Encapsidation of the poxvirus genome

描述（由申请人提供）：痘病毒，如牛痘病毒，在DNA病毒中是独一无二的，它只在被感染细胞的细胞质内复制。因此，病毒生命周期的进展与宿主细胞的细胞器、细胞骨架和细胞质环境的相互作用密切相关。痘病毒的细胞质复制也与它们几乎完全脱离宿主的遗传自主性有关。在病毒编码的约200个基因产物中，有介导基因表达、基因组复制和成熟以及病毒粒子组装复杂过程的基因产物。从本质上讲，一种多产的病毒感染需要病毒基因组被有效地复制并包装成新生的、具有传染性的病毒粒子。正是基因组的这种封装过程，位于复制和形态发生的界面，是这一探索性应用的重点。病毒DNA的合成是由一系列病毒编码的蛋白质完成的，随后是由病毒编码的分解酶将串联中间体加工成成熟的单体基因组。这些单体基因组被认为在IV膜闭合之前被浓缩并转运到未成熟病毒粒子（IV）中。未成熟病毒粒子由脂质双分子层分隔，并被由病毒D13蛋白组成的外部晶格包围。未成熟病毒粒子的内部似乎包含一个非结构化的蛋白质池，这些蛋白质将在以后形成成熟病毒粒子的内部核心。条件致死病毒突变体的遗传分析已经确定了两种与基因组衣壳化直接相关的病毒蛋白：I6和A32。在体外，I6蛋白与代表病毒基因组端粒发夹的DNA探针结合，具有很强的特异性。这些约50 bp的端粒，由于短发夹环的存在而共价关闭，高度富含a - t（约90%），并且在两条DNA链上都含有螺旋外碱基（EHBs）。I6与端粒探针的结合取决于EHBs的存在。相比之下，A32蛋白被认为属于FtsK-HerA泵送atp酶家族，这是由特征序列基序的守恒所定义的。我们假设基因组通过与多胺结合而浓缩，I6通过与端粒结合来标记成熟基因组的衣壳化，随后I6和A32之间的相互作用导致A32的atp酶活性激活，基因组易位到未成熟的病毒粒子中。这一假设将在两个目标中得到验证。目的：我将探讨病毒基因组的缩合及其与I6的关联；Aim II将探讨A32作为易位atp酶的作用。

DESCRIPTION (provided by applicant): Poxviruses, such as vaccinia virus, are unique among DNA viruses in replicating solely within the cytoplasm of the infected cell. As a result, the progression of the viral life cycle is intimately linked to interactions with the organelles, cytoskeleton and cytoplasmic milieu of the host cell. Cytoplasmic replication of poxviruses is also associated with their nearly complete genetic autonomy from the host. Among the ~ 200 gene products encoded by the virus are those mediate gene expression, genome replication and maturation, and the complex process of virion assembly. At its essence, a productive viral infection requires that the viral genome be efficiently replicated and packaged into nascent, infectious virions. It is this process of genome encapsidation, which lies at the interface of replication and morphogenesis, that it is the focus of this exploratory application. Viral DNA synthesis, which is accomplished by a repertoire of virally encoded proteins, is followed by the processing of concatemeric intermediates into mature, monomeric genomes by a virally encoded resolvase. These monomeric genomes are then thought to be condensed and translocated into immature virions (IV) prior to the closure of the IV membrane. Immature virions are delimited by a lipid bilayer and surrounded by an external lattice composed of the viral D13 protein. The interior of the immature virion appears to contain an unstructured pool of proteins that will later form the internal core of the mature virion. Genetic analyses of conditionally lethal viral mutants have identified two viral proteins as being directly associated with genome encapsidation: I6 and A32. In vitro, the I6 protein binds with great specificity to DNA probes representing the telomeric hairpins of the viral genome. These ~50 bp telomeres, which are covalently closed due to the presence of a short hairpin loop, are highly A-T rich (>90%) and contain extrahelical bases (EHBs) on both DNA strands. The binding of I6 to telomeric probes is dependent upon the presence of the EHBs. The A32 protein, in contrast, is thought to belong to the FtsK-HerA family of pumping ATPases, as defined by the conservation of characteristic sequence motifs. We are hypothesizing that the genome is condensed by association with polyamines, that I6 marks mature genomes for encapsidation by binding to their telomeres, and that subsequent interactions between I6 and A32 lead to activation of A32's ATPase activity and translocation of the genome into the immature virion. This hypothesis will be tested in two Aims. Aim I will explore the condensation of the viral genome and its association with I6; Aim II will explore the role of A32 as a translocating ATPase. PUBLIC HEALTH RELEVANCE: The fear that smallpox might be used as a bioterrorist weapon, and the recognition that monkeypox virus is a cause of human morbidity and mortality, has reinforced the need to study the poxviral life cycle in depth. Our investigations of genome encapsidation will provide fundamental insights into this poorly understood facet of the viral life cycle and define new targets for the development of rational antiviral therapies.