RBM24 stabilizes hepatitis B virus pregenomic RNA but inhibits core protein translation by targeting the terminal redundancy sequence

Abstract

The terminal redundancy (TR) sequence of the 3.5-kb hepatitis B virus (HBV) RNA contains sites that govern many crucial functions in the viral life cycle, including polyadenylation, translation, RNA packaging, and DNA synthesis. In the present study, RNA-binding motif protein 24 (RBM24) is shown to be involved in the modulation of HBV replication by targeting the TR of HBV RNA. In HBV-transfected hepatoma cell lines, both knockdown and overexpression of RBM24 led to decreased HBV replication and transcription. Ectopic expression of RBM24 inhibited HBV replication, which was partly restored by knockdown of RBM24, indicating that a proper level of RBM24 was required for HBV replication. The regulation of RBM24 of HBV replication and translation was achieved by the interaction between the RNA-binding domains of RBM24 and both the 5′ and 3′ TR of 3.5-kb RNA. RBM24 interacted with the 5′ TR of HBV pregenomic RNA (pgRNA) to block 80S ribosome assembly on HBV pgRNA and thus inhibited core protein translation, whereas the interaction between RBM24 and the 3′ TR enhanced the stability of HBV RNA. Finally, the regulatory function of RBM24 on HBV replication was further confirmed in a HBV infection model. In conclusion, the present study demonstrates the dual functions of RBM24 by interacting with different TRs of viral RNA and reveals that RBM24 is an important host gene for HBV replication.

RNA-Binding Motif Protein 24 (RBM24) Is Involved in Pregenomic RNA Packaging by Mediating Interaction between Hepatitis B Virus Polymerase and the Epsilon Element

“…Both knockdown and overexpression of RBM24 decreased the amounts of HBV DNAs and total HBV RNAs ( Fig. 6A), as reported previously (18). Knockdown of RBM24 increased the amounts of core protein and capsids, while significantly decreased amounts of packaged pgRNA and minus-strand DNA [(Ϫ)DNA] were observed (Fig.…”

“…The DDX3 DEAD-box RNA helicase is incorporated into nucleocapsids in an HBV Pol-dependent manner but does not affect pgRNA encapsidation (33,34). We have demonstrated that overexpression of RBM24 inhibits translation of core (18), and in the present study, RBM24 knockdown markedly decreased the pgRNA encapsidation efficiency, whereas overexpression of RBM24 notably increased it ( Fig. 6), suggesting that RBM24 is involved in pgRNA packaging.…”

“…Hsp90 interacts only with Pol (30) and not RNA, and the addition of the Hsp90 complex results in only 30% of HBV Pol binding to (8), suggesting that additional host factors may be required for Pol-interaction. As an RNA-binding protein, RBM24, which has been shown to target the 3=-TR and 5=-TR of HBV RNA (18), was found in our study to directly bind to both and Pol, forming a complex. Knockdown of RBM24 significantly decreased binding between and Pol or RT, which highlights the fact that endogenous RBM24 plays an essential role as a bridge in Pol-binding.…”

“…Plasmids. The HBV replication-competent clone pHY106 (HBV genome genotype A, subtype adw2 [GenBank accession number AF305422.1]) and wild-type RBM24 protein expression plasmids pRBM24, pHA-RBM24, and pFlag-RBM24 were used as described previously (18). Gene expression cassettes for RBM24 lacking both RNP1 and RNP2 were amplified and cloned into pXJ40-Flag to generate pFlag-RBM24ΔRNP1/ΔRNP2 (pFlag-ΔRNP1/2).…”

“…Western blotting. Western blotting was performed as described previously (18). The following antibodies were used: antiactin (catalog number 66009-1; ProteinTech), anti-RBM24 (catalog number ab94567; Abcam), anti-HBc (catalog number B0586; Dako), rabbit antihemagglutinin (anti-HA) (catalog number 3724S; Cell Signaling Technology), mouse anti-HA (catalog number H9658; Sigma), rabbit anti-Flag (catalog number 2368S; Cell Signaling Technology), mouse anti-Flag (catalog number F1804; Sigma), streptavidin-horseradish peroxidase (HRP) (catalog number CT353; U-cytech), anti-mouse secondary antibodies (catalog number 115-035-146; Jackson), and anti-rabbit secondary antibodies (catalog number 111-035-003; Jackson).…”