Molecular Genetics of Parvoviral DNA Replication

描述（由申请方提供）：细小病毒在所有已知生物体中是独特的，具有单链和线性的DNA基因组。基因组的5'和3'末端都是回文的，并且可以折叠成发夹结构，其在复制型（RF）DNA中产生两个病毒复制起点。在同源端粒细小病毒中，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无关。这项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自主细小病毒小鼠微小病毒（MVM），这是一种在细胞培养中生长的遗传上易于处理的病毒。我们将使用我们从MVM中学到的知识来探索新发现的致病性人类博卡病毒HBoV的起始蛋白的功能，目前还没有细胞培养系统。我们建议建立在我们以前的研究MVM DNA复制起始研究的末端发夹的结构和功能，扩展到精确的序列要求NS1结合，熔化和切口在MVM OriL。我们将探索嵌入MVM基因组中的NS1结合位点的功能意义，并询问是否存在类似的情况。我们将开始研究病毒如何激活细胞DNA损伤反应，以及病毒阐述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。遗传学和生物化学的方法将被追求，以确定驱动基因组包装的分子马达，并探索在病毒DNA复制和包装的tehrs发夹的功能。公共卫生相关性：该研究项目旨在了解细小病毒DNA复制和包装的机制，并进一步了解细小病毒作为病原体的知识。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，似乎是儿童最普遍的感染之一，了解其自然历史和复制模式将变得越来越重要，因为我们发现更多关于它与人类的相互作用。因此，在目前的更新申请中，我们建议将我们所知道的关于细小病毒基因组结构和小鼠系统DNA复制的知识用于研究调节人类病毒复制的类似过程。

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.