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Hepatitis B virus spliced variants are associated with an impaired response to interferon therapy
Jieliang Chen- , Min Wu
- , Fan Wang
- , Wen Zhang
- , Wei Wang
- , Xiaonan Zhang
- , Jiming Zhang
- , Yinghui Liu
- , Yi Liu
- , Yanling Feng
- , Ye Zheng
- , Yunwen Hu
- & Zhenghong Yuan
- Scientific Reports 5, Article number: 16459 (2015)
- doi:10.1038/srep16459
- Download Citation
Received:04 August 2015Accepted:14 October 2015Published online:20 November 2015
AbstractDuring hepatitis B virus (HBV) replication, spliced HBV genomes and splice-generated proteins have been widely described, however, their biological and clinical significance remains to be defined. Here, an elevation of the proportion of HBV spliced variants in the sera of patients with chronic hepatitis B (CHB) is shown to correlate with an impaired respond to interferon-α (IFN-α) therapy. Transfection of the constructs encoding the three most dominant species of spliced variants into cells or ectopic expression of the two major spliced protein including HBSP and N-terminal-truncated viral polymerase protein result in strong suppression of IFN-α signaling transduction, while mutation of the major splicing-related sites of HBV attenuates the viral anti-IFN activities in both cell and mouse models. These results have associated the productions of HBV spliced variants with the failure response to IFN therapy and illuminate a novel mechanism where spliced viral products are employed to resist IFN-mediated host defense.
IntroductionHepatitis B virus (HBV), which can cause chronic liver disease that may result in cirrhosis and hepatocellular carcinoma (HCC), is a DNA virus that has a 3.2 kB partially double-stranded, relaxed circular genome organized into overlapping open-reading frames from which viral genes are transcribed. The major HBV messenger RNAs (mRNA) synthesized from the HBV genome include: 3.5 kb (long/short) preC/pre-genomic (pg) RNA that encodes hepatitis B e antigen (HBeAg), core and polymerase (Pol) proteins, 2.4 kb and 2.1 kb sub-genomic RNAs that encode the HBV surface proteins (HBs), and a 0.9 kb mRNA that encodes the HBx protein. Through the 5′ stem-loop structure ε, the 3.5 kb pgRNA can also be packaged and reverse-transcribed by the viral polymerase into relaxed circular DNA to generate the wild-type HBV (wtHBV) particles for secretion into circulation1.
In addition to the unspliced mRNAs, a series of spliced (SP) HBV (spHBV) RNAs have been widely described in model systems and in HBV-infected livers2,3,4,5. The most frequently detected spHBV variant is a 2.2 kb molecule termed SP1, which is generated through the removal of a 1.3 kb intron from the pgRNA and accounts for up to 30% of pgRNAs3,4,5,6,7,8. spHBV RNAs can be incorporated into the nucleocapsids and then reverse transcribed into HBV DNA to generate defective HBV (dHBV) particles9,10,11,12. The level of dHBV particles in the sera of patients with chronic hepatitis B (CHB) was shown to be related with liver disease13,14 and was enhanced prior to development of HCC15. spHBV RNAs also serve as the translation templates for a number of non-canonical HBV proteins. HBV splice-generated protein (HBSP) is one of the major spHBV RNAs-encoded proteins which was firstly identified by Soussan et al. in the livers of CHB patients16, and has been reported to be associated with viral replication, liver diseases and cancer development17,18,19. In spite of these evidences suggesting that HBV splicing is a common event during HBV infection and may be involved in the pathogenicity or persistence of HBV, the clinical and biological significance of HBV splicing needs to be further defined.
Interferon-α (IFN-α), a cytokine with antiviral and immunomodulatory activities, has been approved for treatment of HBV infection since 1990s. However, IFN therapy is effective in only 30–40% of CHB patients20,21. One possible explanation for the failure to respond to IFN therapy in CHB patients is that HBV has developed strategies to counteract the IFN signaling transduction22,23,24,25,26,27. The Pol protein consisting of terminal protein (TP), reverse transcriptase (RT), RNaseH (RH), and a non-conserved spacer domain between the TP and RT domains has been shown to be multifunctional, not only playing essential role during viral replication but also having modulatory functions by interacting with a number of host factors28. Foster et al. found that the expression of the TP region of Pol inhibits cellular responses to IFN-α22,23, and we further observed that the TP and RH domains of Pol inhibit IFN-activated STAT1 serine 727 phosphorylation and STAT1 and STAT2 nuclear translocation, respectively25. Since Pol is believed to be produced at a relatively low level during viral replication, the real physiological relevance of these findings remains to be classified.
Considering that most of the spHBV variants contain sequences that could be translated into proteins which contain domains with anti-IFN activities derived from the open reading frame (ORF) of Pol gene11,29, we hypothesized that there may be a correlation between HBV spliced variants and IFN responses. To test this hypothesis, we collected serum samples from CHB patients before and after IFN therapy to analyze the relationship between the productions of HBV spliced variants and the failure to respond to IFN therapy. In addition, we examined the expression of HBV spliced variants and investigated the potential role of HBV splicing in counteraction of IFN signaling in cell culture systems and in a mouse model.
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