Ott D E, Chertova E N, Busch L K, Coren L V, Gagliardi T D, Johnson D G
AIDS Vaccine Program, SAIC/Frederick, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201, USA.
J Virol. 1999 Jan;73(1):19-28. doi: 10.1128/JVI.73.1.19-28.1999.
The p6(Gag) protein of human immunodeficiency virus type 1 (HIV-1) is produced as the carboxyl-terminal sequence within the Gag polyprotein. The amino acid composition of this protein is high in hydrophilic and polar residues except for a patch of relatively hydrophobic amino acids found in the carboxyl-terminal 16 amino acids. Internal cleavage of p6(Gag) between Y36 and P37, apparently by the HIV-1 protease, removes this hydrophobic tail region from approximately 30% of the mature p6(Gag) proteins in HIV-1MN. To investigate the importance of this cleavage and the hydrophobic nature of this portion of p6(Gag), site-directed mutations were made at the minor protease cleavage site and within the hydrophobic tail. The results showed that all of the single-amino-acid-replacement mutants exhibited either reduced or undetectable cleavage at the site yet almost all were nearly as infectious as wild-type virus, demonstrating that processing at this site is not important for viral replication. However, one exception, Y36F, was 300-fold as infectious the wild type. In contrast to the single-substitution mutants, a virus with two substitutions in this region of p6(Gag), Y36S-L41P, could not infect susceptible cells. Protein analysis showed that while the processing of the Gag precursor was normal, the double mutant did not incorporate Env into virus particles. This mutant could be complemented with surface glycoproteins from vesicular stomatitis virus and murine leukemia virus, showing that the inability to incorporate Env was the lethal defect for the Y36S-L41P virus. However, this mutant was not rescued by an HIV-1 Env with a truncated gp41(TM) cytoplasmic domain, showing that it is phenotypically different from the previously described MA mutants that do not incorporate their full-length Env proteins. Cotransfection experiments with Y36S-L41P and wild-type proviral DNAs revealed that the mutant Gag dominantly blocked the incorporation of Env by wild-type Gag. These results show that the Y36S-L41P p6(Gag) mutation dramatically blocks the incorporation of HIV-1 Env, presumably acting late in assembly and early during budding.
人类免疫缺陷病毒1型（HIV-1）的p6（Gag）蛋白作为Gag多蛋白的羧基末端序列产生。该蛋白的氨基酸组成富含亲水性和极性残基，但在羧基末端的16个氨基酸中发现有一片相对疏水的氨基酸区域。在HIV-1 MN中，p6（Gag）在Y36和P37之间的内部切割，显然是由HIV-1蛋白酶进行的，从大约30%的成熟p6（Gag）蛋白中去除了这个疏水尾部区域。为了研究这种切割的重要性以及p6（Gag）这部分的疏水性，在次要蛋白酶切割位点和疏水尾部进行了定点突变。结果表明，所有单氨基酸替代突变体在该位点的切割均减少或无法检测到，但几乎所有突变体的感染性都与野生型病毒相近，这表明该位点的加工对病毒复制并不重要。然而，有一个例外，Y36F的感染性是野生型的300倍。与单替代突变体不同，在p6（Gag）这个区域有两个替代的病毒Y36S-L41P不能感染易感细胞。蛋白质分析表明，虽然Gag前体的加工正常，但双突变体不能将Env整合到病毒颗粒中。该突变体可以被水泡性口炎病毒和鼠白血病病毒的表面糖蛋白互补，表明无法整合Env是Y36S-L41P病毒的致命缺陷。然而，该突变体不能被具有截短的gp41（TM）细胞质结构域的HIV-1 Env拯救，表明它在表型上与先前描述的不整合全长Env蛋白的MA突变体不同。用Y36S-L41P和野生型前病毒DNA进行的共转染实验表明，突变体Gag显著阻断了野生型Gag对Env的整合。这些结果表明Y36S-L41P p6（Gag）突变显著阻断了HIV-1 Env的整合，推测其作用于组装后期和出芽早期。
