady-state RT turnover. In contrast, addition of NC to preformed ternary complexes resulted in efficient primer extension and increased RT processivity at specific DNA template sites. NC stimulated polymerization (2-4 times) through eight of 13 sites examined in the cRRE region of HIV-1 env and increased the rate of polymerization through the D3/CTS region of HIV-1 pol 10 times. The data suggest that NC affects RT processivity by facilitating polymerization through regions of template secondary structure. Thus, NC functions as a single-strand binding (SSB)-like accessory replication factor for RT in vitro and may be part of a multicomponent retroviral replication complex.