RUI: Functional Characterization of a Geminivirus AC4 N-myristoylated Protein

双病毒感染世界各地广泛的具有重要经济意义的作物品种，并且越来越多地出现混合感染，导致协同相互作用，其特点是症状严重和产量损失巨大。部分原因是由于混合感染，这些病毒可能在复制过程中进行重组，从而产生比其系谱更具毒性的毒株。受这些病毒严重影响的作物之一是木薯（Manihot esculenta Crantz），这是一种块根作物，是全球超过5亿人的主食。这种新的木薯病毒被命名为东非木薯花叶喀麦隆病毒（EACMCV），是从喀麦隆的木薯地里分离出来的。我们已经证明EACMCV编码的AC4蛋白是一种症状决定因素，并抑制真核植物中病毒诱导的基因沉默（VIGS），这是一种抗病毒机制。这些研究也表明AC4作用于血浆和细胞质膜。突变分析表明，这些特性依赖于AC4编码的n -肉豆蔻酰基序，这是一个膜结合基序，表明AC4必须在膜区域内才能发挥作用。这些独特的特征和它的小尺寸（10 kDa）使AC4成为一个潜在的很好的探针，用于解剖和阐明病毒感染后植物细胞膜内发生的生理和生化事件。本项目的具体目的是：1)确定AC4蛋白在EACMCV发病机制中的作用；2)研究AC4与酵母双杂交筛选中发现的蛋白之间的相互作用；3)对AC4相互作用蛋白进行功能表征。由于质膜在进出细胞的运输中起重要作用，AC4可能在病毒进入和/或退出中起重要作用。该项目将鉴定和表征与AC4相互作用的膜蛋白，因为这些蛋白可能在双病毒感染中发挥作用，这对于寻求控制这些病毒的新方法至关重要。这个项目的一个重要组成部分是对本科生进行分子生物学和生物技术方面的培训，这些学生大多来自代表性不足的少数群体。这些培训将使这些学生毕业后在快速增长的生物技术就业市场上具有竞争力。

Geminiviruses infect a broad range of economically important crop species all over the world and are increasingly being shown to occur in mixed infections, resulting in synergistic interactions, characterized by severe symptoms and huge yield losses. Partly because of mixed infections, these viruses undergo recombination, presumably during replication, resulting in more virulent strains than their pedigree. One of the crops severely affected by these viruses is cassava (Manihot esculenta Crantz), a root crop that serves as a staple food to more than 500 million people worldwide. This new cassava virus, named East African cassava mosaic Cameroon virus (EACMCV) was isolated from cassava fields in Cameroon. We have shown that the EACMCV encoded AC4 protein is a symptom determinant and suppresses virus induced gene silencing (VIGS), an antivirus mechanism exhibited by eukaryotic plants. These studies also showed that AC4 targets the plasma and cytosolic membranes. Mutantional analyses showed that these properties depend on the AC4 encoded N-myristoylation motif, a membrane-binding motif, suggesting, that to function, AC4 must be within a membrane locale. These unique features and its small size (10 kDa) make AC4 a potentially good probe to dissect and elucidate physiological and biochemical events occurring within the plant cell membrane following virus infection. The specific aims of this project are to: 1) Determine the role of AC4 protein in EACMCV pathogenesis, 2) Investigate the interaction between AC4 and the proteins that have been identified in a yeast two-hybrid screen, and 3) Functionally characterize the AC4-interacting proteins.Since the plasma membrane plays an important role in trafficking in and out of the cell, AC4 likely plays an important role in virus entry and/or exit. This project will identify and characterize membrane proteins that interact with AC4 since such proteins might play a role in geminivirus infection, which would be crucial as new approaches are sought to control these viruses. An important component of this project is the training of undergraduate students, mostly from underrepresented minority groups, in molecular biology and biotechnology. This training will make these students competitive in the fast growing biotechnology job market upon graduation.