Evidence for pH-Dependent Protease Activity in the Adeno-Associated Virus Capsid
Evidence for pH-Dependent Protease Activity in the Adeno-Associated Virus Capsid
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DOI：
10.1128/jvi.01717-12
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发表时间：
2012-11-01
影响因子：
5.4
通讯作者：
McKenna, Robert
中科院分区：
文献类型：
--
作者：
Salganik, Maxim;Venkatakrishnan, Balasubramanian;McKenna, Robert
高度纯化的腺相关病毒（AAV）衣壳在pH 5.5下体外孵育诱导衣壳蛋白在几个氨基酸位置显著自裂。pH为7.5时未见自裂现象。对其他AAV血清型的检测显示至少有两种不同的ph诱导裂解模式，这表明不同的血清型进化出了不同的蛋白酶裂解位点。相比之下，用外部蛋白酶底物孵育AAV血清型表明，纯化的AAV衣壳在中性pH下具有强大的蛋白酶活性，而在pH 5.5下则没有，这与衣壳蛋白自裂解所见相反。一些证据表明，蛋白酶活性是AAV衣壳固有的，而不是由于污染蛋白质。对照病毒制剂在外部底物上没有蛋白酶活性，AAV病毒制剂滤液也没有蛋白酶活性污染衣壳。此外，n端Edman测序在AAV1和AAV9中发现了独特的自切割位点，这些位点附近的氨基酸突变消除了切割。最后，AAV2 （E563A）中一个保守的ph敏感结构区域的氨基酸突变消除了外部底物上的蛋白酶活性，但似乎不影响自裂。综上所述，我们的数据表明AAV衣壳具有一个或多个对pH诱导敏感的蛋白酶活性位点。此外，酸性ph值与晚期核内体中所见的ph值相似，诱导衣壳的结构变化，从而诱导自溶蛋白酶活性。ph依赖性蛋白酶活性可能在病毒感染中起作用。
Incubation of highly purified adeno-associated virus (AAV) capsids in vitro at pH 5.5 induced significant autocleavage of capsid proteins at several amino acid positions. No autocleavage was seen at pH 7.5. Examination of other AAV serotypes showed at least two different pH-induced cleavage patterns, suggesting that different serotypes have evolved alternative protease cleavage sites. In contrast, incubation of AAV serotypes with an external protease substrate showed that purified AAV capsid preparations have robust protease activity at neutral pH but not at pH 5.5, opposite to what is seen with capsid protein autocleavage. Several lines of evidence suggested that protease activity is inherent in AAV capsids and is not due to contaminating proteins. Control virus preparations showed no protease activity on external substrates, and filtrates of AAV virus preparations also showed no protease activity contaminating the capsids. Further, N-terminal Edman sequencing identified unique autocleavage sites in AAV1 and AAV9, and mutagenesis of amino acids adjacent to these sites eliminated cleavage. Finally, mutation of an amino acid in AAV2 (E563A) that is in a conserved pH-sensitive structural region eliminated protease activity on an external substrate but did not seem to affect autocleavage. Taken together, our data suggested that AAV capsids have one or more protease active sites that are sensitive to pH induction. Further, it appears that acidic pHs comparable to those seen in late endosomes induce a structural change in the capsid that induces autolytic protease activity. The pH-dependent protease activity may have a role in viral infection.