Point mutation flanking a CTL epitope ablates in vitro and in vivo recognition of a full-length viral protein.
Point mutation flanking a CTL epitope ablates in vitro and in vivo recognition of a full-length viral protein.
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CTL 表位侧翼的点突变会消除全长病毒蛋白的体外和体内识别。
DOI：
10.4049/jimmunol.158.7.3227
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发表时间：
1997
影响因子：
4.4
通讯作者：
L. Eisenlohr
中科院分区：
文献类型：
--
作者：
A. Yellen;E. Wherry;G. Dubois;L. Eisenlohr
CD8+T细胞(T(CD8+))识别病毒抗原是由MHC-I类分子在细胞表面展示的短肽(表位)。利用一组重组痘苗病毒，我们证明了全长流感核蛋白(NP)中H-2Kd限制性表位147-155两侧的单点突变可以影响甚至破坏该表位的呈现，而对远端表位的呈现没有影响。最严重的阻断突变(第146位Ala到Pro)不能抑制NP(147-155)在截短的微型基因背景下的呈递，这意味着该肽不是功能加工中间产物。氨基末端的脯氨酸替换也显著减少了NP(50-57)的呈递(H-2Kk受限)，而相同的突变不影响第三个NP表位。因此，虽然可能存在加工特异性的趋势，但表位本身有助于侧翼序列效应。这些发现与体内启动实验是平行的，在这些实验中，依赖于病毒剂量，微妙的体外阻断效应是绝对的。蛋白酶体/合成肽的共孵育研究支持增强表位破坏在防止呈现方面的作用，多肽醛LLNL的作用也是如此，它从突变的构建体中恢复了NP(147-155)的呈现。该试剂不抑制抗原表位的呈现，即使是野生型NP，这表明它的产生可能是蛋白酶体无关的。这些结果支持表位侧翼序列的点突变可以作为病毒免疫逃避的机制的观点，揭示了所涉及的机制，并表明体外检测可能不是侧翼序列效应的敏感指标。
CD8+ T cells (T(CD8+)) recognize viral Ags as short peptides (epitopes) displayed at the cell surface by MHC class I molecules. Using a panel of recombinant vaccinia viruses, we show that single-point mutations flanking either side of an H-2Kd-restricted epitope, residues 147-155, within full-length influenza nucleoprotein (NP) can impact, even ablate, presentation of that epitope, while having no effect on presentation of distal epitopes. The most severe blocking mutation (Ala to Pro at position 146) did not inhibit NP(147-155) presentation in the context of a truncated minigene, implying that this peptide is not a functional processing intermediate. An amino-terminal proline replacement also significantly reduced presentation of NP(50-57) (H-2Kk restricted), while the same mutation did not affect a third NP epitope. Thus, while trends in processing specificity may exist, the epitope itself contributes to flanking sequence effects. These findings were paralleled by in vivo priming experiments in which, depending on viral dose, subtle in vitro blocking effects were absolute. Proteasome/synthetic peptide coincubation studies support a role for enhanced epitope destruction in preventing presentation, as did the effect of the peptide aldehyde, LLnL, which restored presentation of NP(147-155) from the mutated constructs. This reagent did not inhibit epitope presentation, even from wild-type NP, suggesting that its production may be proteasome independent. These results support the notion that point mutation of epitope flanking sequence can serve as a mechanism for viral immune evasion, shed light on the mechanisms involved, and suggest that in vitro assays may not be sensitive indicators of flanking sequence effects.