Short hairpin RNA induces methylation of hepatitis B virus covalently closed circular DNA in human hepatoma cells

Park, Hyun Kyung; Min, Bo Young; Kim, Nam Young; Jang, Eun Sun; Shin, Cheol Min; Park, Young Soo; Hwang, Jung‐Won; Jeong, Sook‐Hyang; Kim, Nayoung; Lee, Dong Hoon; Kim, Jin-Wook

doi:10.1016/j.bbrc.2013.04.108

Cited by 13 publications

(23 citation statements)

References 37 publications

(54 reference statements)

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“…A similar trend is found in hepatitis B virus (HBV) (51). In this case, it has been observed that cccDNA methylation in regulatory regions can regulate the expression of their genes (52-54), but if methylation by RNA silencing (TGS) is induced, it can suppress viral transcription and adversely affect its replication mechanism (55,56). Similarly, different minichromosome populations were also observed in duck hepatitis B virus infection.…”

Section: Discussionmentioning

confidence: 54%

Two Populations of Viral Minichromosomes Are Present in a Geminivirus-Infected Plant Showing Symptom Remission (Recovery)

Ceniceros-Ojeda

Rodríguez-Negrete

Rivera-Bustamante

2016

J Virol

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Geminiviruses are important plant pathogens characterized by circular, single-stranded DNA (ssDNA) genomes. However, in the nuclei of infected cells, viral double-stranded DNA (dsDNA) associates with host histones to form a minichromosome. In phloem-limited geminiviruses, the characterization of viral minichromosomes is hindered by the low concentration of recovered complexes due to the small number of infected cells. Nevertheless, geminiviruses are both inducers and targets of the host posttranscriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) machinery. We have previously characterized a "recovery" phenomenon observed in pepper plants infected with pepper golden mosaic virus (PepGMV) that is associated with a reduction of viral DNA and RNA levels, the presence of virus-related siRNAs, and an increase in the levels of viral DNA methylation. Initial micrococcal nuclease-based assays pinpointed the presence of different viral chromatin complexes in symptomatic and recovered tissues. Using the pepper-PepGMV system, we developed a methodology to obtain a viral minichromosome-enriched fraction that does not disturb the basic chromatin structural integrity, as evaluated by the detection of core histones. Using this procedure, we have further characterized two populations of viral minichromosomes in PepGMV-infected plants. After further purification using sucrose gradient sedimentation, we also observed that minichromosomes isolated from symptomatic tissue showed a relaxed conformation (based on their sedimentation rate), are associated with a chromatin activation marker (H3K4me3), and present a low level of DNA methylation. The minichromosome population obtained from recovered tissue, on the other hand, sedimented as a compact structure, is associated with a chromatin-repressive marker (H3K9me2), and presents a high level of DNA methylation. IMPORTANCEViral minichromosomes have been reported in several animal and plant models. However, in the case of geminiviruses, there has been some recent discussion about the importance of this structure and the significance of the epigenetic modifications that it can undergo during the infective cycle. Major problems in this type of studies are the low concentration of these complexes in an infected plant and the asynchronicity of infected cells along the process; therefore, the complexes isolated in a given moment usually represent a mixture of cells at different infection stages. The recovery process observed in PepGMV-infected plants and the isolation procedure described here provide two distinct populations of minichromosomes that will allow a more precise characterization of the modifications of viral DNA and its host proteins associated along the infective cycle. This structure could be also an interesting model to study several processes involving plant chromatin. G eminiviruses are important plant pathogens consisting of circular, single-stranded DNA (ssDNA) genomes ranging from 2.7 to 5.2 kb packed into 1 or 2 icosahedral twine-shaped part...

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Section: Discussionmentioning

confidence: 54%

Two Populations of Viral Minichromosomes Are Present in a Geminivirus-Infected Plant Showing Symptom Remission (Recovery)

Ceniceros-Ojeda

Rodríguez-Negrete

Rivera-Bustamante

2016

J Virol

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“…Several reports demonstrated de novo methylation by siRNA in plants [8][9][10]. In human cells, small RNA was used to promote DNA methylation, shRNA for long interspersed element-1 (LINE-1) and hepatitis B virus, and siRNA for HIV-1 promoter region [11][12][13]. There are many types of IRS such as Alu elements, LINE-1, and human endogenous retrovirus [14][15][16][17].…”

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confidence: 99%

Alu siRNA to Increase Alu Element Methylation and Prevent DNA Damage

et al. 2018

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Global DNA hypomethylation promoting genomic instability leads to cancer and deterioration of human health with age. Aim: To invent a biotechnology that can reprogram this process. Methods: We used Alu siRNA to direct Alu interspersed repetitive sequences methylation in human cells. We evaluated the correlation between DNA damage and Alu methylation levels. Results: We observed an inverse correlation between Alu element methylation and endogenous DNA damage in white blood cells. Cells transfected with Alu siRNA exhibited high Alu methylation levels, increased proliferation, reduced endogenous DNA damage and improved resistance to DNA damaging agents. Conclusion: Alu methylation stabilizes the genome by preventing accumulation of DNA damage. Alu siRNA could be useful for evaluating reprograming of the global hypomethylation phenotype in cancer and aging cells. DNA methylation at interspersed repetitive sequences (IRSs) plays an important role in maintaining genome stability. Cells with IRS hypomethylation exhibit increased mutation rates [1,2]. Here, we tested whether DNA damage, an alteration in the chemical structure of DNA and a precursor to mutation [3], plays a role in mediating how global hypomethylation promotes genomic instability. Our recent study found that global hypomethylation is associated with plasma 8-hydroxy-2 -deoxyguanosine (8-OHdG) in biliary atresia patients [4]. Moreover, urinary 8-OHdG, DNA strand breaks and global DNA hypomethylation are associated with low serum folate [5] and oxidative stress [6]. Therefore, we hypothesized that the human genome may use DNA methylation in IRSs to prevent DNA damage.This study developed a technology to add DNA methylation at Alu elements. The human genome contains greater than one million Alu elements [7]. Several reports demonstrated de novo methylation by siRNA in plants [8][9][10]. In human cells, small RNA was used to promote DNA methylation, shRNA for long interspersed element-1 (LINE-1) and hepatitis B virus, and siRNA for HIV-1 promoter region [11][12][13]. There are many types of IRS such as Alu elements, LINE-1, and human endogenous retrovirus [14][15][16][17]. To increase DNA methylation, we tested siRNA to unique sequences and several IRS sequences. Our preliminary trial demonstrated that only Alu siRNA could increase methylation of the target sequences. Here, we evaluated whether Alu siRNA is a useful tool to explore the role of global hypomethylation in genomic instability [18].Alu hypomethylation may play a role in causing genomic instability in cancer and aging cells. Genomic instability, the main enabling characteristic of cancer and aging processes [18,19], may mainly be promoted by IRS hypomethylation. IRS hypomethylation is commonly observed both in aging [14,20] and cancer cells [21]. IRS

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“…HBx may play a role, because it recruits DNMT3A to the regulatory promoters [48]. Small RNAs may be a plausible candidate, as suggested by our in vitro study in which short hairpin RNA induced methylation of the target site in HBV [49].…”

Section: Epigenetic Control Of Hbv Transcription: Cccdna Methylationmentioning

confidence: 76%

Epigenetic Regulation of Hepatitis B Virus Replication

Moon¹,

Kim²

2020

Chromatin and Epigenetics

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Hepatitis B virus (HBV) is the most important cause of chronic viral hepatitis worldwide. The genome of HBV is 3.2 kb partially double-stranded DNA, which is translocated to the nuclei of infected hepatocytes and converted to complete double-stranded DNA, aka covalently closed circular DNA (cccDNA). Typical course of chronic HBV infection results in inactive carrier state with clearance of viral particles in the bloodstream. However, the cccDNA can be detected in the hepatocytes from inactive carriers by sensitive methods. It has been increasingly known that epigenetic mechanisms contribute to the control of HBV replication in the inactive stage of HBV infection. Histone modification and DNA methylation have been identified in the HBV cccDNA, leading to modification of transcriptional activity. The understanding of epigenetic control of transcription will shed light on the development of new therapeutic strategy against HBV cccDNA.

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Short hairpin RNA induces methylation of hepatitis B virus covalently closed circular DNA in human hepatoma cells

Cited by 13 publications

References 37 publications

Two Populations of Viral Minichromosomes Are Present in a Geminivirus-Infected Plant Showing Symptom Remission (Recovery)

Two Populations of Viral Minichromosomes Are Present in a Geminivirus-Infected Plant Showing Symptom Remission (Recovery)

Alu siRNA to Increase Alu Element Methylation and Prevent DNA Damage

Epigenetic Regulation of Hepatitis B Virus Replication

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