Osteopetrosis-Associated Transmembrane Protein 1 Recruits RNA Exosome To Restrict Hepatitis B Virus Replication

Abstract

Hepatitis B virus (HBV) chronically infects approximately 350 million people worldwide, and 600,000 deaths are caused by HBV-related hepatic failure, liver cirrhosis, and hepatocellular carcinoma annually. It is important to reveal the mechanism underlying the regulation of HBV replication. This study demonstrated that osteopetrosis-associated transmembrane protein 1 (Ostm1) plays an inhibitory role in HBV replication. Ostm1 represses the levels of HBeAg and HBsAg proteins, HBV 3.5-kb and 2.4/2.1-kb RNAs, and core-associated DNA in HepG2, Huh7, and NTCP-HepG2 cells. Notably, Ostm1 has no direct effect on the activity of HBV promoters or the transcription of HBV RNAs; instead, Ostm1 binds to HBV RNA to facilitate RNA decay. Detailed studies further demonstrated that Ostm1 binds to and recruits the RNA exosome complex to promote the degradation of HBV RNAs, and knockdown of the RNA exosome component exonuclease 3 (Exosc3) leads to the elimination of Ostm1-mediated repression of HBV replication. Mutant analyses revealed that the N-terminal domain, the transmembrane domain, and the C-terminal domain are responsible for the repression of HBV replication, and the C-terminal domain is required for interaction with the RNA exosome complex. Moreover, Ostm1 production is not regulated by interferon-α (IFN-α) or IFN-γ, and the expression of IFN signaling components is not affected by Ostm1, suggesting that Ostm1 anti-HBV activity is independent of the IFN signaling pathway. In conclusion, this study revealed a distinct mechanism underlying the repression of HBV replication, in which Ostm1 binds to HBV RNA and recruits RNA exosomes to degrade viral RNA, thereby restricting HBV replication. IMPORTANCE Hepatitis B virus (HBV) is a human pathogen infecting the liver to cause a variety of diseases ranging from acute hepatitis to advanced liver diseases, fulminate hepatitis, liver cirrhosis, and hepatocellular carcinoma, thereby causing a major health problem worldwide. In this study, we demonstrated that Ostm1 plays an inhibitory role in HBV protein production, RNA expression, and DNA replication. However, Ostm1 has no effect on the activities of the four HBV promoters; instead, it binds to HBV RNA and recruits RNA exosomes to promote HBV RNA degradation. We further demonstrated that the anti-HBV activity of Ostm1 is independent of the interferon signaling pathway. In conclusion, this study reveals a distinct mechanism underlying the repression of HBV replication and suggests that Ostm1 is a potential therapeutic agent for HBV infection.

An enrichment protocol and analysis pipeline for long read sequencing of the hepatitis B virus transcriptome

“…4a ). This observation is consistent with exosome mediated degradation of HBV RNAs [ 43 ]. Mapping the polyadenylation length (Supplementary Information: extract-seq.sh and Acounter.R) showed a median of 29–31 for all HBV RNAs with the exception of preC-S, where the median length was >200 nucleotides, reflecting a population of short transcripts that terminate at the PAS ( Fig.…”

“…4a). This observation is consistent with exosome mediated degradation of HBV RNAs [43]. Mapping the polyadenylation length (Supplementary Information: extract-seq.…”

Expression pattern of the V5‐Ostm1 protein in bacterial artificial chromosome transgenic mice

“…In order to evaluate post-translational modification of the V5-tagged Ostm1 mouse protein, which primary sequence predicts a total of 10 N-glycosylation consensus sites (Pandruvada et al, 2016 Figure 3b). As previously described (Chalhoub et al, 2003;Ma et al, 2020), these results confirm that (Figure 4b). Together, these results defined more precisely neuronal Ostm1 protein cellular expression in the central and peripheral nervous system that can explain the F I G U R E 1 Generation, analysis and expression of bacterial artificial chromosome (BAC) V5-Ostm1 transgenic mice.…”

“…In lumbar spinal cord, a strong cytosolic signal is mostly restricted to motor neurons from ventral horn compared to the broad nuclear neuron‐specific Neu‐N protein expression (Figure 3b). As previously described (Chalhoub et al, 2003; Ma et al, 2020), these results confirm that Ostm1 localization is restricted to the cytosolic cellular compartment and excluded from the nuclear fraction. In retina, V5 immunofluorescence delineates transgenic Ostm1 protein expression in granular cell layer (GCL), inner nuclear layer (INL) including amacrine and bipolar cells, in outer segments (OS) of photoreceptors and in retinal pigmented epithelium (RPE) (Figure 4a).…”

Exosomal Non-Coding RNAs: Regulatory and Therapeutic Target of Hepatocellular Carcinoma

“…Targeted excision of the RNA exosomal component Xonuclease 3 (Exosc3) can eliminate Ostm1-mediated HBV replication inhibition. C-terminal domain provides a targeted site for HBV-related treatments as the site of RNA exosomes ( 62 ). HBV X protein (HBx), a multifunctional viral regulator, participates in the regulation of the virus life cycle and the progress of HBV-related HCC.…”