Investigation of the expression and replication of the HBV genome as well as the full viral life cycle has been hampered by the lack of an in vitro tissue culture system in which HBV is propagated. The HepG2.2. 15 cell line is a putative HBV cellular model and it have been used for antiviral research for many years (9, 3, 7) ,however,HBV replication in HepG2.2.15 cells is very different from HBV natural replication in vivo. HBV covalently closed circular DNA(cccDNA),a key viral replicative intermediates in viral life cycle,is absent in this cell line. Here we constructed a HBV replicon containing a 1.3× copy of the HBV genome,and established a transient transfection cellular model of HBV replication.
A 915 bp fragment was obtained by PCR. This fragment was inserted to pCR2.1 vector. Plasmids contai-ning forward fragment were identified by restriction endonuclease analysis and named as pHBV0.3,sequence analysis showed that there was no mutation in the inserted fragment. A 2852 bp fragment was obtained from pHBV-dimer by digestion with AatⅡ and NsiⅠ,and inserted into pHBV0.3,plasmids containing 1.0 fold of HBV genome was constructed and named as pHBV1.0. A 921 bp fragment was obtained from pHBV-dimer by digestion with NsiⅠand BglⅡ,and inserted into pHBV1.0; plasmid pHBV1.3 was identified by restriction endonuclease analysis. After digestion with AatⅡ,There were two fragments of 3 200 bp and 3 856 bp (Fig. 1).
Figure 1. Restriction endonuclease analysis of plasmid pHBV1.3. pHBV1.3 was digested with restriction endonuclease AatⅡ,the cleaved fragments were separate by electrophoresis in a 1% agarose gel,The feagments are 3 200 bp and 3 856 bp (lane3). Plasmid pHBV1.3 is 7 056 bp (lane2). The molecular weight standard is 1 kb DNA ladder (lane1).
Plasmids were transfected into Huh7 cells and allowed to replicate for 96 h.Quantities of HBsAg and HBeAg present in the supernatant were measured by ELISA; quantity of HBV DNA in supernatant was determined by real-time PCR; intracellular replicative intermediates and transcripts were analyzed by Southern blot and Northern blot respectively. The levels of HBsAg and HBeAg reached a plateau within 72 h. Further incubation did not increase the concentration of HBsAg and HBeAg in the culture supernatant. There was a well consistency of the levels between HBsAg and HBeAg in supernatants (Table 1.). Meanwhile,the levels of HBVDNA in the culture supernatant were 6.5×102copies/mL,1.2×103 copies/mL,2.5×105 copies/mL and 2.7×105 copies/mL at 24 h,48 h,72 h,and 96 h respectively. Furthermore,the intensity of the hybridi-zation signal,for intracellular replicative intermediates and int-racellular transcripts also reached a maximum within 72 h (Fig. 2). The results indicated that the HBV replicon could effectively initiate HBV replication and expression of HBV proteins.
Table 1. HBV products in culture supernatant after transfection with HBV replicon(x±s)
Figure 2. Intracellular HBV replicative intermediates (A) and intracellular HBV transcripts RNA (B) in Huh7 cells transfected with a HBV replicom. A: Southern blot analysis of 5μg of total DNA extracted from Huh7 cells transfected with pHBV1.3. and probed by a 32P-labeled HBV-specific DNA probe. Bands corresponding to the expected size of double-stranded (DS) and single-stranded (SS) HBV DNAs are indicated. B: Northern blot analysis of 20μg of total RNA was extracted from transfected cells. and hybridized with 32P-labeled HBV-and GAPDH-specific probe. Bands corresponding to the expected size of 3.5kb and 2.1/2.4kb HBV transcripts are indicated. Lane1-4 : 24h,48h,72h and 96h. post transfectionre spectively.
Huh7 cells transfected with pHBV1.3 were passaged at an interval of 4 days. Southern blot analysis shown that intracellular HBV DNA replicative intermediates could be found for at least twelve days. The results indicate that the HBV replicon can persist for an extended time,even after cell division.
A cell-based antiviral assay was used to test the adefovir activity. Huh7 cells were transfected with pHBV1.3,and treated with different concentrations of adefovir on the next day. Southern blot analysis indicated 10 mol/L of adefovir could completely inhibit HBV replication in Huh7 cells transfected with plasmid pHBV1.3,and the inhibition is dose-dependent (Fig. 3).
Figure 3. Antiviral test of in vitro system transfected with HBV replicon. Huh7 cells were transfected with plasmid pHBV1.3.and then treated with 0.01,0.1,1 and 10μmol/L of adefovir for 72 h. At the end of the treatment,total DNA was isolated and analyzed by Southern blot. Lane1-lane4: total DNA from Huh7 cells treated with 0.01,0.1,1 and 10μmol/L of adefovir respectively.