Journal: Virology

Article Title: Hepatitis B virus regulatory HBx protein binding to DDB1 is required but is not sufficient for maximal HBV replication

doi: 10.1016/j.virol.2012.01.021

Figure Lengend Snippet: (A) Schematic representation of HBx protein showing minimum domain required for binding to DDB1 [adapted from (Keasler and Slagle, 2008)]. (B) Schematic of wildtype and point mutant HBx proteins. DDB1 binding determined previously (Becker et al., 1998; Lin-Marq et al., 2001). (C) Quantitation of capsid-associated viral DNA as described in Materials and Methods. Mean copy number from cells transfected with pHBV was set to 100% and compared to others. Error bars (SE) from three independent experiments. Statistical significance compared to pHBV is noted by an asterisk (p<0.05). (D) Western blot detection of wildtype and point mutant HBx proteins with rabbit anti-HBx.

Article Snippet: The membrane was incubated with either rabbit anti-DDB1 (Zymed; 1:1000) or anti-tubulin (Santa Cruz; 1:1000).

Techniques: Binding Assay, Mutagenesis, Quantitation Assay, Transfection, Western Blot

Journal: Virology

Article Title: Hepatitis B virus regulatory HBx protein binding to DDB1 is required but is not sufficient for maximal HBV replication

doi: 10.1016/j.virol.2012.01.021

Figure Lengend Snippet: (A) Schematic representation of HBx protein and truncation mutants. Gray box indications the region of HBx known to bind DDB1 (see Fig. 1A). “+” indicates DDB1 binding, as reported previously (Becker et al., 1998). (B) Quantitation of capsid-associated viral DNA. Mean copy number from cells transfected with pHBV was set to 100% and compared to others. Error bars (SEM) are from three independent experiments. Statistical significance compared to pHBV is noted by an asterisk (p<0.05).

Article Snippet: The membrane was incubated with either rabbit anti-DDB1 (Zymed; 1:1000) or anti-tubulin (Santa Cruz; 1:1000).

Techniques: Binding Assay, Quantitation Assay, Transfection

Journal: Virology

Article Title: Hepatitis B virus regulatory HBx protein binding to DDB1 is required but is not sufficient for maximal HBV replication

doi: 10.1016/j.virol.2012.01.021

Figure Lengend Snippet: Representative co-IP/western blot for wild type HBx with the DDB1/Cul4A complex. HepG2 cells were co-transfected with the plasmids shown, and at 48 hr post-transfection cells were lysed for IP using the indicated antibody. Western blots for myc-Cul4A, DDB1, or HBx. * indicates non-specific background band.

Article Snippet: The membrane was incubated with either rabbit anti-DDB1 (Zymed; 1:1000) or anti-tubulin (Santa Cruz; 1:1000).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Transfection

Journal: Virology

Article Title: Hepatitis B virus regulatory HBx protein binding to DDB1 is required but is not sufficient for maximal HBV replication

doi: 10.1016/j.virol.2012.01.021

Figure Lengend Snippet: (A) Schematic representation of the floxed DDB1 locus (DDB1F/F) before and after excision by the Cre recombinase protein, which is under the control of the Albumin promoter (Alb-Cre+/−). (B) Western blot analysis of DDB1 and tubulin loading control in liver lysates. Numbers below each lane indicate results of densitometer scanning for DDB1 normalized to tubulin for that lane. (C) Immunostaining for DDB1 protein of liver sections of 6-wk old mice. (D) Sequential IP/western blot detection HBx expression in liver lysates.

Article Snippet: The membrane was incubated with either rabbit anti-DDB1 (Zymed; 1:1000) or anti-tubulin (Santa Cruz; 1:1000).

Techniques: Western Blot, Immunostaining, Expressing

Journal: Virology

Article Title: Hepatitis B virus regulatory HBx protein binding to DDB1 is required but is not sufficient for maximal HBV replication

doi: 10.1016/j.virol.2012.01.021

Figure Lengend Snippet: (A) HBx-DDB1 cccDNA model. During HBV replication, the partially double-stranded DNA genome is repaired to form cccDNA. This model predicts that HBx (which does not bind DNA) is tethered to the cccDNA via DDB1, and that the carboxyl portion of HBx is needed to recruit transcription factors (TFs) for the transcription of viral mRNAs, as described in the text. (B) HBx-DDB1 Displacement/Recruitment model. DDB1 functions as an adaptor for the CUL4A E3 ubiquitin ligase complex, where it recruits DDB1-CUL4 Accessory Factors (DCAFs), which bind substrate proteins that are ubiquinated and degraded by the proteosome. This model proposes that during HBV replication, the HBx interaction with DDB1 may either displace one or more DCAFs to block the degradation of DCAF substrates that benefit HBV replication, and/or recruit other substrates either for protection from degradation (positive factor) or for degradation (negative factor).

Article Snippet: The membrane was incubated with either rabbit anti-DDB1 (Zymed; 1:1000) or anti-tubulin (Santa Cruz; 1:1000).

Techniques: Blocking Assay