MOLECULAR BASIS FOR PARAINFLUENZA 3 INFECTION

人副流感病毒3型（HPF 3），副粘病毒属成员 非节段负链RNA病毒家族，是重要的病原体 儿童下呼吸道疾病。 这种病毒会导致几种 在发达国家和发展中国家， 世界欠发达地区。 尽管这在医学上很重要 病毒，我们对基本过程的了解存在很大差距 导致病毒生长，包括转录机制 和复制。 病毒与宿主细胞相互作用的大部分生物学原理， 包括对病毒诱导的膜融合的要求和 这种病毒在两种细胞中引起持续感染倾向 文化和动物（包括人），仍然是模糊的。 的总目标 本项目旨在阐明HPF 3感染的分子机制 细胞并引起细胞病理学，产生子代病毒，并建立 坚持不懈 这一目标将主要通过使用 明确的体外系统来研究膜融合的过程， RNA基因组的转录和复制以及持续感染。 此外，将设计一种新的体外系统来研究 病毒转录和复制中的顺式作用序列。 的 本提案的具体目标是：（1）确定 HPF 3诱导的膜融合的要求，特别是通过评估 两种病毒糖蛋白，融合蛋白（F）和 血凝素-神经氨酸酶蛋白（HN）。 细胞融合的诱导 在中性pH下，导致合胞体的形成， 副粘病毒感染的细胞培养的特点，并可能是重要的 由这些病毒引起的疾病的发病机理。 拟议的研究 将持续利用细胞独特的融合特性 感染了HPF 3，这是我演示的直接结果， 膜融合需要HPF 3的F和HN蛋白。 (2)确定参与转录的病毒和细胞蛋白质 和HPF 3的复制。 对于这些研究，将利用 最近，一种定义明确的体外转录/复制测定系统 在我的实验室开发的，一个系统，支持完整的 转录、复制和组装成HPF 3的核衣壳 基因组(3)来分析特定的病毒核苷酸序列， 病毒RNA聚合酶功能。 为了实现这一目标，将设计各种方法， 在体外将合成RNA组装成核衣壳。 上的特定位点 病毒序列将通过限制酶消化而改变， 定点突变，以评估特定病毒基因组的作用， 序列在核衣壳组装和病毒RNA聚合酶功能中的作用。（四） 为了扩展HPF 3持久性的体外模型的表征， 感染，以分析的因素负责持久性 这个病毒。病毒基因组和宿主的相对贡献 将评估细胞与持续感染表型的关系， 病毒蛋白稳定性和细胞表面表达的改变 在持续感染的细胞中， 将检查持续性感染。 这些研究应该有助于 深入了解HPF 3生命周期中的重要分子事件， 应该有助于设计未来的预防和治疗。

Human parainfluenza virus type 3 (HPF3), a member of the paramyxovirus family of non-segmented negative-strand RNA viruses, is an important agent of lower respiratory tract disease in children. This virus causes several of the most significant childhood viral diseases in both developed and underdeveloped areas of the world. Despite the medical importance of this virus, there are great gaps in our knowledge of the fundamental processes leading to growth of the virus, including the mechanisms of transcription and replication. Much of the biology of virus-host cell interactions, including the requirements for virus-induced membrane fusion and the propensity of this virus to cause persistent infections in both cell culture and animals (including man), remains obscure. The overall goal of this project is to elucidate the molecular mechanisms whereby HPF3 infects cells and causes cytopathology, produces progeny virus, and establishes persistence. This goal will be accomplished primarily by using well-defined in vitro systems to study the processes of membrane fusion, transcription and replication of the RNA genome, and persistent infection. In addition, a new in vitro system will be designed to study the role of cis-acting sequences in transcription and replication of the virus. The specific objectives of the current proposal are: (1) To determine the requirements for HPF3-induced membrane fusion, specifically by assessing the role of each of the two viral glycoproteins, the fusion protein (F) and the hemagglutinin-neuraminidase protein (HN). The induction of cell fusion at neutral pH, resulting in the formation of syncytia, is a characteristic feature of paramyxovirus infection in cell culture, and may be important in the pathogenesis of diseases caused by these viruses. The proposed studies will take advantage of the unique fusion properties of cells persistently infected with HPF3, and are a direct outgrowth of my demonstration that both the F and the HN proteins of HPF3 are required for membrane fusion. (2) To determine the viral and cellular proteins involved in transcription and replication of HPF3. For these studies, advantage will be taken of the well-defined in vitro transcription/replication assay system recently developed in my laboratory, a system which supports the complete transcription, replication and assembly into nucleocapsids of the HPF3 genome. (3) To analyze the specific viral nucleotide sequences that control viral RNA polymerase function. To achieve this, methods will be devised to assemble synthetic RNA into nucleocapsids in vitro. Specific sites on the viral sequence will be altered by restriction enzyme digestion and site-directed mutagenesis, to assess the roles of particular , viral genome sequences in nucleocapsid assembly and viral RNA polymerase function. (4) To extend the characterization of an in vitro model of HPF3 persistent infection, in order to analyze the factors responsible for persistence of this virus. The relative contributions of the viral genome and the host cell to the persistently infected phenotype will be assessed, and potential alterations in the stability and cell-surface expression of viral proteins in the persistently infected cells that may contribute to the maintenance of the persistent infection will be examined. These studies should lend insight into important molecular events in the life cycle of HPF3, and should assist in the design of future prevention and therapy.