| Object:GB virus C (GBV-C), also known as hepatitis G virus(HGV), is an apparently nonpathogenic virus that replicates in T and B lymphocytes.Several findings reported that the patients who coinfected with human immunodeficiency virus(HIV) and GBV-C had a better prognosis. Higher GBV-C viral load is associated with a greater suppression of HIV-1 replication. However it remains controversial. Other studies showed there were no beneficial effects of the GBV-C coinfection with HIV. GBV-C and HIV are distributed worldwide. GBV-C is a single-stranded RNA virus belonging to flaviviridae, but it is accepted that GBV-C by itself is not a causative agent of hepatitis or any other disease. GBV-C is also transmitted through sexual contacts. We do be interested in the role and mechanism of GBV-C in the regulation of HIV type 1 long terminal repeat (HIV-LTR) promoter activity on HIV. We conducted the present study to investigate the effect of GBV-C on HIV long terminal repeat (HIV-LTR) promoter activity, clarify GBV-C infection regulates HIV transcription, estimate the role of GBV-C in the regulation of HIV-LTR promoter activity, try to illuminate the key point and mechanism which GBV-C inhibit HIV reproduction. Methods:1.The total HIV LTR sequence was devided into 11 fragment. Using the 3' end of base sequence which match next flagment of the HIV-I LTR as template and connected with PCR, we have got the total HIV-LTR, and then determined by cleavage and sequence analysis. 2.We designed KpnI and Xho–site within terminal region of LTR, cleaved pCAT plasmid and LTR with KpnI and Xho and connected the fragments with T4 DNA ligase ,and then determined by cleavage and sequence analysis.3. Jurkat cell lines was transfected with pLTR-1CAT .The CAT activity of all cells was measured using ELISA assay and showed highly expression.4.Jurkat cell lines were choosed as target cell in this study. They were cultured in RPMI 1640 Medium supplemented with 10% fetal calf serum.The study was divided into 4 groups. Group 1 (control group): Jurkat cell lines was transfected with pLTR-1CAT only, Group 2: Jurkat cell lines was transfected with pLTR-1CAT and GBV-C infection simultaneity, Group 3:Jurkat cell lines was transfected with pLTR-1CAT after infected with GBV-C for 24 hours, Group 4:Jurkat cell lines was infected with GBV-C after transfected with pLTR-1CAT for 24 hours. The Contents of GBV-C were determined with RT-PCR. The CAT activity of all cells was measured using ELISA Assay. All data were presented as means and±SD value and analyzed by one-way ANOVA. Results:1. We constructed HIV LTR expression vector via gene engineering method. pLTR-1CAT was made up of LTR and reporter gene --chloramphenicol acetyltransferase(CAT) and determined by cleavage and sequence analysis.2. The CAT activity of Group 2,3,4 were lower than that of control group(P<0.05). The CAT activity of Group 3 were lower than other groups at each time point(P<0.05).There was no difference between the CAT activity of group 4 and group 2 at 48,96 hour(P>0.05).Conclusion: The study suggested that GBV-C could decrease CAT activity via inhibiting LTR promoter transcription. HIV-LTR might serve as a key point deactivated by GBV-C and its product. GBV-C could inhibit HIV replication by dereasing trancription of HIV regulation protein and structure protein. |
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