TRANSPORT OF VIRAL PROTEINS IN MAMMALIAN CELLS

我们实验室的总体目标是了解 控制病毒蛋白表达的方向和速度 哺乳动物细胞从它们的合成部位运输到它们的 最终目的地。我们正在用两种方式来解决这个问题：(一)通过 对一系列基因工程突变和嵌合病毒的分析 在其特定的不同阶段有缺陷的蛋白质 运输途径(II)通过分离哺乳动物细胞的突变体 在将病毒蛋白运送到特定目的地方面存在缺陷。 最近，两种类型的氨基酸序列已被证明是必要的 足以将蛋白质引导到这些非常不同的细胞中 地点。其中一种--疏水信号序列--会导致新生 多肽被转移到粗糙的细胞膜上 内质网，它们可能从那里进入高尔基体 然后到细胞表面或到细胞泡状胞质 细胞器，如溶酶体。另一个最新发现的原因是 被运输到细胞核并保留在细胞核中的蛋白质。这些 亲核信号，就像疏水信号一样，在顺序上变化很大 从蛋白质到蛋白质；在它们最简单的形式中，它们由一小段 主要由碱性氨基酸组成的区域。 这个应用程序的目标是了解两种病毒蛋白(SV40 T抗原和流感病毒血凝素)移动到其特定的 目的地：致癌的SV40T抗原被运送到 转化细胞的胞核和质膜。感染病毒 血凝素(HA)是一种典型的完整膜蛋白， 沿着传统的胞吐途径高效地进入细胞 浮出水面。特别是，我们将(I)分离和鉴定细胞 不能将HA沿细胞表面运输到细胞表面的突变体 常规胞吐途径(II)研究其性质和 一组嵌合的SV40T抗原的细胞内转运 来自另一种蛋白质(流感病毒)的疏水序列 血凝素)N端(III)检验了SV40T的假设 与质膜相关的抗原到达细胞表面，而不是 通过传统的分泌途径，但通过细胞运输 细胞核(IV)分析透明质酸沿胞吐途径的转运 突变的CHO细胞系，显示出明显改变的模式 糖基化。

The general aim of our laboratory is to understand the mechanisms which govern the direction and speed with which viral proteins expressed in mammalian cells are transported from their sites of synthesis to their final destinations. We are approaching this problem in two ways (i) by analyzing a series of genetically-engineered mutant and chimeric viral proteins that are defective at different stages of their particular transport pathways (ii) by isolating mutants of mammalian cells that are defective in transporting viral proteins to specific destinations. Recently, two types of amino acid sequences have been shown to be necessary and sufficient to guide proteins to these very different cellular locations. One of these - hydrophobic signal sequences - causes nascent polypeptides to be translocated across the membrane of the rough endoplasmic reticulum, from where they may travel to the Golgi apparatus and then to the cell surface or to cellular vesicular cytoplasmic organelles such as lysosomes. The other, more recently discovered, causes proteins to be transported to and retained in the cell nucleus. These karyophilic signals, like hydrophobic signals, vary greatly in sequence from protein to protein; in their simplest forms they consist of a short tract of largely basic amino acids. The goal of this application is to understand how two viral proteins (SV40 T antigen and influenze virus hemagglutinin) move to their specific destinations: the oncogenic SV40 T antigen is transported both to the nucleus and the plasma membrane of transformed cells. Influenze virus hemagglutinin (HA) is a prototypical integral membrane protein that moves with high efficiency along the conventional exocytotic pathway to the cell surface. In particular we will (i) isolate and characterize cellular mutants that are unable to transport HA to the cell surface along the conventional exocytotic pathway (ii) investigate the properties and intracellular transport of a set of chimeric SV40 T antigens and contain a hydrophobic sequence derived from another protein (influenza virus hemagglutinin) at their N-terminus (iii) test the hypothesis that SV40 T antigen associated with the plasma membrane reaches the cell surface, not by the conventional secretory pathway, but by transport through the cell nucleus (iv) analyze the transport of HA along the exocytotic pathway in lines of mutant CHO cells that display grossly altered patterns of glycosylation.