Enhancing the proteolytic maturation of human immunodeficiency virus type 1 envelope glycoproteins
Enhancing the proteolytic maturation of human immunodeficiency virus type 1 envelope glycoproteins
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DOI：
10.1128/jvi.76.6.2606-2616.2002
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发表时间：
2002-03-01
影响因子：
5.4
通讯作者：
Moore, JP
中科院分区：
文献类型：
--
作者：
Binley, JM;Sanders, RW;Moore, JP
在病毒感染的细胞中，人类免疫缺陷病毒I型包膜糖蛋白(Env)前体gp160被细胞蛋白酶切割成融合活性的gp120-gp41异二聚体，其中两个亚单位非共价关联。然而，当重组Env以全长gp160或立即被N端截断到跨膜区的可溶性gp140的形式高水平表达时，切割效率会很低。我们探索了几种获得完全裂解的Env作为疫苗抗原的方法。在体外实验中，我们检测了纯化的Env能否被纯化的酶消化。纤溶酶有效地切割了env前体，但也在gp120的第二个位置切割，最有可能的是V3环。相反，可溶性形式的呋喃对gp120-gp41裂解位点是特异的，但切割效率较低。Env与全长或可溶形式的Furin共表达促进了Env的切割，但也降低了Env的表达。当对Env裂解位点(REKR)进行突变，以确定它是否可以被细胞蛋白酶利用时，发现几个突变体的处理效率比野生型蛋白更高。最佳切割位点顺序为RRRRRR、RRRRKR和RRRKKR。这些突变没有显著改变Env蛋白介导融合的能力，因此它们没有从根本上扰乱Env结构。此外，与野生型Env不同，共表达Furin并不能显著降低裂解位点突变体的表达。因此，共表达Env裂解位点突变体和Furin是获得高水平表达加工型Env的有效方法。
In virus-infected cells, the envelope glycoprotein (Env) precursor, gp160, of human immunodeficiency virus type I is cleaved by cellular proteases into a fusion-competent gp120-gp41 heterodimer in which the two subunits are noncovalently associated. However, cleavage can be inefficient when recombinant Env is expressed at high levels, either as a full-length gp160 or as a soluble gp140 truncated immediately N-terminal to the transmembrane domain. We have explored several methods for obtaining fully cleaved Env for use as a vaccine antigen. We tested whether purified Env could be enzymatically digested with purified protease in vitro. Plasmin efficiently cleaved the Env precursor but also cut at a second site in gp120, most probably the V3 loop. In contrast, a soluble form of furin was specific for the gp120-gp41 cleavage site but cleaved inefficiently. Coexpression of Env with the full-length or soluble form of furin enhanced Env cleavage but also reduced Env expression. When the Env cleavage site (REKR) was mutated in order to see if its use by cellular proteases could be enhanced, several mutants were found to be processed more efficiently than the wild-type protein. The optimal cleavage site sequences were RRRRRR, RRRRKR, and RRRKKR. These mutations did not significantly alter the capacity of the Env protein to mediate fusion, so they have not radically perturbed Env structure. Furthermore, unlike that of wild-type Env, expression of the cleavage site mutants was not significantly reduced by furin coexpression. Coexpression of Env cleavage site mutants and furin is therefore a useful method for obtaining high-level expression of processed Env.