Restitution of gene expression and histone acetylation signatures altered by hepatitis B virus through antiviral microRNA-like molecules in nontransformed murine hepatocytes

Abstract

BackgroundVirus-host interactions result in altered gene expression profiles in host cell nuclei and enable virus particle production, thus obligatorily involving changes in their epigenomes. Neither such epigenome changes nor their response to antiviral treatment have been extensively studied to date, although viral infections are known to contribute to the long-term development of severe secondary diseases, for example, hepatocellular carcinoma. This may be causally linked to virus-induced persistent plastic chromatin deformations.ResultsWe studied whether impaired hepatitis B virus (HBV) replication can lead to the restitution of epigenome signatures hypothesizing that hepatocytes alternatively could adopt a ‘memory’ state of the infection; that is, the chromatin could persist in a HBV-induced configuration potentially inheritable between dividing hepatocytes. We therefore determined epigenomic signatures and gene expression changes altered by HBV and the effects of suppressed HBV replication in nontransformed hepatocytes of newborn mice. Further we investigated differential histone acetyltransferase and histone deacetylase activities in HBV-negative and HBVpositive hepatocytes, as well as the effects of HBV suppression on gene expression and the chromatin landscape. We show that the expression of several genes and the chromatin landscape become altered upon HBV infection, including global hypoacetylation of H2A.Z and H3K9. Reporter assays monitoring the activities of histone acetyltransferases or histone deacetylases, respectively, suggest that hypoacetylation most probably depends on elevated sirtuin deacetylase activity, but not on class I/II histone deacetylases. Using Micrococcus nuclease to study the chromatin accessibility in met murine-D3 and hepatitis B virus met murine hepatocytes, we demonstrate that the observed differences in H2A.Z/H3K9 acetylation lead to global chromatin structure changes. At all selected sites examined by chromatin immunoprecipitation and quantitative real-time PCR, these effects can be partly restituted via the nucleoside analog reverse transcriptase inhibitor 3TC or using anti-HBV microRNA-like molecules.ConclusionsIncreased sirtuin activity might lead to global histone hypoacetylation signatures, which could contribute to the HBV-induced pathomechanism in nontransformed hepatocytes. Using several techniques to suppress HBV replication, we observed restituted gene expression and chromatin signature patterns reminiscent of noninfected hepatocytes. Importantly, ectopic expression of antiviral short-hairpin RNA, but not microRNA-like molecules, provoked intolerable off-target effects on the gene expression level.Electronic supplementary materialThe online version of this article (doi:10.1186/1868-7083-6-26) contains supplementary material, which is available to authorized users.

Virus–host interplay in hepatitis B virus infection and epigenetic treatment strategies

“…Similarly, another study reported hypermethylation of the CDKN2A promoter associated with in preneoplastic lesions, which was interpreted as that CDKN2A hypermethylation could be an early event during HCC development . More recently obtained data, however, do not support this conclusion, as DNA methylation pattern differences at any of the same sites examined could not be detected in earlier stages of malignant transformation . Notably, the latter study examined mouse homologous sites for CDKN2A and GSTP1 that were mentioned in the respective literature.…”

“…Obligatorily, the deregulation of gene expression is a phenotypic consequence of epigenome plasticity modulated by HBV. However, analyses of putative HBV‐induced promoter DNA methylation patterns in various models or human specimens yielded conflicting results . Only a few studies, which focus on a limited number of selected genes, are available to date describing HBV‐induced changes in the epigenome of hepatocytes.…”

Sirtuins as Potential Therapeutic Targets for Hepatitis B Virus Infection

“…Notably, current evidence shows that the expression of all SIRT proteins can be modulated by HBV. In particular, expression levels of SIRT1 (6), SIRT2 (7), SIRT5 (8), and SIRT7 were found to be increased in HBV-infected cells (8,9), whereas those of SIRT3, SIRT4, and SIRT6 were decreased in HBV-infected hepatocytes (10)(11)(12)21). Of note, studies have also demonstrated that changes in the expression level of SIRT1 (14), SIRT2 (7), SIRT3 (11), SIRT4 (10), and SIRT7 are associated with a nonstructural viral protein, HBx (9).…”

“…Sirtuins (SIRTs) are nicotinamide adenine dinucleotide (NAD) + -dependent histone deacetylases that regulate various biological processes, including stress responses, apoptosis, and metabolism (4,5). In particular, emerging evidence shows that SIRTs play a significant role in chronic HBV infection (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16).…”

Episomal HBV persistence within transcribed host nuclear chromatin compartments involves HBx

“…For the big picture, interactions between both viral and host genomes must be interpreted in the context of the entirety and complexity of both epigenomes. Due to the overwhelming complexity of already known epigenetic modifications as well as to their partially transient and often dynamic nature, most studies have focused on specific epigenetic changes of the HBV genome [ 26 – 29 ]. However, the interplay of HBV and the higher-order nuclear architecture of the host cell nucleus remains almost unexplored to date.…”