Hepatitis B virus-mediated changes of apolipoprotein mRNA abundance in cultured hepatoma cells

Abstract

An inverse correlation between hepatitis B virus (HBV) and steady-state levels of apolipoprotein AI and CIII mRNAs was observed in two hepatoma cell lines. Analysis of a third line containing an inducible viral genome implicated viral pregenomic RNA in apolipoprotein mRNA reduction. We conclude that HBV alters infected cells despite the absence of overt cytopathogenicity.

FIG. 1.

Comparison of apolipoprotein mRNA levels in cells that do or do not contain replicating HBV. (a) Relative levels of apoAI, apoB, apoCIII, and actin mRNAs present in HepG2 and HepG2.2.15 cells were compared by RT-PCR. Amplified products were separated by gel electrophoresis and detected by staining with ethidium bromide. The image contrast was reversed to improve the visibility of faint bands. The identity of each product was confirmed by DNA sequencing. (b) Relative levels of apoAI, apoB, apoCIII, and actin mRNAs present in McA-RH-7777 and Q7-21 cells were compared by RT-PCR. Rat-specific primers were used for apoB and apoCIII; the human-based primers for apoAI and actin were capable of detecting the rat homologues. The identities of the amplified rat products were confirmed by DNA sequencing.

FIG. 2.

Inverse correlation between viral RNA levels and apolipoprotein mRNAs. Total RNA was prepared from cells incubated at confluence for increasing lengths of time, so as to yield increasing levels of viral nucleic acids and protein. (a) RT-PCR was used to detect HepG2.2.15 cell mRNAs for apoAI, apoB, apoCIII, α₁-antitrypsin (AAT), actin, and viral RNA (HBV). Note that HBV primers specifically detect the pregenomic RNA and not the subgenomic mRNAs. The image contrast was reversed to improve the visibility of faint bands. (b) Plot of densitometric data illustrating the relative changes in steady-state levels of mRNAs over time in culture.

FIG. 3.

Induction of HBV pregenomic RNA synthesis results in apolipoprotein mRNA reduction. (a) Organization of the HBV transgene present in AD38 cells. Only the 3.5-kb pregenomic transcript is under the control of the tetracycline-regulated promoter. (b) RT-PCR detection of apolipoprotein mRNAs at 2, 4, and 6 days after the induction of HBV by withdrawal of tetracycline (−Tet). At left, controls were cells at the start of the time course (day 0) or cells that had been incubated for 6 days in the presence of tetracycline (+Tet). The image contrast was reversed to improve the visibility of faint bands.