Browsing by Subject "Viral DNA"

Now showing 1 - 3 of 3
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    Characterization of the Termini of Cytoplasmic Hepatitis B Virus Deproteinated Relaxed Circular DNA
    (American Society for Microbiology, 2020-12-09) Cai, Dawei; Yan, Ran; Xu, Jerry Z.; Zhang, Hu; Shen, Sheng; Mitra, Bidisha; Marchetti, Alexander; Kim, Elena S.; Guo, Haitao; Microbiology and Immunology, School of Medicine
    The biosynthesis of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) requires the removal of the covalently linked viral polymerase from the 5' end of the minus strand [(-)strand] of viral relaxed circular DNA (rcDNA), which generates a deproteinated rcDNA (DP-rcDNA) intermediate. In the present study, we systematically characterized the four termini of cytoplasmic HBV DP-rcDNA by 5'/3' rapid amplification of cDNA ends (RACE), 5' radiolabeling, and exonuclease digestion, which revealed the following observations: (i) DP-rcDNA and rcDNA possess an identical 3' end of (-)strand DNA; (ii) compared to rcDNA, DP-rcDNA has an extended but variable 3' end of plus strand [(+)strand] DNA, most of which is in close proximity to direct repeat 2 (DR2); (iii) DP-rcDNA exhibits an RNA primer-free 5' terminus of (+)strand DNA with either a phosphate or hydroxyl group; and (iv) the 5' end of the DP-rcDNA (-)strand is unblocked at nucleotide G1828, bearing a phosphate moiety, indicating the complete removal of polymerase from rcDNA via unlinking the tyrosyl-DNA phosphodiester bond during rcDNA deproteination. However, knockout of cellular 5' tyrosyl-DNA phosphodiesterase 2 (TDP2) did not markedly affect rcDNA deproteination or cccDNA formation. Thus, our work sheds new light on the molecular mechanisms of rcDNA deproteination and cccDNA biogenesis.IMPORTANCE The covalently closed circular DNA (cccDNA) is the persistent form of the hepatitis B virus (HBV) genome in viral infection and an undisputed antiviral target for an HBV cure. HBV cccDNA is converted from viral genomic relaxed circular DNA (rcDNA) through a complex process that involves removing the covalently bound viral polymerase from rcDNA, which produces a deproteinated-rcDNA (DP-rcDNA) intermediate for cccDNA formation. In this study, we characterized the four termini of cytoplasmic DP-rcDNA and compared them to its rcDNA precursor. While rcDNA and DP-rcDNA have an identical 3' terminus of (-)strand DNA, the 3' terminus of (+)strand DNA on DP-rcDNA is further elongated. Furthermore, the peculiarities on rcDNA 5' termini, specifically the RNA primer on the (+)strand and the polymerase on the (-)strand, are absent from DP-rcDNA. Thus, our study provides new insights into a better understanding of HBV rcDNA deproteination and cccDNA biosynthesis.
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    "D" matters in recombinant AAV DNA packaging
    (Elsevier, 2021) Zhang, Junping; Guo, Ping; Xu, Yinxia; Mulcrone, Patrick L.; Samulski, R. Jude; Xiao, Weidong; Pediatrics, School of Medicine
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    Hepatitis B virus X protein counteracts high mobility group box 1 protein-mediated epigenetic silencing of covalently closed circular DNA
    (PLOS, 2022-06-09) Kim, Elena S.; Zhou, Jun; Zhang, Hu; Marchetti, Alexander; van de Klundert, Maarten; Cai, Dawei; Yu, Xiaoyang; Mitra, Bidisha; Liu, Yuanjie; Wang, Mu; Protzer, Ulrike; Guo, Haitao; Microbiology and Immunology, School of Medicine
    Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), serving as the viral persistence form and transcription template of HBV infection, hijacks host histone and non-histone proteins to form a minichromosome and utilizes posttranslational modifications (PTMs) "histone code" for its transcriptional regulation. HBV X protein (HBx) is known as a cccDNA transcription activator. In this study we established a dual system of the inducible reporter cell lines modelling infection with wildtype (wt) and HBx-null HBV, both secreting HA-tagged HBeAg as a semi-quantitative marker for cccDNA transcription. The cccDNA-bound histone PTM profiling of wt and HBx-null systems, using chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR), confirmed that HBx is essential for maintenance of cccDNA at transcriptionally active state, characterized by active histone PTM markers. Differential proteomics analysis of cccDNA minichromosome established in wt and HBx-null HBV cell lines revealed group-specific hits. One of the hits in HBx-deficient condition was a non-histone host DNA-binding protein high mobility group box 1 (HMGB1). Its elevated association to HBx-null cccDNA was validated by ChIP-qPCR assay in both the HBV stable cell lines and infection systems in vitro. Furthermore, experimental downregulation of HMGB1 in HBx-null HBV inducible and infection models resulted in transcriptional re-activation of the cccDNA minichromosome, accompanied by a switch of the cccDNA-associated histones to euchromatic state with activating histone PTMs landscape and subsequent upregulation of cccDNA transcription. Mechanistically, HBx interacts with HMGB1 and prevents its binding to cccDNA without affecting the steady state level of HMGB1. Taken together, our results suggest that HMGB1 is a novel host restriction factor of HBV cccDNA with epigenetic silencing mechanism, which can be counteracted by viral transcription activator HBx.