Molecular Genetics of Parvoviral DNA Replication

描述（由申请人提供）：细小病毒在所有已知生物体中是独一无二的，其DNA基因组既是单链又是线性的。基因组的 5' 和 3' 末端都是回文的，可以折叠成发夹结构，从而在复制型 (RF) DNA 中产生两个病毒复制起点。在同端粒细小病毒中，这些发夹是末端重复的一部分，而对于异端粒亚组，末端通常在序列或结构上彼此不相关。该研究计划的长期目标是在分子水平上了解异端粒细小病毒如何诱使宿主细胞复制和包装这些明显的外来分子。我们将继续探索的主要模型是小鼠自主细小病毒（MVM），这是一种在细胞培养物中生长的遗传易处理的病毒。我们将利用从MVM中学到的知识来探索新发现的致病性人类博卡病毒HBoV的启动蛋白的功能，目前还没有细胞培养系统。我们建议以之前关于 MVM DNA 复制起始的研究为基础，研究末端发夹的结构和功能，并将其扩展到 MVM OriL 上 NS1 结合、熔解和切口的精确序列要求。我们将探讨嵌入 MVM 基因组中的 NS1 结合位点的功能意义，并询问类似的情况是否也适用于 HBoV。我们将开始研究病毒如何激活细胞 DNA 损伤反应，以及病毒所产生的独特假染色质是否是其逃避或利用这些先天宿主防御要素的策略的一部分。将采用遗传和生化方法来识别驱动基因组包装的分子马达，并探索末端发夹在病毒 DNA 复制和包装中的功能。公共卫生相关性：该研究项目旨在加深对细小病毒 DNA 复制和包装机制的了解，并加深我们对细小病毒作为病原体的了解。这些也是使用这些病毒作为针对传染病和癌症的疫苗载体的必要先决条件。新发现的人类细小病毒 HBoV 似乎是儿童时期最流行的感染之一，随着我们对它与人类相互作用的了解越来越多，了解其自然史和复制模式将变得越来越重要。因此，在目前的更新申请中，我们建议将我们对细小病毒基因组结构和小鼠系统 DNA 复制的了解运用到对调节人类病毒复制的类似过程的研究中。

DESCRIPTION (provided by applicant): Parvoviruses are unique among all known organisms in having DNA genomes which are both single-stranded and linear. Both 5' and 3' ends of the genome are palindromic and can fold into hairpin structures that give rise to the two viral replication origins in replicative-form (RF) DNA. In the homotelomeric parvoviruses these hairpins are part of a terminal repeat, whereas for the heterotelomeric subgroup, the termini are typically unrelated to one another in either sequence or structure. The long term goal of this research program is to understand, at the molecular level, how heterotelomeric parvoviruses inveigle their host cell into replicating and packaging such apparently alien molecules. The major model we will continue to explore is the autonomous parvovirus Minute Virus of Mice (MVM), a genetically tractable virus that grows in cell culture. We will use what we learn from MVM to explore the function of the initiator protein of the newly-discovered pathogenic human bocavirus HBoV, for which there is currently no cell culture system. We propose to build on our previous research on MVM DNA replication initiation to study the structure and function of the terminal hairpins, extending this to the precise sequence requirements for NS1 binding, melting and nicking at MVM OriL. We will explore the functional significance of NS1 binding sites embedded in the MVM genome, and ask whether a similar situation pertains for HBoV. We will initiate studies on how the virus activates cellular DNA damage responses and whether the unique pseudochromatin that the virus elaborates is part of its strategy to evade, or employ, these elements of innate host defense. Genetic and biochemical approaches will be pursued to identify the molecular motor that drives genome packaging, and to explore the function(s) of the teminal hairpins in viral DNA replication and packaging. PUBLIC HEALTH RELEVANCE: This research program aims to develop an understanding of the mechanisms underlying parvoviral DNA replication and packaging, and further our knowledge of parvoviruses as pathogens. These are also necessary prerequisites for the use of these viruses as vaccine vectors against infectious diseases and cancer. The newly discovered human parvovirus, HBoV, appears to be one of the most prevalent infections of childhood, and understanding its natural history and mode of replication will become of increasing importance as we discover more about its interaction with the human population. In the present renewal application, therefore, we propose to parlay what we know about parvoviral genome structure and DNA replication from the murine system into a study of similar processes that regulate replication of the human virus.