FUNCTION OF THE BACULOVIRUS MEMBRANE FUSION PROTEIN

病毒包膜蛋白与宿主细胞的最初相互作用是 对大多数动物病毒的感染至关重要。 无脊椎动物作为许多病毒病原体的中间宿主， 人类，但对它们之间的相互作用知之甚少。 病毒和无脊椎动物细胞。杆状病毒是一种大的DNA病毒， 昆虫，因此，作为一个重要的模式系统的研究， 病毒与无脊椎动物细胞的相互作用。杆状病毒包膜 糖蛋白gp64是一种中和性抗原， 与两种蜱介导的包膜蛋白的酸序列同一性 脊椎动物的正粘病毒样病毒（Thogoto和Dhori虫媒病毒）。 这表明这些虫媒病毒和杆状病毒具有共同的 感染无脊椎动物细胞的机制。然而，这些杆状病毒和 虫媒病毒包膜蛋白没有显示明显的序列或结构 与正粘病毒如流感病毒的HA蛋白相似。 最近的研究（本实验室）表明，杆状病毒gp64蛋白是 pH依赖性膜融合蛋白，表明gp64介导 病毒体形成过程中病毒体包膜与内体膜的融合 通过内吞作用进入。在拟议的研究中，gp64的功能 蛋白质将通过多种互补方法进行详细检查。 蛋白质功能和功能域将被识别， 其特征在于特别关注膜融合和细胞受体 识别.将使用以下方法：1）直接 蛋白质的诱变和功能测定将鉴定功能性的 蛋白质的结构域。2)抗gp64单克隆抗体（MAb）将在 产生并筛选用于中和膜融合或病毒体 传染性将绘制由中和MAb识别的表位。第三章 结构内含有离散突变的重组杆状病毒 产生gp64蛋白的功能结构域并用于 检查对病毒感染性、感染周期和病毒体的影响 成熟4)与gp64蛋白（或 出芽病毒上的其他蛋白质）将被鉴定、克隆， 测序 类似于上述（1和2）的方法将被 用于鉴定与病毒包膜结合所需的结构域 proteins.总的来说，这些研究将提供有价值的新见解， 无脊椎动物病毒使用的高度保守的分子机制， 识别并进入宿主细胞。

The initial interactions of viral envelope proteins with host cells are critical for the establishment of infection by most animal viruses. Invertebrates serve as intermediate hosts for numerous viral pathogens of humans, but relatively little is known about the interactions between viruses and invertebrate cells. Baculoviruses are large DNA viruses of insects and as such, serve as an important model system for studies of the interactions of viruses with invertebrate cells. The baculovirus envelope glycoprotein, gp64, is a neutralizing antigen and shows striking amino acid sequence identity with the envelope proteins of two tick vectored orthomyxo-like viruses (Thogoto and Dhori arboviruses) of vertebrates. This indicates that these arboviruses and baculoviruses share a common mechanism for infecting invertebrate cells. However, these baculovirus and arbovirus envelope proteins show no apparent sequence or structural similarity to the HA proteins of orthomyxoviruses, such as influenza. Recent studies (this laboratory) show that the baculovirus gp64 protein is a pH dependent membrane fusion protein, indicating that gp64 mediates fusion of the virion envelope with the endosome membrane during virion entry by endocytosis. In the proposed studies, the function of the gp64 protein will be examined in detail by multiple and complementary methods. Protein functions and functional domains will be identified and characterized with a specific focus on membrane fusion and cell receptor recognition. The following approaches will be used: 1) Directed mutagenesis of the protein and functional assays will identify functional domains of the protein. 2) Anti-gp64 monoclonal antibodies (MAbs) will be generated and screened for neutralization of membrane fusion or virion infectivity. Epitopes recognized by neutralizing MAbs will be mapped. 3) Recombinant baculoviruses containing discrete mutations within structural and functional domains of the gp64 protein will be generated and used to examine the effects on viral infectivity, the infection cycle, and virion maturation. 4) Cellular receptors that interact with the gp64 protein (or other proteins on the budded virus) will be identified, cloned, and sequenced. Approaches similar to those described above (1 and 2) will be utilized to identify domains required for binding to viral envelope proteins. In total, these studies will provide valuable new insight into the highly conserved molecular mechanisms used by invertebrate viruses to recognize and enter host cells.