The hypervariable domain of the murine leukemia virus surface protein tolerates large insertions and deletions, enabling development of a retroviral particle display system
The hypervariable domain of the murine leukemia virus surface protein tolerates large insertions and deletions, enabling development of a retroviral particle display system
复制标题
DOI：
10.1128/jvi.73.3.1802-1808.1999
复制
发表时间：
1999-03-01
影响因子：
5.4
通讯作者：
Pinter, A
中科院分区：
文献类型：
--
作者：
Kayman, SC;Park, H;Pinter, A
鼠 C 型逆转录病毒的表面蛋白 (SU) 具有不含半胱氨酸但富含脯氨酸的中央高变结构域。 Friend亲嗜性鼠白血病病毒SU的这个41个氨基酸区域被证明对插入和缺失具有高度的耐受性。该区域的 N 端 30 个氨基酸或 C 端 22 个氨基酸被 7 个氨基酸序列 ASAVAGA 取代的病毒具有完全感染性。在高变结构域的 N 端、中心和 C 端插入这 7 个氨基酸序列对包膜蛋白 (Env) 功能几乎没有影响，而在 N 端之后的 10 个氨基酸位置处的插入部分破坏了 Env、大型复杂结构域（252 个氨基酸的单链抗体结合结构域 [scFv] 或HIV-1 SU 的 96 个氨基酸 V1/V2 结构域（包含八个 N 连接糖基化位点和两个二硫键）在插入高变结构域的中心或 C 端部分时不会干扰 Env 功能。插入高变结构域 C 端区域的 scFv 结构域可介导抗原与病毒颗粒的结合，证明它折叠成活性构象并展示在病毒粒子的表面上。通过使用对插入序列呈现的构象表位具有特异性的单克隆抗体，可以实现表达 V1/V2 序列的病毒体的正向和负向富集。这些结果表明，Friend ecotropic SU 的高变结构域不包含任何对 Env 功能至关重要的特定序列或结构，并证明该结构域的插入可用于将颗粒展示方法扩展到需要在真核细胞中表达以进行糖基化和正确折叠的复杂蛋白质结构域。
The surface proteins (SU) of murine type-C retroviruses have a central hypervariable domain devoid of cysteine and rich in proline. This 41-amino-acid region of Friend ecotropic murine leukemia virus SU was shown to be highly tolerant of insertions and deletions. Viruses in which either the N-terminal 30 amino acids or the C-terminal 22 amino acids of this region were replaced by the 7-amino-acid sequence ASAVAGA were fully infectious. Insertions of this 7-amino-acid sequence at the N terminus, center, and the C terminus of the hypervariable domain had little effect on envelope protein (Env) function, while this insertion at a position 10 amino acids following the N terminus partially destabilized the association between the SU and transmembrane subunits of Env, Large, complex domains (either a 252-amino-acid single-chain antibody binding domain [scFv] or a 96-amino-acid V1/V2 domain of HIV-1 SU containing eight N-linked glycosylation sites and two disulfides) did not interfere with Env function when inserted in the center or C-terminal portions of the hypervariable domain. The scFv domain inserted into the C-terminal region of the hypervariable domain was shown to mediate binding of antigen to viral particles, demonstrating that it folded into the active conformation and was displayed on the surface of the virion. Both positive and negative enrichment of virions expressing the V1/V2 sequence were achieved by using a monoclonal antibody specific for a conformational epitope presented by the inserted sequence, These results indicated that the hypervariable domain of Friend ecotropic SU does not contain any specific sequence or structure that is essential for Env function and demonstrated that insertions into this domain can be used to extend particle display methodologies to complex protein domains that require expression in eukaryotic cells for glycosylation and proper folding.