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，因为它只在受感染细胞的细胞质内复制。因此，病毒生命周期的进展与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独立性有关。在病毒编码的约200个基因产物中，有参与基因表达、基因组复制和成熟以及复杂的病毒粒子组装过程的基因产物。从本质上讲，有效的病毒感染需要有效地复制病毒基因组，并将其包装成新生的具有感染性的病毒粒子。正是这种位于复制和形态发生交界处的基因组封装过程，是这一探索性应用的重点。病毒DNA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解旋酶将连接的中间产物加工成成熟的单体基因组。这些单体基因组随后被认为在IV膜关闭之前被浓缩并转移到未成熟的病毒粒子(IV)中。未成熟的病毒粒子由脂质双层分隔，并被病毒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无结构的蛋白质池，这些蛋白质稍后将形成成熟病毒粒子的内部核心。对条件致死病毒突变的遗传分析已经确定了两种与基因组封装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探针具有极高的特异性。这些~50bp的端粒由于短发夹环的存在而共价闭合，高度富含A-T(&gt；90%)，并在两条DNA链上含有螺旋外碱基(EHb)。I6与端粒探针的结合取决于EHBs的存在。相反，A32蛋白被认为属于FtsK-Hera泵ATPase家族，其定义是特征序列基序的保守性。我们假设基因组是通过与多胺结合而浓缩的，I6通过与它们的端粒结合来标记成熟基因组的囊化，并且I6和A32之间随后的相互作用导致A32‘S ATPase活性的激活和基因组的移位到未成熟的病毒粒子中。这一假设将在两个目标上得到检验。目的I将探讨病毒基因组的浓缩及其与I6的关系；AIM II将探讨A32作为转位ATPase的作用。 公共卫生相关性：对天花可能被用作生物恐怖武器的恐惧，以及对猴痘病毒是人类发病和死亡的原因的认识，加强了深入研究天花病毒生命周期的必要性。我们对基因组包裹的研究将提供对病毒生命周期这一鲜为人知的方面的基本见解，并为合理的抗病毒疗法的开发确定新的靶点。

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.