Hepatitis B Virus-Encoded MicroRNA Controls Viral Replication

Abstract

MicroRNAs (miRNAs) are a class of small, single-stranded, noncoding, functional RNAs. Hepatitis B virus (HBV) is an enveloped DNA virus with virions and subviral forms of particles that lack a core. It was not known whether HBV encodes miRNAs. Here, we identified an HBV-encoded miRNA (called HBV-miR-3) by deep sequencing and Northern blotting. HBV-miR-3 is located at nucleotides (nt) 373 to 393 of the HBV genome and was generated from 3.5-kb, 2.4-kb, and 2.1-kb HBV in a classic miRNA biogenesis (Drosha-Dicer-dependent) manner. HBV-miR-3 was highly expressed in hepatoma cell lines with an integrated HBV genome and HBV ϩ hepatoma tumors. In patients with HBV infection, HBV-miR-3 was released into the circulation by exosomes and HBV virions, and HBV-miR-3 expression had a positive correlation with HBV titers in the sera of patients in the acute phase of HBV infection. More interestingly, we found that HBV-miR-3 represses HBsAg, HBeAg, and replication of HBV. HBV-miR-3 targets the unique site of the HBV 3.5-kb transcript to specifically reduce HBc protein expression, levels of pregenomic RNA (pgRNA), and HBV replication intermediate (HBV-RI) generation but does not affect the HBV DNA polymerase level, thus suppressing HBV virion production (replication). This may explain the low levels of HBV virion generation with abundant subviral particles lacking core during HBV replication, which may contribute to the development of persistent infection in patients. Taken together, our findings shed light on novel mechanisms by which HBV-encoded miRNA controls the process of self-replication by regulating HBV transcript during infection.IMPORTANCE Hepatitis B is a liver infection caused by the hepatitis B virus (HBV) that can become a long-term, chronic infection and lead to cirrhosis or liver cancer. HBV is a small DNA virus that belongs to the hepadnavirus family, with virions and subviral forms of particles that lack a core. MicroRNA (miRNA), a small (ϳ22-nt) noncoding RNA, was recently found to be an important regulator of gene expression. We found that HBV encodes miRNA (HBV-miR-3). More importantly, we revealed that HBV-miR-3 targets its transcripts to attenuate HBV replication. This may contribute to explaining how HBV infection leads to mild damage in liver cells and the subsequent establishment/maintenance of persistent infection. Our findings highlight a mechanism by which HBV-encoded miRNA controls the process of self-replication by regulating the virus itself during infection and might provide new biomarkers for diagnosis and treatment of hepatitis B.

The Regulatory Role of MicroRNA in Hepatitis-B Virus-Associated Hepatocellular Carcinoma (HBV-HCC) Pathogenesis

“…HBV genome replication and survival is self-managed as a result of modulating (up/down) both host cell miRNA expression, as well as coding for its own miRNA [27]. The hepatitis B virus x protein (HBx), for example, blocks p53 stimulated miR-34 expression in hepatocytes leading to the upregulation of macrophage-derived chemokine (CCL22) that stimulates regulatory T-cells (Tregs) that, in turn, block effector T-cells allowing HBV expression to increase [28,29] HBV genome transcription or by directly binding to HBV transcripts [13] (see Scheme 1, pathway 1).…”

“…Recently it was also discovered that the HBV virion generates its own HBV-miR-2/-3 [37]. HBV-miR-3 suppresses the hepatitis B core protein (HBc) mRNA to self-regulate HBV expression downwards [27], possibly in order to promote its survival by way of avoiding the host immune system [28,38] (see Scheme 1, pathway 6).…”

Circulating miRNA expression profile and bioinformatics analysis in patients with occult hepatitis B virus infection

“…In turn, cellular miRNAs could be changed by HBV infection . In addition, HBV‐encoded miRNAs could also regulate the viral infection status and the virus life cycle . These interactions between host and virus determine the clinical outcome of HBV infection.…”

The Interplay Between Viral-Derived miRNAs and Host Immunity During Infection

“…The corresponding study identified and confirmed the expression of HBV-miR-3. HBV-miR-3 downregulated HBV protein and HBV replication by reducing the expression of HBcAg, a positive regulator of HBV transcription and pregenomic RNA (pgRNA), which inhibits HBV replication overall (90).…”