Ganaie Safder S, Chen Aaron Yun, Huang Chun, Xu Peng, Kleiboeker Steve, Du Aifang, Qiu Jianming
Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA.
Institute of Preventive Veterinary Medicine, Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou, China.
J Virol. 2018 Mar 28;92(8). doi: 10.1128/JVI.02050-17. Print 2018 Apr 15.
Human parvovirus B19 (B19V) expresses a single precursor mRNA (pre-mRNA), which undergoes alternative splicing and alternative polyadenylation to generate 12 viral mRNA transcripts that encode two structural proteins (VP1 and VP2) and three nonstructural proteins (NS1, 7.5-kDa protein, and 11-kDa protein). Splicing at the second 5' donor site (D2 site) of the B19V pre-mRNA is essential for the expression of VP2 and the 11-kDa protein. We previously identified that -acting intronic splicing enhancer 2 (ISE2) that lies immediately after the D2 site facilitates the recognition of the D2 donor for its efficient splicing. In this study, we report that ISE2 is critical for the expression of the 11-kDa viral nonstructural protein. We found that ISE2 harbors a consensus RNA binding motif protein 38 (RBM38) binding sequence, 5'-UGUGUG-3'. RBM38 is expressed during the middle stage of erythropoiesis. We first confirmed that RBM38 binds specifically with the ISE2 element The knockdown of RBM38 significantly decreases the level of spliced mRNA at D2 that encodes the 11-kDa protein but not that of the D2-spliced mRNA that encodes VP2. Importantly, we found that the 11-kDa protein enhances viral DNA replication and virion release. Accordingly, the knockdown of RBM38 decreases virus replication via downregulating 11-kDa protein expression. Taken together, these results suggest that the 11-kDa protein facilitates B19V DNA replication and that RBM38 is an essential host factor for B19V pre-mRNA splicing and for the expression of the 11-kDa protein. B19V is a human pathogen that can cause fifth disease, arthropathy, anemia in immunocompromised patients and sickle cell disease patients, myocarditis, and hydrops fetalis in pregnant women. Human erythroid progenitor cells (EPCs) are most susceptible to B19V infection and fully support viral DNA replication. The exclusive tropism of B19V for erythroid-lineage cells is dependent not only on the expression of viral receptors and coreceptors on the cell surface but also on the intracellular host factors that support B19V replication. Our present study shows that B19V uses a host factor, RNA binding motif protein 38 (RBM38), for the processing of its pre-mRNA during virus replication. Specifically, RBM38 interacts with the intronic splicing enhancer 2 (ISE2) element of B19V pre-mRNA and promotes 11-kDa protein expression, thereby regulating the 11-kDa protein-mediated augmentation of B19V replication. The identification of this novel host-pathogen interaction will provide mechanistic insights into B19V replication and aid in finding new targets for anti-B19V therapeutics.
人细小病毒B19（B19V）表达一种单一的前体mRNA（pre-mRNA），该前体mRNA经过可变剪接和可变聚腺苷酸化，产生12种病毒mRNA转录本，这些转录本编码两种结构蛋白（VP1和VP2）和三种非结构蛋白（NS1、7.5 kDa蛋白和11 kDa蛋白）。B19V pre-mRNA第二个5'供体位点（D2位点）的剪接对于VP2和11 kDa蛋白的表达至关重要。我们之前鉴定出位于D2位点之后的顺式作用内含子剪接增强子2（ISE2）有助于识别D2供体以实现其有效剪接。在本研究中，我们报告ISE2对于11 kDa病毒非结构蛋白的表达至关重要。我们发现ISE2含有一个共有RNA结合基序蛋白38（RBM38）结合序列，即5'-UGUGUG-3'。RBM38在红细胞生成的中期表达。我们首先证实RBM38与ISE2元件特异性结合。敲低RBM38会显著降低编码11 kDa蛋白的D2位点处剪接mRNA的水平，但不会降低编码VP2的D2剪接mRNA的水平。重要的是，我们发现11 kDa蛋白可增强病毒DNA复制和病毒粒子释放。因此，敲低RBM38会通过下调11 kDa蛋白表达来降低病毒复制。综上所述，这些结果表明11 kDa蛋白促进B19V DNA复制，并且RBM38是B19V pre-mRNA剪接和11 kDa蛋白表达所必需的宿主因子。B19V是一种人类病原体，可引起传染性红斑、关节病、免疫功能低下患者和镰状细胞病患者的贫血、心肌炎以及孕妇的胎儿水肿。人类红系祖细胞（EPC）对B19V感染最为敏感，并能完全支持病毒DNA复制。B19V对红系细胞的独特嗜性不仅取决于细胞表面病毒受体和共受体的表达，还取决于支持B19V复制的细胞内宿主因子。我们目前的研究表明，B19V在病毒复制过程中利用宿主因子RNA结合基序蛋白38（RBM38）来加工其pre-mRNA。具体而言，RBM38与B19V pre-mRNA的内含子剪接增强子2（ISE2）元件相互作用并促进11 kDa蛋白表达，从而调节11 kDa蛋白介导的B19V复制增强。这种新型宿主-病原体相互作用的鉴定将为B19V复制提供机制性见解，并有助于寻找抗B19V治疗的新靶点。
