Viral and Host Determinants of Parvovirus Replication

人类bocavavirus 1 （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病毒中是独特的，因为它在衣壳蛋白编码区之后的3'非编码区表达140个核苷酸的非编码RNA。bocavavirus编码的小RNA （BocaSR）仅在细胞核中表达，在病毒非结构蛋白的表达和病毒DNA复制中起重要作用。HBoV1在气液界面培养的终末分化人气道上皮（HAE-ALI）中感染和复制，模拟人气道的自然感染。重要的是，HBoV1基因组在非分裂气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Pol κ， BocaSR特异性地与病毒非结构蛋白相互作用。另一方面，HBoV1是HAE-ALI中腺相关病毒（adeno-associated virus， AAV）复制的辅助病毒，BocaSR在促进AAV Rep基因表达和DNA复制中也起着关键作用。我们的中心假设是HBoV1已经进化到表达一种非编码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，这模仿了由无错误Pol η和Pol κ驱动的DNA修复过程。在这项应用中，我们旨在揭示bocasr调控病毒非结构蛋白表达和细胞核内病毒DNA复制的机制，并了解病毒在DNA合成中对无错误Pol η和Pol κ的操纵。BocaSR是在所有小DNA病毒中发现的第一个BocaSR，与其他已知的RNA聚合酶III （Pol III）转录的病毒非编码RNA不同，它具有调节病毒基因表达和直接参与病毒DNA在细胞核内复制的独特特性。研究bocasr对细小病毒复制的调控作用将有助于了解其他病毒非编码rna在病毒复制中的功能。此外，了解细小病毒如何利用DNA修复机制在非分裂细胞中进行病毒基因组复制，将有助于我们了解小DNA病毒如何克服屏障，在细胞周期外复制其基因组。解剖HBoV1基因组复制的病毒和宿主决定因素将有助于开发预防由HBoV1引起的急性呼吸道感染的治疗方法。最后，深入了解AAV DNA复制将有助于找到更好的方法来生产重组AAV （rAAV）载体，并提高rAAV基因递送的效率，特别是对非分裂细胞的递送。

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.