ADENOVIRUS 2 CODED EARLY GLYCOPROTEIN

蛋白质通常位于细胞内的特定位点， 因此调控信息必须存在于蛋白质的结构中 以确保他们到达正确的目的地。 我们的主要目标 是识别这些信息，使用19 kDa血浆作为模型， 腺病毒1型E3早期区编码的膜糖蛋白gp 19 K 或者Ad 5，并使用遗传学方法。 Gp 19 K是一种丰富的蛋白质， 结构是已知的，并且可以通过以下方法容易地构建活的病毒突变体： 体外定点诱变。 抗血清可用于 纯化的gp 19 K以及对应于gp 19 K的合成肽。 Gp 19 K具有一个17个氨基酸的N-末端“信号”序列，两个高甘露糖 寡糖和20个氨基酸的疏水跨膜结构域 随后是在C-末端的15个氨基酸的极性结构域。 我们有 在信号序列中具有测序损伤的分离的病毒突变体， 糖基化位点和C-末端疏水和/或极性结构域。 我们建议在加工和 运输 现在有一些突变体的数据。 以协作 研究中，突变体将作为病毒抗原类的独特模型进行研究 1 MHC抗原相互作用。 其他质粒突变体，一些测序， 可用于构建额外的病毒突变体。 我们提出了两个新的和 应用细菌遗传学标准技术的简单方法， 分离gp 19 K中随机错义突变。 E3区编码其他几种膜蛋白，其结构如下： 与GP 19 K不同。 我们建议进行基因研究，类似于 gp 19 K，以确定膜结合和转运 这些蛋白质的信号。 其中一些基因的病毒突变体， 包括与gp 19 K的融合体， 很容易建造。 针对其中每一种的肽抗血清 蛋白是可用的，并且有效的抗血清定向融合蛋白 将产生由表达载体合成的蛋白质。

Proteins generally are localized at specific sites within the cell, and therefore regulatory information must exist in the structure of the protein to assure that they arrive at the correct destination. Our major objective is to identify this information, using as a model the 19 kDa plasma membrane glycoprotein (gp19K) coded by early region E3 adenovirus 1 (Ad2) or Ad5, and using a genetic approach. Gp19K is an abundant protein whose structure is known, and viable virus mutants can easily be constructed by in vitro site directed mutagenesis. Antisera are available against purified gp19K as well as synthetic peptides corresponding to gp19K. Gp19K has a 17 amino acid N-terminal "signal" sequence, two high-mannose oligosaccharides, and a 20 amino acid hydrophobic transmembrane domain followed by a 15 amino acid polar domain at the C-terminus. We have isolated virus mutants with sequenced lesions in the signal sequence, the glycosylation sites, and the C-terminal hydrophobic and/or polar domains. We propose to characterize these mutants in terms of processing and transport. Data are now available with some mutants. In a collaborative study, the mutants will be studied as a unique model for viral antigenclass 1 MHC antigen interaction. Other plasmid mutants, some sequenced, are available to construct additional virus mutants. We propose two novel and simple methods that apply standard techniques in bacterial genetics to isolate random missense mutations in gp19K. Region E3 encodes several other membrane proteins whose structures are fundamentally different from gp19K. We propose genetic studies, similar to those with gp19K, to identify the membrane-association and transport signals for these proteins. Virus mutants in some of the these genes, including fusions to gp19K, are currently available and additional mutants are easily constructed. Peptide antisera targeted to each of these proteins are available, and potent antisera directed fusion proteins synthesized by expression vectors will be generated.