X Protein of hepatitis B virus potently activates HIV-1 subtype C long terminal repeat promoter: implications for faster spread of HIV-1 subtype C

Abstract

Several genetic subtypes of HIV have been identified throughout the world, but it is predominantly subtype C that is responsible for causing the epidemic in India, some regions of south Asia and Africa. Besides HIV-1 infection, individuals are also co-infected with other pathogens such as hepatitis B virus (HBV), and other bacterial and yeast pathogens. HBV encodes the X gene (HBx) whose product is known to activate several heterologous promoters, including the HIV-1 long terminal repeat (LTR) promoter [1–4]. HIV-1 gene expression is controlled by the LTR promoter, which is rich in various transcription factor binding sites [5]. The HIV-1 LTR sequence of genetic subtype B is significantly different than subtype C, and it is therefore reasonable to assume that HBx may have different effects on promoter activity [5]. In order to study the possible impact of HBx protein on HIV-1 subtype-specific LTR-mediated activation, we co-transfected HIV-1 LTR-B and C reporter constructs (pBlue-3′-LTR-B and C-Luc) along with HBx encoding DNA, pSG5.HBx (465 nucleotide long X gene is placed under SV40 promoter that allows intracellular expression) [6] into human 293 cells using lipofectin (Invitrogen, Carlsbad, California, USA) for 48 h. Cell lysates were prepared and the amounts of luciferase were determined according to the manufacturer's instructions (Promega Biotech, Madison, Wisconsin, USA); the results are shown in Fig. 1a (mean of three independent experiments). To ensure uniform transfection efficiency we included an internal reporter control plasmid (pSV-β-gal; Promega). Control cells or cells transfected with 100 ng pSG5.HBx alone showed background luciferase activity. LTR-B and LTR-C reporter plasmids showed basal promoter activity, and the latter showed approximately twofold more basal activity. When co-transfected with 100 ng of either LTR-B or LTR-C plasmids along with 100 ng pSG5.HBx construct, the latter combination (LTR-C plus HBx) showed more than sevenfold more activation when compared with basal activity, whereas it was less than twofold with LTR-B plus HBx combination. When 100 ng of each LTR and 100 ng of pSG5.HBx DNA were used for transfection, the pattern and the extent of activation remained unchanged. When co-transfected with HBx plus B-Tat (cytomegalovirus promoter driven Tat derived from pNL4-3DNA) plus reporter construct (LTR-B-luciferase), a strong synergistic effect was observed. It is noteworthy that the level of LTR-B promoter activation by the HBx protein is equivalent to what we observed with LTR plus B-TAT interaction (mean ± SD from three experiments, Fig. 1b).Fig. 1: Amounts of luciferase determined from prepared cell lysates according to the manufacturer's instructions. (a) Human 293 cells were grown in Dulbecco's modified essential medium plus 10% fetal bovine serum, grown to 80% confluence in a 12-well plate. They were co-transfected with the indicated amounts of pSG5.HBx [6] and HIV-1 subtype B and C specific long terminal repeat (LTR)-luciferase constructs using lipofectin as described by the manufacturer. Twenty-four hours after transfection, cell lysates were prepared and the amounts of luciferase from equivalent fractions were determined as described by us earlier [3]. (b) Various combinations of plasmid DNA as indicated (100 ng each) were co-transfected and luciferase activity was determined as described in (a).This is the first study that suggests that the HBx protein might play an important role in upregulating HIV-1 subtype-C LTR-mediated gene expression compared with subtype-LTR-B-driven expression. These observations have strong implications for the increased replication of subtype C virus. Acknowledgements HIV-1 subtype-B and C-LTR-luciferase vectors and pNL4-3 DNA plasmid DNA were obtained from the AIDS Research and Reference Reagent Program of National Institutes of Health, Bethesda, Maryland, USA. Plasmid pSG5.HBx was a gift from Vijay Kumar, ICGEB, New Delhi, India. Sponsorship: This work was supported by a grant from the Department of Biotechnology, Government of India to the National Institute of Immunology, New Delhi, and to the corresponding author (A.C.B).