الفهرس 
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Abstract
Around 250 million people are chronically infected with HBV worldwide, and at a high risk to develop liver cirrhosis, fibrosis and HCC, resulting in 650000 deaths per year. Beside the available prophylactic vaccine, there are some antivirals like INF-α and drugs that target HBV multifunction polymerase known as NAs.
HBV has the ability to hide its genome in the nucleus as a minichromosome named cccDNA, and to take over the cellular machinery including its enzymes and transcription factors to build up its transcripts and express its proteins, and thus producing new virions. This form of the DNA is responsible for the chronicity of the HBV.
The aim of the current study was to characterize the role of host DNA damage response in the HBV rcDNA to cccDNA conversion by monitoring the role of PML-NBs in the formation of cccDNA relevant to HBV core protein. This study addressed complementary unresolved questions in cccDNA biology and pave the way for new curative treatments of CHB that target the cccDNA persistence reservoir.
In the present study after demonstrating the interaction between HBV core protein and the endogenous PML proteins in HepaRG cell line, interaction between each of the single PML isoforms and the HBV core protein was investigated. H1299 shPML cell line was co-transfected with two plasmids, one carrying the HBV core protein gene and the other one carrying each of the six nuclear PML isoforms. Cell lysates were prepared and the flag-tagged transfected PML proteins were pulled down with a flag antibody in a co-immunoprecipitation experiment aiming to find if the HBV core protein was pulled down together with the transfected PML isoforms. After running Western Blots no signal for the core protein was detected in all lanes of the PML isoforms indicating that there was no interaction between the HBV core protein and the each of the six PML isoforms.
Immunofluorescence staining was carried out to detect co-localization of the HBV core protein with both endogenous PML proteins and single PML isoforms. After 24 hours, the cells were PFA- fixed on the glass slides and then stained with the primary and the secondary antibodies. Immunofluorescent microscopy was used to detect the fluorescent signals produced by the antibodies and then a merge was applied to detect if there is any co-localization. Very strong co-localization signal was detected with the endogenous PML and the core protein followed by PML IV isoform and PML II isoform. A weaker co-localization signal was detected with the rest of the PML isoforms and the HBV core protein.
Furthermore, cccDNA, as determined by real time PCR, decreased to 89.9% in the absence of the endogenous PML proteins in HepG2 shPML cell line as compared to 100% in the presence of the endogenous PML in HepG2 cell line. While the total HBV DNA decreased to 78% in the absence of the endogenous PML protein as compared to 100% in the presence of the endogenous PML in both HepG2 cell line and HepG2 shPML cell line respectively indicating that PML proteins might play a role in the replication of HBV and the formation of the cccDNA which is responsible for the chronicity of the HBV.
Summary, Conclusion and Recommendations
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The study was extended to investigate the role of single PML isoform in relation to cccDNA and total HBV DNA. HepG2 shPML cell line was first transfected with the single PML isoform and then infected with HBV. After 10 days incubation, cell lysates were prepared and real time PCR was carried out to quantitate the amount of cccDNA and total HBV DNA in relation to single PML isoforms. Inconsistent results were obtained with no indication that the presence or the absence of any of the six PML isoforms has an impact on the amount of HBV DNA or cccDNA.
Conclusion:
Interaction between HBV core protein and endogenous PMLs was detected in HepaRG cells.
Colocalization of the core protein and endogenous PMLs, PMLII and PML IV; and infrequent colocalization with other PML isoforms could be interpreted from the Immunofluorescence data.
Inconsistent results were obtained on performing quantitative PCR for the detection of the complete HBV DNA and cccDNA in single isoforms transfected HepG2 shPML cells.
Recommendations:
Generation of stable single PML isoform expressing HepG2 cell line and then repetition of the:
qPCR experiments.
Interaction between the HBV Core protein and the single PML isoforms.