Expression and Functional Analysis of Hepatitis B Virus DNA Polymerase

PART I: Expression of chimeric DNA polymerase of human and duck hepatitis B virus Hepatitis B Virus (HBV) DNA polymerase is a multifunctional protein with four domains, terminal protein (TP), spacer, reverse transcriptase (RT), and RNaseH. Since HBV DNA polymerase is very unstable and rapidly degradable, it has been very difficult to study the biochemical aspect of HBV DNA polymerase. In the present study, several chimeras of DNA polymerase by substituting DHBV sequence in the HBV DNA polymerase were constructed to have insights for the nature of HBV DNA polymerase. Also rabbit polyclonal antiserum specific for each domain of HBV DNA polymerase was produced. RNAs of chimeric DNA polymerases, and 2.4 and 2.1 Kbp of surface RNAs were produced by all chimeras but the expression proteins of chimeric polymerases were not detected. The distribution of HBV polyemerase was exclusively localized in cytoplasm and little co-localized between HBV polymerase and the organelles markers such as endoplasmic reticulum, Golgi and peroxisome. When the several chimeric constructs and polymerase deficient mutant that provides pgRNA and HBV proteins except polymerase, were cotransfected to hepatoma cells, all chimeras were incompatible for replication. Interestingly core particle formation varied in chimeric DNA polymerase construct transfected cells. This result suggests that the primary sequence of chimeric DNA polymerase may affect the capability core particle formation or the level of pregenomic RNA. PART II: Down-regulation of hepatitis B virus replication by splicing event Various spliced HBV transcripts have been described in human liver tissues and in HBV-transfected hepatoma cell lines, but their functions are unknown. To analyze the expression and the function of HBV DNA polymerase, HBV DNA polymerase construct was tranfected into hepatoma cells and then several spliced HBV RNAs from expressed HBV DNA polymerase RNA were founded. Since the spliced RNA encoded new viral protein, the polymerase-surface (PS) fusion protein was analyzed in detail. By confocal microscopy with monoclonal antibody specific for surface antigen and polyclonal antiserum specific for TP domain of HBV DNA polymerase, the exclusive expression of PS fusion protein in perinuclear region was detected. Also, intracellular distribution of PS fusion protein was overlapped with nuclear pore complex and vimentin, which is known as intermediate filament providing a perinuclear stable core area, as well as endoplasmic reticulum. Also, HBV small antigen secretion was drastically inhibited even with a small amount of this construct. Since it is possible that PS fusion protein expression might be involved in HBV life cycle by modulating gene regulation through nuclear/cytoplasmic transport like Rev protein of HIV, the spliced RNA only expressing mutant that did not express PS protein to elucidate the modulatory effects of the spliced RNA or the PS fusion protein on HBV life cycle. Core particle formation was blocked by the expression of spliced construct. Also, when the PS fusion RNA expression was increased, HBV DNA synthesis was decreased gradually by endogenous polymerase assay and Southern blot analysis. These data indicate that PS fusion RNA down-regulates HBV DNA synthesis, which distinguishes the HBV large surface RNA with PS fusion RNA. Taken together, these data suggest that PS fusion RNA may modulate the HBV replication, which is different from the reported previously.

Author dc:creator

• 박, 길순

Contributors dc:contributor

• 김, 경민
• 대학원 의학과
• 200124283

• Hepatitis B Virus DNA polymerase
• Chimera
• replication
• core particle formation
• splicing
• polymerase-surface (PS) fusion protein
• 중합효소-표면 fusion RNA
• 복제
• 세포골격

Language dc:language

en

Identifier

http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000000652
000000000652

OAI identifier oai:identifier

oai:repository.ajou.ac.kr:201003/1404