THE NONMYRISTYLATED PR160GAG-POL POLYPROTEIN OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 INTERACTS WITH PR55GAG AND IS INCORPORATED INTO VIRUS-LIKE PARTICLES
THE NONMYRISTYLATED PR160GAG-POL POLYPROTEIN OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 INTERACTS WITH PR55GAG AND IS INCORPORATED INTO VIRUS-LIKE PARTICLES
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DOI：
10.1128/jvi.66.11.6304-6313.1992
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发表时间：
1992-11-01
影响因子：
5.4
通讯作者：
MORROW, CD
中科院分区：
文献类型：
--
作者：
PARK, J;MORROW, CD
人类1型免疫缺陷病毒的pol基因通过gag和pol基因之间的核糖体移码表达。所得蛋白为Gag- pol多蛋白，其含量为Gag蛋白的5 - 10%。Gag-Pol多蛋白被整合到病毒粒子中，并提供病毒蛋白酶、逆转录酶和整合酶，这些都是病毒传染性所必需的。一般认为Gag- pol多蛋白通过与Gag蛋白的相互作用被并入病毒粒子，但其具体机制尚不清楚。为了进一步研究这一问题，我们构建了过表达Gag-Pol多蛋白（Pr160gag-pol）的人类免疫缺陷病毒1型原病毒基因组。将该前病毒基因组（pGPpr-）转染到COS-1细胞中，可表达全长Pr160gag-pol多蛋白。虽然大部分Pr160gag-pol被限制在细胞内，但在细胞上清液中检测到低水平的逆转录酶活性。pGPpr-与仅表达Pr55gag前体（pGAG）的第二质粒共转染后，转染细胞的培养基中Pr160gag-pol水平显著升高。利用蔗糖密度梯度的沉降分析表明，Pr160gag-pol大部分存在于与病毒粒子密度相对应的组分中，这表明Pr160gag-pol多蛋白是与Pr55gag病毒样颗粒一起释放的。为了进一步表征释放的要求，构建了一个突变来表达无花柱化的Pr160gag-pol多蛋白。与Pr55gag共表达表明，未分化的Pr160gag-pol也被纳入病毒粒子中。亚细胞分离实验表明，在低离子强度和高离子强度条件下，Pr160gag-pol和Pr160gag-pol在膜和细胞质中的分布相似。综上所述，这些结果表明Pr160gag-pol多蛋白的肉豆蔻化并不需要与包装成病毒样颗粒所需的Pr55gag相互作用。
The expression of the pol gene of human immunodeficiency vims type 1 occurs via a ribosomal frameshift between the gag and pol genes. The resulting protein, a Gag-Pol polyprotein, is produced at a level 5 to 10% of that of the Gag protein. The Gag-Pol polyprotein is incorporated into virions and provides viral protease, reverse transcriptase, and integrase, which are essential for infectivity. It is generally believed that the Gag-Pol polyprotein is incorporated into virions via interaction with the Gag protein, although the details of the mechanism are unknown. To further study this problem, we have constructed a human immunodeficiency virus type 1 proviral genome which overexpresses the Gag-Pol polyprotein (Pr160gag-pol). Transfection of this proviral genome (pGPpr-) into COS-1 cells resulted in the expression of full-length Pr160gag-pol polyprotein. Although the majority of the Pr160gag-pol was confined to the cells, low levels of reverse transcriptase activity were detectable in the cell supernatants. The cotransfection of pGPpr- with a second plasmid which expresses only the Pr55gag precursor (pGAG) resulted in a significantly higher level of Pr160gag-pol in the medium of transfected cells. Sedimentation analysis using sucrose density gradients demonstrated that most Pr160gag-pol was found in fractions corresponding to the density of virion particles, indicating that the Pr160gag-pol polyprotein was released in association with a Pr55gag viruslike particle. To further characterize the requirements for the release, a mutation was constructed to express an unmyristylated Pr160gag-pol polyprotein. Coexpression with Pr55gag demonstrated that the unmyristylated Pr160gag-pol was also incorporated into virion particles. Subcellular fractionation experiments revealed that the distributions of the Pr160gag-pol myr- and Pr160gag-pol in the membrane and cytosol were similar under low- or high-ionic-strength conditions. Taken together, these results suggest that myristylation of the Pr160gag-pol polyprotein is not required for the interaction with the Pr55gag necessary for packaging into a viruslike particle.