Molecular Mechanisms of Parvovirus Gene Expression and Replication

描述(申请人提供)：人类细小病毒B19(B19V)感染会导致怀孕中期的孕妇胎儿积水。它还导致严重的血液疾病，包括红细胞转换率高的患者的一过性再生障碍性危象，以及免疫缺陷和免疫受损患者的慢性贫血，在某些情况下，这可能是致命的。目前，还没有专门的抗病毒药物或疫苗(可防止高危人群感染B19V)。B19V复制高度受限于 骨髓和胎肝中的人红系祖细胞(EPC)。B19V感染引起的胎儿积水和血液病主要是由于B19V复制的内皮祖细胞被直接杀伤所致。在DNA病毒中，B19V在处理其前体mRNA(Pre-mRNA)方面有一个独特的特征，即所有病毒的mRNAs都是从一个Pre-mRNA交替处理而来的。B19V Pre-mRNA的选择性剪接受多个剪接增强子控制，在调节B19V mRNAs的选择性多聚腺苷基化中起关键作用，并产生丰富的小病毒mRNAs，编码两种小的非结构病毒蛋白，即7.5 kDa和11 kDa。11-kDa蛋白与Grb2蛋白相互作用，Grb2蛋白将促红细胞生成素受体介导的信号连接到内皮祖细胞中的Ras/MEK/ERK通路，并在病毒DNA复制中发挥重要作用。重要的是，B19V感染可诱导DNA损伤反应(DDR)。ATR和DNA-PKcs的激活促进了病毒DNA的复制。B19V不使用宿主复制机制来复制其单链DNA基因组；相反，它似乎诱导了DDR，并随后利用宿主DNA修复机制来促进自身的复制。此外，B19V在内皮祖细胞中的复制在低氧条件下显著增加，并通过下调MEK/ERK信号转导。在过去的几年里，我们已经建立了两个实验细胞系统，消除了研究B19V复制的两个关键障碍：一个是高效的B19V感染系统，涉及低氧条件下内皮祖细胞的体外扩增(它模拟了人骨髓和胎肝中内皮祖细胞的微环境)；另一个是反向遗传学方法，涉及将B19V双链DNA形式的基因组导入在低氧条件下培养的UT7/EPO-S1细胞。使用这两个系统，以及一种新建立的体外检测病毒DNA复制的方法，我们将：i)确定B19V Pre-mRNA替代处理的潜在机制；ii)阐明11-kDa蛋白通过颠覆MEK/ERK信号促进B19V复制的机制；以及iii)确定DDR促进B19V DNA复制的机制。我们的长期目标是确定在生理相关的环境中，即B19V在低氧条件下体外扩增的内皮祖细胞中，单个启动子转录的细小病毒前mRNA的改变处理以及细小病毒DNA复制的关键分子机制。

DESCRIPTION (provided by applicant): Human parvovirus B19 (B19V) infection causes hydrops fetalis in pregnant women during the second- trimester. It also causes severe hematological diseases, including transient aplastic crisis in patients with a high turn-over rate f red blood cells; and chronic anemia in immunodeficient and immunocompromised patients which, in some cases, can be fatal. At present, no specific antiviral drugs or vaccines (that prevent B19V infection in high-risk groups) are available. B19V replication is highly restricted to human erythroid progenitor cells (EPCs) in the bone marrow and fetal liver. B19V infection-mediated hydrops fetalis and hematological disorders mainly result from the direct killing of the EPCs in which B19V replicates. Among DNA viruses, B19V has a unique feature in the processing of its precursor mRNA (pre-mRNA) in that all the viral mRNAs are alternatively processed from a single pre-mRNA. Alternative splicing of the B19V pre-mRNA, which is controlled by multiple splicing enhancers, plays a key role in regulating alternative polyadenylation of B19V mRNAs, and generates abundant small viral mRNAs for encoding two small non- structural viral proteins, i.e. 7.5-kDa and 11-kDa. The 11-kDa protein interacts with Grb2, a protein that links the signals mediated by the erythropoietin receptor to the Ras/MEK/ERK pathway in EPCs, and plays an important role in viral DNA replication. Importantly, B19V infection induces a DNA damage response (DDR). Activation of ATR and DNA-PKcs facilitates viral DNA replication. B19V does not use the host replication machinery to replicate its ssDNA genome; rather, it appears to induce a DDR and subsequently co-opts the host DNA repair mechanism to facilitate its own replication. Furthermore, B19V replication in EPCs is markedly increased under hypoxic conditions and is mediated via the down-regulation of MEK/ERK signaling. Over the past few years, we have established two experimental cell systems that remove two critical barriers to the study of B19V replication: an efficient system of productive B19V infection involving the ex vivo- expansion of EPCs under hypoxic conditions (which mimic the microenvironment of EPCs in human bone marrow and fetal liver), and a reverse genetics approach that involves transfection of the B19V dsDNA-form genome into UT7/Epo-S1 cell line cells cultured under hypoxic conditions. Using these two systems, as well as a newly-established in vitro assay to measure viral DNA replication, we will: i) determine the mechanisms underlying the alternative processing of B19V pre-mRNA; ii) elucidate the mechanisms by which the 11-kDa protein subverts MEK/ERK signaling to promote B19V replication; and iii) determine the mechanisms underlying DDR-facilitated B19V DNA replication. Our long-term goal is to identify the key molecular mechanisms underlying the alterative processing of the single promoter-transcribed parvoviral pre-mRNA, as well as parvovirus DNA replication, in a physiologically- relevant setting, i.e., EPCs ex vivo-expanded under hypoxic conditions for B19V in this application.