title: Gene expression analysis from human immunodeficiency virus type 1 subtype C promoter and construction of bicistronic reporter vectors.
abstract: We report the cloning and sequence analysis of the long terminal repeat (LTR) of several primary HIV-1 subtype C strains of India. Phylogenetically, all the LTRs and the paired env sequences clustered with subtype C reference strains. The LTRs demonstrated extensive polymorphism in the transcription factor binding sites (TFBS) within the enhancer and the modulator regions. We generated reporter vectors under the control of a select subset of the subtype C LTRs. The reporter vectors are distinguished by the simultaneous expression of two independent reporter genes, secreted alkaline phosphatase (SEAP) and enhanced green fluorescence protein (EGFP), in response to Tat. Expression of EGFP was facilitated by engineering an internal ribosome entry site (IRES) into the expression cassette. Although subtype C strains cause a large majority of the global infections, and important differences in the transcription factor binding sites have been identified in the subtype C promoter, few reporter vectors containing subtype C-LTR have been described. We analyzed gene expression from the C-LTR reporter vectors in different cell lines under diverse experimental conditions and compared it to the B-LTR reporter vector. The reporter vectors were responsive to Tat derived from diverse viral subtypes. Furthermore, a positive correlation was observed between the expression of the reporter genes and the viral structural protein p24 when the cells were infected with viral molecular clones. The LTR reporters we developed could be of significant use in the study of viral transactivation, in the evaluation of biological properties of viral subtypes, and in the screening for antiviral inhibitors.
authors.affiliation: Molecular Virology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India.
authors.forename: Nagadenahalli Byrareddy
authors.initials: NB
authors.lastname: Siddappa
authors.forename: Venkatesh Prasanna
authors.initials: VP
authors.lastname: Kashi
authors.forename: Mohanram
authors.initials: M
authors.lastname: Venkatramanan
authors.forename: Anangi
authors.initials: A
authors.lastname: Balasiddaiah
authors.forename: Narayana
authors.initials: N
authors.lastname: Jayasuryan
authors.forename: Anita
authors.initials: A
authors.lastname: Mahadevan
authors.forename: Anita
authors.initials: A
authors.lastname: Desai
authors.forename: Kadappa S
authors.initials: KS
authors.lastname: Satish
authors.forename: Susarla K
authors.initials: SK
authors.lastname: Shankar
authors.forename: Vasanthapuram
authors.initials: V
authors.lastname: Ravi
authors.forename: Udaykumar
authors.initials: U
authors.lastname: Ranga
mesh_terms: D000328:Adult; D000469:Alkaline Phosphatase / Q000235:genetics; D002460:Cell Line; D005260:Female; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D015756:Genes, tat; D014644:Genetic Variation; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D016655:HIV Core Protein p24 / Q000235:genetics / Q000378:metabolism*; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D015497:HIV-1 / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007194:India; D008213:Lymphocyte Activation; D008297:Male; D008875:Middle Aged; D010802:Phylogeny; D011401:Promoter Regions, Genetic / Q000235:genetics*
chemical_list: D016655:HIV Core Protein p24; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000469:Alkaline Phosphatase