CHARACTIZATION OF MOSQUITO FURIN ENDOPROTEASE

蚊子弗林内切蛋白酶的表征

• 批准号: 6258297
• 负责人: HANS W HELDNER
• 金额: $ 14.87万
• 依托单位: UNIVERSITY OF TEXAS SAN ANTONIO
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-09-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6258297
• 关键词: Aedes; Sindbis virus; antisense nucleic acid; arthropod borne communicable disease; communicable disease transmission; disease /disorder prevention /control; disease vectors; endopeptidases; enzyme activity; gene expression; glycoproteins; host organism interaction; in situ hybridization; nucleic acid sequence; polymerase chain reaction; prohormone convertase; tissue /cell culture; virulence; virus infection mechanism; virus protein; virus replication

Alphaviruses are maintained in nature in a cycle that involves mosquito and vertebrate hosts. Several alphaviruses are significant human pathogens, and are among the primary causes of mosquito-borne viral encephalitis in the Americas. Due to the lack of effective vaccines to many of these viruses, research into novel non-vaccine based antiviral strategies has been initiated. These strategies include the engineering of virus resistance through genetic manipulation of the mosquito vector. To accomplish this goal it will be necessary to characterize virus/host interactions that are required for virus replication in the mosquito, and to devise intervention strategies that target these interactions directly. The broad objective of this proposal is to characterize virus mosquito, and to devise intervention strategies that target these interactions directly. The broad objective of this proposal is to characterize virus/mosquito interactions that may provide targets for intervention in the natural virus maintenance cycle. The specific objective of this application is to identify and characterize the mosquito furin endoprotease, and to investigate the role of this enzyme in alphavirus replication. Furin-mediated cleavage of the viral glycoprotein precursor, PE2, occurs during virus replication in vertebrate and arthropod cells and is required for normal virus maturation processes. Inhibition of PE2 cleavage has been linked to attenuation of viral virulence in a model vertebrate host and to an arthropod cell-specific growth restriction. A furin enzyme (Dfurin1) of Drosophila melanogaster has been characterized, however, no mosquito furin enzyme has been identified or studied directly. The experiments described in this proposal will utilize genetic reagents based on the Durin1 gene to construct and characterize a cDNA clone encoding the mosquito homologue of the furin enzyme. Genetic constructs based on this clone will be used to express and characterize the furin gene product, and to characterize the distribution of mosquito tissues in which the furin gene is transcribed at levels detectable by in situ hybridization. The potential for targeting the PE2/furin interaction a an antiviral strategy will be addressed by investigating the replication, dissemination, and tissue tropism phenotypes of PE2 cleavage-defective Sindbis viruses within living mosquitos, and by investigating the effects that furin specific antisense RNA expression has upon Sindbis virus replication within cultured mosquito cells.

甲病毒在自然界中以涉及蚊子和脊椎动物宿主的循环方式维持。几种甲病毒是重要的人类病原体，也是美洲蚊媒病毒性脑炎的主要原因之一。由于许多此类病毒缺乏有效的疫苗，因此已经开始研究新型非疫苗抗病毒策略。这些策略包括通过蚊子载体的基因操作来工程化病毒抗性。 为了实现这一目标，有必要描述病毒在蚊子中复制所需的病毒/宿主相互作用的特征，并设计直接针对这些相互作用的干预策略。该提案的总体目标是描述病毒蚊子的特征，并制定直接针对这些相互作用的干预策略。该提案的主要目标是描述病毒/蚊子相互作用的特征，这可能为自然病毒维持周期的干预提供目标。本应用的具体目的是鉴定和表征蚊子弗林蛋白酶内切酶，并研究该酶在甲病毒复制中的作用。弗林蛋白酶介导的病毒糖蛋白前体 PE2 的裂解发生在脊椎动物和节肢动物细胞中的病毒复制过程中，并且是正常病毒成熟过程所必需的。 PE2 裂解的抑制与模型脊椎动物宿主中病毒毒力的减弱以及节肢动物细胞特异性生长限制有关。黑腹果蝇的弗林蛋白酶（Dfurin1）已被表征，然而，还没有直接鉴定或研究蚊子弗林酶。本提案中描述的实验将利用基于 Durin1 基因的遗传试剂来构建和表征编码弗林酶的蚊子同源物的 cDNA 克隆。基于该克隆的遗传构建体将用于表达和表征弗林蛋白酶基因产物，并表征蚊子组织的分布，其中弗林蛋白酶基因以可通过原位杂交检测的水平转录。通过研究 PE2 裂解缺陷型 Sindbis 病毒在活蚊子中的复制、传播和组织向性表型，以及研究弗林蛋白酶特异性反义 RNA 表达的影响，将解决以 PE2/弗林蛋白酶相互作用作为抗病毒策略的潜力。辛德比斯病毒在培养的蚊子细胞内复制。