REGULATION OF ADENOVIRUS PROTEINASE BY A PEPTIDE AND DNA

在某些情况下出现的抗病毒治疗的潜在靶点 病毒感染是病毒编码的蛋白酶。这些酶， 对于合成传染性病毒来说是必不可少的，都需要进行 病毒特异性前体蛋白参与成熟、组装 以及复制致病的人类病毒，如腺病毒， 脊髓灰质炎病毒、脑炎病毒、甲型和丙型肝炎病毒， 巨细胞病毒和人类免疫缺陷病毒。病毒编码 蛋白水解酶对其病毒编码底物具有高度的特异性。如果 可以开发出同样特定的抑制剂，并针对感染的 细胞，它们应该干扰病毒复制，而不是正常的 细胞新陈代谢。我们的模型系统是HeLa细胞在体内的感染 用人腺病毒2型(AD2)培养。 我们发现在体外最大的AD2蛋白水解酶活性，有三种成分 是必需的-腺病毒L3 23K基因的蛋白产物，AN11 起源于病毒粒子C末端的氨基酸多肽(PVlc) 前体蛋白PVI和病毒DNA。辅因子增加了k(CAT)， 使用pVlc是300倍，使用ad2 DNA也是6000倍。我们解决了 PVlc与pVlc复合酶2.6的三维结构 Angstrom分辨率。这种蛋白质的折叠是独一无二的。推定的 活性中心中含有Cys-His-Glu三联体和氧阴离子空穴 木瓜酶中类似木瓜酶的排列。如果这是活动站点，则此 蛋白质将代表一类新的半胱氨酸蛋白酶和一种新的， 第五组含有催化三联体的酶。 在这里，我们建议在生物化学和结构层面上理解 腺病毒蛋白水解酶的活性是如何调节的。我们会 定位和表征活性部位，通过以下方式阐明作用机制 其中两个辅因子刺激了蛋白酶活性，并表征了 感染后不同时间的酶活性。我们刚刚展示了 这种β-肌动蛋白可以作为辅因子，并将表征 在体外和体内与酶的相互作用。 尽管这些实验本身很重要，但它们的结果将 也可在后续拨款中使用，以设计不同类型的 作为抗病毒药物的蛋白水解酶抑制剂。

Among potential targets for antiviral therapy that arise during certain viral infections are the virus-coded proteinases. These enzymes, essential for the synthesis of infectious virus, are required to process virus-specific precursor proteins involved in the maturation, assembly and replication of such pathogenic human viruses as adenovirus, poliovirus, encephalitis virus, hepatitis A and C viruses, cytomegalovirus, and human immunodeficiency virus. virus-coded proteinases are highly specific for their virus-coded substrates. If equally specific inhibitors can be developed and targeted to infected cells, they should interfere with virus replication and not with normal cellular metabolism. Our model system is the infection of HeLa cells in culture by human adenovirus serotype 2 (Ad2). We showed for maximal Ad2 proteinase activity in vitro, three components are required- the protein product of the adenovirus L3 23K gene, an 11 amino acid peptide (pVlc) that originates from the C-terminus of virion precursor protein pvI, and the viral DNA. The cofactors increase k(cat), 300-fold with pVlc and 6000-fold with Ad2 DNA as well. We solved the three-dimensional structure of the proteinase complexed with pVlc at 2.6 Angstrom resolution. The fold of the protein is unique. A putative active site contains a Cys-His-Glu triplet and oxyanion hole in an arrangement similar to that in papain. If that is the active site, this protein would represent a new class of cysteine proteinases and a new, fifth group of enzymes that contain catalytic triads. Here we propose to understand at the biochemical and structural levels how the activity of the adenovirus proteinase is regulated. We shall locate and characterize the active site, elucidate the mechanisms by which the two cofactors stimulate proteinase activity and characterize the enzyme during different times after infection. We have just shown that beta-actin can serve as a cofactor and will characterize that interaction with the enzyme in vitro and in vivo. Although important in themselves, the results of these experiments will also be used in a subsequent grant to design different types of proteinase inhibitors to act as antiviral agents.