Dubay J W, Dubay S R, Shin H J, Hunter E
Department of Microbiology, University of Alabama at Birmingham 35294, USA.
J Virol. 1995 Aug;69(8):4675-82. doi: 10.1128/JVI.69.8.4675-4682.1995.
Endoproteolytic cleavage of the glycoprotein precursor to the mature SU and TM proteins is an essential step in the maturation of retroviral glycoproteins. Cleavage of the precursor polyprotein occurs at a conserved, basic tetrapeptide sequence and is carried out by a cellular protease. The glycoprotein of the human immunodeficiency virus type 1 contains two potential cleavage sequences immediately preceding the N terminus of the TM protein. To determine the functional significance of these two potential cleavage sites, a series of mutations has been constructed in each site individually, as well as in combinations that altered both sites simultaneously. A majority of the mutations in either potential cleavage site continued to allow efficient cleavage when present alone but abrogated cleavage of the precursor when combined. Despite being transported efficiently to the cell surface, these cleavage-defective glycoproteins were unable to initiate cell-cell fusion and viruses containing them were not infectious. Viruses that contained glycoproteins with a single mutation, and that retained the ability to be processed, were capable of mediating a productive infection, although infectivity was impaired in several of these mutants. Protein analyses indicated that uncleaved glycoprotein precursors were inefficiently incorporated into virions, suggesting that cleavage of the glycoprotein may be a prerequisite to incorporation into virions. The substitution of a glutamic acid residue for a highly conserved lysine residue in the primary cleavage site (residue 510) had no effect on glycoprotein cleavage or function, even though it removed the only dibasic amino acid pair in this site. Peptide sequencing of the N terminus of gp41 produced from this mutant glycoprotein demonstrated that cleavage continued to take place at this site. These results, demonstrating that normal cleavage of the human immunodeficiency virus type 1 glycoprotein can occur when no dibasic sequence is present at the cleavage site, raise questions about the specificity of the cellular protease that mediates this cleavage and suggest that cleavage of the glycoprotein is required for efficient incorporation of the glycoprotein into virions.
糖蛋白前体裂解为成熟的表面单位（SU）和跨膜单位（TM）蛋白是逆转录病毒糖蛋白成熟过程中的关键步骤。前体多蛋白的裂解发生在一个保守的碱性四肽序列处，由细胞蛋白酶催化。1型人类免疫缺陷病毒的糖蛋白在TM蛋白N端之前紧邻有两个潜在的裂解序列。为确定这两个潜在裂解位点的功能意义，分别在每个位点构建了一系列突变，同时也构建了使两个位点同时改变的组合突变。任一潜在裂解位点的大多数突变单独存在时仍能高效裂解，但组合时则会消除前体的裂解。尽管这些裂解缺陷型糖蛋白能有效转运至细胞表面，但却无法引发细胞间融合，含有它们的病毒也无感染性。含有单个突变且保留加工能力的糖蛋白的病毒能够介导有效感染，不过其中一些突变体的感染性有所受损。蛋白质分析表明，未裂解的糖蛋白前体难以有效整合到病毒粒子中，这表明糖蛋白的裂解可能是整合到病毒粒子中的前提条件。在主要裂解位点（第510位残基）将一个高度保守的赖氨酸残基替换为谷氨酸残基，对糖蛋白的裂解或功能并无影响，即便该位点唯一的双碱性氨基酸对被去除。对该突变型糖蛋白产生的gp41 N端进行肽段测序表明，裂解仍在此位点发生。这些结果表明，当裂解位点不存在双碱性序列时，1型人类免疫缺陷病毒糖蛋白仍可正常裂解，这引发了关于介导此裂解的细胞蛋白酶特异性的疑问，并提示糖蛋白的裂解是其有效整合到病毒粒子中的必要条件。
