Rational site-directed mutations of the LLP-1 and LLP-2 lentivirus lytic peptide domains in the intracytoplasmic tail of human immunodeficiency virus type 1 gp4l indicate common functions in cell-cell fusion but distinct roles in virion envelope incorporation
Rational site-directed mutations of the LLP-1 and LLP-2 lentivirus lytic peptide domains in the intracytoplasmic tail of human immunodeficiency virus type 1 gp4l indicate common functions in cell-cell fusion but distinct roles in virion envelope incorporation
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DOI：
10.1128/jvi.77.6.3634-3646.2003
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发表时间：
2003-03-01
影响因子：
5.4
通讯作者：
Montelaro, RC
中科院分区：
文献类型：
--
作者：
Kalia, V;Sarkar, S;Montelaro, RC
两个高度保守的阳离子两亲性α-螺旋基序，命名为慢病毒裂解肽1和2（LLP-1和LLP-2），已被表征在慢病毒的跨膜（TM）包膜糖蛋白（Env）的羧基末端。虽然这些结构域具有多种性质，但其结构和功能意义尚不清楚。为了确定Env LLP结构域对Env表达、加工和掺入以及对病毒复制和合胞体诱导的特定贡献，对原发性双嗜性感染性人类免疫缺陷病毒1型（HIV-1）分离株（ME 46）的定点LLP突变体进行了检查。对LLP-1或LLP-2结构域（分别为MX 1或MX2）或两个结构域（MX4）中的高度保守精氨酸残基进行置换。LLP结构域改变的HIV-1突变体表现出不同的表型。LLP-1突变体（MX 1和MX4）是复制缺陷型的，感染性平均降低85%，这与gp 41掺入病毒粒子的明显减少有关，而转染的293 T细胞中Env表达或加工没有显著减少。相反，MX2病毒具有复制能力，并将Env的完整补体掺入其病毒体中，表明LLP-1结构域在Env掺入中的不同作用。有趣的是，有复制能力的MX2病毒在T细胞系中诱导合胞体的能力受损。这种细胞-细胞融合的缺陷与细胞表面Env表达、寡聚化或构象水平的明显缺陷无关。然而，在基于定量内切酶的细胞-细胞融合测定中，与野生型Env相比，合胞体形成的缺乏与MX2 Env融合性降低约90%相关。LLP-1突变体MX 1和MX4 Env也表现出融合性平均降低80%。总之，这些结果首次证明了高度保守的LLP结构域在Env掺入和融合性中执行关键但不同的功能。
Two highly conserved cationic amphipathic alpha-helical motifs, designated lentivirus lytic peptides 1 and 2 (LLP-1 and LLP-2), have been characterized in the carboxyl terminus of the transmembrane (TM) envelope glycoprotein (Env) of lentiviruses. Although various properties have been attributed to these domains, their structural and functional significance is not clearly understood. To determine the specific contributions of the Env LLP domains to Env expression, processing, and incorporation and to viral replication and syncytium induction, site-directed LLP mutants of a primary dualtropic infectious human immunodeficiency virus type 1 (HIV-1) isolate (ME46) were examined. Substitutions were made for highly conserved arginine residues in either the LLP-1 or LLP-2 domain (MX1 or MX2, respectively) or in both domains (MX4). The HIV-1 mutants with altered LLP domains demonstrated distinct phenotypes. The LLP-1 mutants (MX1 and MX4) were replication defective and showed an average of 85% decrease in infectivity, which was associated with an evident decrease in gp41 incorporation into virions without a significant decrease in Env expression or processing in transfected 293T cells. In contrast, MX2 virus was replication competent and incorporated a full complement of Env into its virions, indicating a differential role for the LLP-1 domain in Env incorporation. Interestingly, the replication-competent MX2 virus was impaired in its ability to induce syncytia in T-cell lines. This defect in cell-cell fusion did not correlate with apparent defects in the levels of cell surface Env expression, oligomerization, or conformation. The lack of syncytium formation, however, correlated with a decrease of about 90% in MX2 Env fusogenicity compared to that of wild-type Env in quantitative luciferase-based cell-cell fusion assays. The LLP-1 mutant MX1 and MX4 Envs also exhibited an average of 80% decrease in fusogenicity. Altogether, these results demonstrate for the first time that the highly conserved LLP domains perform critical but distinct functions in Env incorporation and fusogenicity.