RNA Binding Motif Protein RBM45 Regulates Expression of the 11-Kilodalton Protein of Parvovirus B19 through Binding to Novel Intron Splicing Enhancers.

Jianke Wang,Fang Cheng,Safder S Ganaie,Peng Xu,Steve Kleiboeker,Kang Ning,Jianming Qiu,Shipeng Cheng,Xiaomei Wang,Xiang-Jin Meng

doi:10.1128/mbio.00192-20

Abstract

During infection of human parvovirus B19 (B19V), one viral precursor mRNA (pre-mRNA) is transcribed by a single promoter and is alternatively spliced and alternatively polyadenylated. Here, we identified a novel cis-acting sequence (5'-GUA AAG CUA CGG GAC GGU-3'), intronic splicing enhancer 3 (ISE3), which lies 72 nucleotides upstream of the second splice acceptor (A2-2) site of the second intron that defines the exon of the mRNA encoding the 11-kDa viral nonstructural protein. RNA binding motif protein 45 (RBM45) specifically binds to ISE3 with high affinity (equilibrium dissociation constant [KD ] = 33 nM) mediated by its RNA recognition domain and 2-homo-oligomer assembly domain (RRM2-HOA). Knockdown of RBM45 expression or ectopic overexpression of RRM2-HOA in human erythroid progenitor cells (EPCs) expanded ex vivo significantly decreased the level of viral mRNA spliced at the A2-2 acceptor but not that of the mRNA spliced at A2-1 that encodes VP2. Moreover, silent mutations of ISE3 in an infectious DNA of B19V significantly reduced 11-kDa expression. Notably, RBM45 also specifically interacts in vitro with ISE2, which shares the octanucleotide (GGGACGGU) with ISE3. Taken together, our results suggest that RBM45, through binding to both ISE2 and ISE3, is an essential host factor for maturation of 11-kDa-encoding mRNA.IMPORTANCE Human parvovirus B19 (B19V) is a human pathogen that causes severe hematological disorders in immunocompromised individuals. B19V infection has a remarkable tropism with respect to human erythroid progenitor cells (EPCs) in human bone marrow and fetal liver. During B19V infection, only one viral precursor mRNA (pre-mRNA) is transcribed by a single promoter of the viral genome and is alternatively spliced and alternatively polyadenylated, a process which plays a key role in expression of viral proteins. Our studies revealed that a cellular RNA binding protein, RBM45, binds to two intron splicing enhancers and is essential for the maturation of the small nonstructural protein 11-kDa-encoding mRNA. The 11-kDa protein plays an important role not only in B19V infection-induced apoptosis but also in viral DNA replication. Thus, the identification of the RBM45 protein and its cognate binding site in B19V pre-mRNA provides a novel target for antiviral development to combat B19V infection-caused severe hematological disorders.

Highlights

During infection of human parvovirus B19 (B19V), one viral precursor mRNA is transcribed by a single promoter and is alternatively spliced and alternatively polyadenylated
Knockdown of RNA binding motif protein 45 (RBM45) exhibits a phenotype in splicing of the 11-kDa-encoding mRNA only and not that of the VP2-encoding mRNA, highlighting the specific role of RBM45, which is similar to the role of the RNA binding motif protein 38 (RBM38) [37], in splicing of viral pre-mRNA at the A2-2 acceptor for production of the 11-kDaencoding mRNA
We propose an RBM45 working model (Fig. 11) in which RBM45 functions as a scaffold protein that facilitates the interaction between the U1 snRNP that binds to the D2 and the U2 snRNP that binds to the A2-2 acceptor, which defines the intron between the D2 and A2-2 splice sites

Summary

Introduction

During infection of human parvovirus B19 (B19V), one viral precursor mRNA (pre-mRNA) is transcribed by a single promoter and is alternatively spliced and alternatively polyadenylated. During B19V infection, only one viral precursor mRNA (pre-mRNA) is transcribed by a single promoter of the viral genome and is alternatively spliced and alternatively polyadenylated, a process which plays a key role in expression of viral proteins. The identification of the RBM45 protein and its cognate binding site in B19V pre-mRNA provides a novel target for antiviral development to combat B19V infection-caused severe hematological disorders. The 11-kDa protein interacts with Grb (growth factor receptor bound protein 2) through the three SH3-binding motifs of the 11-kDa protein This interaction disrupts the extracellular signal-regulated kinase-1 signaling and upregulates viral DNA replication [38]

Methods

Results

Conclusion