Gene expression analysis from human immunodeficiency virus type 1 subtype C promoter and construction of bicistronic reporter vectors.
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.
Siddappa
Nagadenahalli Byrareddy
NB
Molecular Virology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India.
Kashi
Venkatesh Prasanna
VP
Venkatramanan
Mohanram
M
Balasiddaiah
Anangi
A
Jayasuryan
Narayana
N
Mahadevan
Anita
A
Desai
Anita
A
Satish
Kadappa S
KS
Shankar
Susarla K
SK
Ravi
Vasanthapuram
V
Ranga
Udaykumar
U
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*
D016655:HIV Core Protein p24; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000469:Alkaline Phosphatase