Detection of the hepatitis B virus (HBV) covalently-closed-circular DNA (cccDNA) in mice transduced with a recombinant AAV-HBV vector

International audienceHepatitis B Virus (HBV) persists in infected hepatocytes as an episomal covalently-closed-circular DNA mini-chromosome, called cccDNA. As the main nuclear transcription template, HBV cccDNA is a key replication intermediate in the viral life cycle. Little is known about the mechanisms involved in its formation, maintenance and fate under antiviral therapies. This is mainly due to the lack of small immune-competent animal models able to recapitulate the entire HBV replication cycle, including formation of HBV cccDNA. Here we report that HBV cccDNA can be detected by Southern blot analyses in the liver of C57BL6 mice transduced with AAV-HBV. HBV cccDNA persists in the liver of these animals together with the AAV-HBV episome. We also set up a PCR strategy to distinguish the HBV cccDNA from the AAV-HBV episome. These suggest that the AAV-HBV/mouse model might be relevant to test drugs targeting HBV cccDNA regulation and persistence

Conditional FKBP12.6 Overexpression in Mouse Cardiac Myocytes Prevents Triggered Ventricular Tachycardia Through Specific Alterations in Excitation- Contraction Coupling

International audienceBackground— Ca 2+ release from the sarcoplasmic reticulum via the ryanodine receptor (RyR2) activates cardiac myocyte contraction. An important regulator of RyR2 function is FKBP12.6, which stabilizes RyR2 in the closed state during diastole. β-Adrenergic stimulation has been suggested to dissociate FKBP12.6 from RyR2, leading to diastolic sarcoplasmic reticulum Ca 2+ leakage and ventricular tachycardia (VT). We tested the hypothesis that FKBP12.6 overexpression in cardiac myocytes can reduce susceptibility to VT in stress conditions. Methods and Results— We developed a mouse model with conditional cardiac-specific overexpression of FKBP12.6. Transgenic mouse hearts showed a marked increase in FKBP12.6 binding to RyR2 compared with controls both at baseline and on isoproterenol stimulation (0.2 mg/kg IP). After pretreatment with isoproterenol, burst pacing induced VT in 10 of 23 control mice but in only 1 of 14 transgenic mice ( P <0.05). In isolated transgenic myocytes, Ca 2+ spark frequency was reduced by 50% ( P <0.01), a reduction that persisted under isoproterenol stimulation, whereas the sarcoplasmic reticulum Ca 2+ load remained unchanged. In parallel, peak I Ca,L density decreased by 15% ( P <0.01), and the Ca 2+ transient peak amplitude decreased by 30% ( P <0.001). A 33.5% prolongation of the caffeine-evoked Ca 2+ transient decay was associated with an 18% reduction in the Na + -Ca 2+ exchanger protein level ( P <0.05). Conclusions— Increased FKBP12.6 binding to RyR2 prevents triggered VT in normal hearts in stress conditions, probably by reducing diastolic sarcoplasmic reticulum Ca 2+ leak. This indicates that the FKBP12.6-RyR2 complex is an important candidate target for pharmacological prevention of VT