Chun Huang, Jianming Qiu, Peng Xu, Aaron Yun Chen, Steve Kleiboeker, Safder S Ganaie, Aifang Du
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, and 11-kDa). Splicing at the second 5' donor site (D2) of the B19V pre-mRNA is essential for the expression of VP2 and 11-kDa. We have previously identified that a cis-acting intronic splicing enhancer 2 (ISE2) that lies immediately after the D2 site facilitates recognition of the D2 donor for its efficient splicing. In this study, we report that ISE2 is critical for expression of the 11-kDa viral non-structural 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 the RBM38 binds specifically with the ISE2 element in vitro. Knockdown of RBM38 significantly decreases the level of the spliced mRNA at D2 that encodes 11-kDa protein and, thereafter, expression of the 11-kDa protein, but not the D2-spliced mRNA that encodes VP2. Importantly, we found that the 11-kDa protein enhances viral DNA replication and virion release. Accordingly, knockdown of RBM38 decreases virus replication via downregulating 11-kDa 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.IMPORTANCE B19V is a human pathogen that can cause fifth disease, arthropathy, anemia in immune compromised 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 to erythroid lineage cells is not only dependent on the expression of viral receptor and co-receptors on the cell surface but also on the intracellular host factors that support B19V replication. Our current 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 expression, thereby regulates 11-kDa-mediated augmentation of B19V replication. Identification of novel host-pathogen interactions will provide the mechanistic insights into B19V replication and find new targets for anti-B19V therapeutics.
