Viral and Host Determinants of Parvovirus Replication

人类博卡病毒1型(HBoV1)是一种自主的人类细小病毒，可引起幼儿急性呼吸道感染。HBoV1在所有小DNA病毒中是独一无二的，因为它表达一个140个核苷酸的非编码RNA，该RNA来自衣壳蛋白编码区之后的3‘非编码区。这种博卡病毒编码的小RNA(BocaSR)只在细胞核中表达，在病毒非结构蛋白的表达和病毒DNA的复制中都发挥着重要的作用。HBoV1在气液界面培养的终末分化的人呼吸道上皮细胞(HAE-ALI)中感染和复制，模拟人类呼吸道的自然感染。重要的是，HBoV1基因组在未分裂的呼吸道上皮细胞中的复制利用无错误的Y家族DNA修复DNA聚合酶(POL)η和POLκ，并且BOCASR与病毒非结构蛋白特异性地相互作用。另一方面，HBoV1是腺相关病毒(AAV)在HAE-ALI中复制的辅助病毒，BocaSR也在促进AAV Rep基因表达和DNA复制中发挥关键作用。我们的中心假设是，HBoV1已经进化到表达一个非编码的RNA，以促进病毒非结构蛋白的表达及其在许可病毒DNA复制中的功能，这模拟了由无错误的POLη和POLκ驱动的DNA修复过程。在这一应用中，我们旨在揭示BocaSR调控病毒非结构蛋白表达和核内病毒DNA复制的机制，并了解病毒对无错误的POLη和POLκ在DNA合成中的操纵。BocaSR是在所有小DNA病毒中发现的第一个新的病毒，它不同于其他已知的RNA聚合酶III(Pol III)转录的病毒非编码RNA，其独特的性质是调节病毒基因的表达，并在病毒核内的DNA复制中发挥直接作用。对BocaSR调控细小病毒复制的研究将有助于理解其他病毒非编码RNA在病毒复制中的功能。此外，关于细小病毒如何在未分裂的细胞中使用DNA修复机制进行病毒基因组复制的知识将有助于我们了解小型DNA病毒如何克服障碍，在细胞周期外复制其基因组。剖析HBoV1基因组复制的病毒和宿主决定因素将有助于开发预防HBoV1引起的急性呼吸道感染的治疗方法。最后，深入了解AAV的DNA复制将有助于找到一种更好的方法来制备重组AAV(RAAV)载体，并提高rAAV基因的传递效率，特别是对未分裂细胞的传递。

Human bocavirus 1 (HBoV1), an autonomous human parvovirus, causes acute respiratory tract infections in young children. HBoV1 is unique among all small DNA viruses in that it expresses a noncoding RNA of 140 nucleotides from the 3' noncoding region after the capsid protein-coding region. This bocavirus-encoded small RNA (BocaSR) is exclusively expressed in the nucleus and plays an important role both in expression of viral nonstructural proteins and in viral DNA replication. HBoV1 infects and replicates in terminally-differentiated human airway epithelium cultured at an air-liquid interface (HAE-ALI), mimicking natural infection of human airways. Importantly, HBoV1 genome replication in the nondividing airway epithelial cells utilizes error-free Y- family DNA repair DNA polymerase (Pol) η and Pol κ, and BocaSR specifically interacts with viral nonstructural proteins. On the other hand, HBoV1 is a helper virus for the replication of adeno-associated virus (AAV) in HAE-ALI, and BocaSR also plays a key role in facilitating AAV Rep gene expression and DNA replication. Our central hypothesis is that HBoV1 has evolved to express a noncoding RNA to facilitate expression of the viral nonstructural proteins and their function in licensing viral DNA replication, which mimics a DNA repair process driven by error-free Pol η and Pol κ. In this application, we aim to reveal the mechanisms underlying the BocaSR-regulated expression of viral nonstructural proteins and viral DNA replication in the nucleus, and to understand the viral manipulation of the error-free Pol η and Pol κ in DNA synthesis. The BocaSR is novel as it is the first one found among all small DNA viruses and differs from other known RNA polymerase III (Pol III) transcribed viral noncoding RNAs by its unique properties in regulating viral gene expression and playing a direct role in viral DNA replication in the nucleus. Studying BocaSR-regulating parvovirus replication will contribute to the understanding of the functions of other viral noncoding RNAs in virus replication. In addition, knowledge in how parvovirus employs the DNA repair machinery for viral genome replication in nondividing cells will help us understand how a small DNA virus overcomes the barrier to replicate its genome outside of the cell cycle. Dissecting the viral and host determinants of HBoV1 genome replication will help the development of therapeutic approaches to prevent acute respiratory tract infections caused by HBoV1. Finally, deep understanding AAV DNA replication will help find a better way to produce recombinant AAV (rAAV) vector and to increase the efficacy of rAAV gene delivery, in particular, to nondividing cells.