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.

WD repeat-containing protein 5 (WDR5) is essential for assembling the VISA-associated complex to induce a type I interferon antiviral response to Sendai virus infection. However, the roles of WDR5 in DNA virus infections are not well described. Here, we report that human cytomegalovirus exploits WDR5 to facilitate capsid nuclear egress. Overexpression of WDR5 in fibroblasts slightly enhanced the infectious virus yield. However, WDR5 knockdown dramatically reduced infectious virus titers with only a small decrease in viral genome replication or gene expression. Further investigation of late steps of viral replication found that WDR5 knockdown significantly impaired formation of the viral nuclear egress complex and induced substantially fewer infoldings of the inner nuclear membrane. In addition, fewer capsids were associated with these infoldings, and there were fewer capsids in the cytoplasm. Restoration of WDR5 partially reversed these effects. These results suggest that WDR5 knockdown impairs the nuclear egress of capsids, which in turn decreases virus titers. These findings reveal an important role for a host factor whose function(s) is usurped by a viral pathogen to promote efficient replication. Thus, WDR5 represents an interesting regulatory mechanism and a potential antiviral target. Human cytomegalovirus (HCMV) has a large (∼235-kb) genome with over 170 open reading frames and exploits numerous cellular factors to facilitate its replication. HCMV infection increases protein levels of WD repeat-containing protein 5 (WDR5) during infection, overexpression of WDR5 enhances viral replication, and knockdown of WDR5 dramatically attenuates viral replication. Our results indicate that WDR5 promotes the nuclear egress of viral capsids, the depletion of WDR5 resulting in a significant decrease in production of infectious virions. This is the first report that WDR5 favors HCMV, a DNA virus, replication and highlights a novel target for antiviral therapy.

The secondary structures of hepatitis C virus (HCV) RNA and the cellular proteins that bind to them are important for modulating both translation and RNA replication. However, the sets of RNA-binding proteins involved in the regulation of HCV translation, replication and encapsidation remain unknown. Here, we identified RNA binding motif protein 24 (RBM24) as a host factor participated in HCV translation and replication. Knockdown of RBM24 reduced HCV propagation in Huh7.5.1 cells. An enhanced translation and delayed RNA synthesis during the early phase of infection was observed in RBM24 silencing cells. However, both overexpression of RBM24 and recombinant human RBM24 protein suppressed HCV IRES-mediated translation. Further analysis revealed that the assembly of the 80S ribosome on the HCV IRES was interrupted by RBM24 protein through binding to the 5′-UTR. RBM24 could also interact with HCV Core and enhance the interaction of Core and 5′-UTR, which suppresses the expression of HCV. Moreover, RBM24 enhanced the interaction between the 5′- and 3′-UTRs in the HCV genome, which probably explained its requirement in HCV genome replication. Therefore, RBM24 is a novel host factor involved in HCV replication and may function at the switch from translation to replication.

Feasible and effective cell models for hepatitis B virus (HBV) infection are required for investigating the complete lifecycle of this virus, including the early steps of viral entry. Resistance to dimethyl sulfoxide/polyethylene glycol (DMSO/PEG), hNTCP expression, and a differentiated state are the limiting factors for successful HBV infection models. In the present study, we used a hepatoma cell line (Huh7D hNTCP ) to overcome these limiting factors so that it exhibits excellent susceptibility to HBV infection. To achieve this goal, different hepatoma cell lines were tested with 2.5% DMSO / 4% PEG8000, and one resistant cell line (Huh7D) was used to construct a stable hNTCP-expressing cell line (Huh7D hNTCP ) using a recombinant lentivirus system. Then, the morphological characteristics and differentiation molecular markers of Huh7D hNTCP cells with or without DMSO treatment were characterized. Finally, the susceptibility of Huh7D hNTCP cells to HBV infection was assessed. Our results showed that Huh7D cells were resistant to 2.5% DMSO / 4% PEG8000, whereas the others were not. Huh7D hNTCP cells were established to express a high level of hNTCP compared to liver extracts, and Huh7D hNTCP cells rapidly transformed into a non-dividing, well-differentiated polarized phenotype under DMSO treatment. Huh7D hNTCP cells fully supported the entire lifecycle of HBV infection. This cell culture system will be useful for the analysis of host-virus interactions, which should facilitate the discovery of antiviral drugs and vaccines.

Encapsidation of pregenomic RNA (pgRNA) is a crucial step in hepatitis B virus (HBV) replication. Binding by viral polymerase (Pol) to the epsilon stem-loop () on the 5=-terminal region (TR) of pgRNA is required for pgRNA packaging. However, the detailed mechanism is not well understood. RNA-binding motif protein 24 (RBM24) inhibits core translation by binding to the 5=-TR of pgRNA. Here, we demonstrate that RBM24 is also involved in pgRNA packaging. RBM24 directly binds to the lower bulge of via RNA recognition submotifs (RNPs). RBM24 also interacts with Pol in an RNA-independent manner. The alanine-rich domain (ARD) of RBM24 and the reverse transcriptase (RT) domain of Pol are essential for binding between RBM24 and Pol. In addition, overexpression of RBM24 increases Pol-interaction, whereas RBM24 knockdown decreases the interaction. RBM24 was able to rescue binding between and mutant Pol lacking -binding activity, further showing that RBM24 mediates the interaction between Pol and by forming a Pol-RBM24-complex. Finally, RBM24 significantly promotes the packaging efficiency of pgRNA. In conclusion, RBM24 mediates Pol-interaction and formation of a Pol-RBM24-complex, which inhibits translation of pgRNA and results in pgRNA packing into capsids/ virions for reverse transcription and DNA synthesis. IMPORTANCE Hepatitis B virus (HBV) is a ubiquitous human pathogen, and HBV infection is a major global health burden. Chronic HBV infection is associated with the development of liver diseases, including fulminant hepatitis, hepatic fibrosis, cirrhosis, and hepatocellular carcinoma. A currently approved vaccine can prevent HBV infection, and medications are able to reduce viral loads and prevent liver disease progression. However, current treatments rarely achieve a cure for chronic infection. Thus, it is important to gain insight into the mechanisms of HBV replication. In this study, we found that the host factor RBM24 is involved in pregenomic RNA (pgRNA) packaging and regulates HBV replication. These findings highlight a potential target for antiviral therapeutics of HBV infection.

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Ceruloplasmin (CP) is mainly synthesized by hepatocytes and plays an essential role in iron metabolism. Previous reports have shown that CP levels correlate negatively with disease progression in patients with chronic hepatitis B. However, the function of CP in the hepatitis B virus (HBV) life cycle and the mechanism underlying the above correlation remain unclear. Here, we report that CP can selectively inhibit the production of extracellular HBV virions without altering intracellular viral replication. HBV expression can also downregulate the expression of CP. Knockdown of CP using small interfering RNA significantly increased the level of extracellular HBV virions in both Huh7 and HepG2.2.15 cells, while overexpression of CP decreased this level. Mechanistically, CP could specifically interact with the HBV middle surface protein (MHB). Using an HBV replication-competent clone unable to express MHBs, we demonstrated that the overexpression of CP did not affect the production of extracellular HBV virions in the absence of MHBs. Furthermore, introduction of an MHB expression construct could rescue the impairment in virion production caused by CP. Taken together, our results suggest that CP may be an important host factor that targets MHBs during the envelopment and/or release of virions.

Sodium taurocholate cotransporting polypeptide (NTCP) is a functional receptor for hepatitis B virus (HBV) entry. However, little is known regarding whether NTCP is involved in regulating the postentry steps of the HBV life cycle. Here, we found that NTCP expression upregulated HBV transcription at the postentry step and that the NTCP-targeting entry inhibitor Myrcludex B (MyrB) effectively suppressed HBV transcription both in an HBV in vitro infection system and in mice hydrodynamically injected with an HBV expression plasmid. Mechanistically, NTCP upregulated HBV transcription via farnesoid X receptor α (FxRα)-mediated activation of the HBV EN2/core promoter at the postentry step in a manner that was dependent on the bile acid (BA)-transport function of NTCP, which was blocked by MyrB. Our findings uncover a novel role for NTCP in the HBV life cycle and provide a reference for the use of novel NTCP-targeting entry inhibitors to suppress HBV infection and replication.