Role Of HBV Posttranscriptional Regulatory Element In Noncytopathic Mechanism And Biological Significance Of Its Heterogeneity

To explore the effect of IFN- v and TNF- a on hepatitis B virus posttranscriptional regulatory elements.The objective eukaryotic expression vectors were constructed by molecular cloning in vitro, and identified by using PCR and sequencing analysis. CAT assays were performed with ELISA kit The eukaryotic expression vectors containing HPRE segment were constructed successfully as confirmed by sequencing analysis. The normalized CAT activity from recombinant pDM138 and control produced in transfected cells incubated in the absence of cytokine were 896.5 ?213.6 and 36.7 ?11.3 pg/ml, respectively. After incubation with IFN- y, TNF- a and IFN- y +TNF- a , the normalized CAT activity from recombinant pDM138 produced in transfected cells were 324.4 + 56.8, 395.8 + 82.3, 436.7 + 97.2 ( t = 5.19,4.39, 6.68, all P<0.01) , respectively. The effect of IFN- y and TNF- a were dose dependent over a range of 0 to lOOOU/ml. These suggested that the expression of HBV gene may be regulated by IFN- y and TNF- a via inhibiting the function of HPRE.To screen the variation points related with response to interferon- a hi HBV post- transcriptional regulatory element. Serum HPRE sequences of 31 Chinese patients with HBV infection were detected by direct sequencing of PCR products and compared with that of Caucasian patients. The T to C variation point at nt 1504 and C to T(G) at nt 1508 in HPRE were found in 21 and 19 patients with chronic hepatitis B, respectively. The C to T variation point at nt 1509 were found in 17 cases; The variation points mentioned above did not exit in HPRE sequence of the Caucasian patients. The relationship between the HPRE variation points and the levels of HBV DNA has not been found. These resultssuggested that variation points exit in the HPRE sequence from Chinese patients, which may correlate with response to interferon- a .To explore the relationship between the HPRE heterogeneity and noncytolytic antiviral mechanism. The objective eukaryotic expression vectors were constructed by molecular cloning and PCR-based site-directed mutagensis in vitro, and identified by using PCR and sequencing analysis. The eukaryotic expression vectors containing HPRE segment and mutating point were constructed successfully as confirmed by sequencing analysis. The activity of CAT gene obviously increased in the T to C mutation at nt 1504 of HPRE and no alteration in the C to T(G) at nt 1508. The mutation at nt 1508 of HPRE may escape the suppression role of IFN- a on HPRE. These results suggested that the heterogeneity of HPRE may affect the function of HPRE and influence the regulative function of IFN- a on HPRE, but no IFN- y or TNF- a .