SARS Replicase Proteins in Pathogenesis and Replication

虽然大多数复制酶基因蛋白在SARS-CoV复制和致病机制中的确切作用尚不清楚，但现有数据表明，它们可能在培养复制以及动物复制和致病过程中发挥关键作用。该项目的总体目标是确定SARS-CoV复制酶蛋白表达和加工的要求，用于病毒在培养中的生长以及在动物中的复制、免疫反应和保护。基因方法将被用来在复制酶基因中引入突变，这些突变将改变蛋白质的加工，使假定的功能蛋白质失活，并确定嵌合蛋白质功能和病毒重组的能力。这些变化的影响将是 在培养中通过评估病毒生长、蛋白质表达和加工以及RNA合成来确定，在动物中通过评估复制、免疫反应和保护来确定。复制酶需求的确定和复制酶突变体的开发将为突变病毒的安全生长、灭活或作为候选减毒活病毒疫苗的进一步研究奠定基础。该计划的具体目标是：1)确定复制酶多蛋白裂解事件和加工中间体在SARS-CoV复制中的作用；2)确定病毒复制中对假定的RNA处理酶(npl4、15和16)的需求；3)确定SARS-CoV与嵌合复制酶蛋白和与MHV重组的能力；以及4)评估复制酶活突变对小鼠和雪貂的复制和免疫反应以及对野生型SARS-CoV攻击的保护作用。

While the precise roles of most replicase gene proteins in SARS-CoV replication and pathogenesis are not known, available data suggest that they likely play critical roles in replication in culture and in replication and pathogenesis in animals. The overall goal of this project is to define the requirements for SARS-CoV replicase protein expression and processing for virus growth in culture and for replication, immune response, and protection in animals. Genetic approaches will be used to introduce mutations in the replicase gene that will alter protein processing, inactivate putative functional proteins, and determine the capacity for chimeric protein function and virus recombination. The effects of these changes will be determined in culture by assessing virus growth, protein expression and processing, and RNA synthesis, and in animals by assessing replication, immune response, and protection. The determination of replicase requirements and the development of replicase mutants will serve as the basis for the development of mutant viruses to be grown safely for inactivation or for further study as candidate live-attenuated live virus vaccines. The Specific Aims of the program are: 1) to determine the role for replicase polyprotein cleavage events and processing intermediates in SARS-CoV replication; 2) to determine the requirement for putative RNA processing enzymes (nspl4, 15, and 16) in viral replication; 3) to determine the ability of SARS-CoV to function with chimeric replicase proteins and to recombine with MHV; and 4) to evaluate the replication and immune response from viable replicase mutants and the protection from wildtype SARS-CoV challenge in mice and ferrets.