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.

Transactivation and signaling functions of Tat are not correlated: biological and immunological characterization of HIV-1 subtype-C Tat protein.

BACKGROUND: Of the diverse subtypes of Human Immunodeficiency Virus Type-1 (HIV-1), subtype-C strains cause a large majority of infections worldwide. The reasons for the global dominance of HIV-1 subtype-C infections are not completely understood. Tat, being critical for viral infectivity and pathogenesis, may differentially modulate pathogenic properties of the viral subtypes. Biochemical studies on Tat are hampered by the limitations of the current purification protocols. Tat purified using standard protocols often is competent for transactivation activity but defective for a variety of other biological functions. Keeping this limitation in view, we developed an efficient protein purification strategy for Tat. RESULTS: Tat proteins obtained using the novel strategy described here were free of contaminants and retained biological functions as evaluated in a range of assays including the induction of cytokines, upregulation of chemokine coreceptor, transactivation of the viral promoter and rescue of a Tat-defective virus. Given the highly unstable nature of Tat, we evaluated the effect of the storage conditions on the biological function of Tat following purification. Tat stored in a lyophilized form retained complete biological activity regardless of the storage temperature. To understand if variations in the primary structure of Tat could influence the secondary structure of the protein and consequently its biological functions, we determined the CD spectra of subtype-C and -B Tat proteins. We demonstrate that subtype-C Tat may have a relatively higher ordered structure and be less flexible than subtype-B Tat. We show that subtype-C Tat as a protein, but not as a DNA expression vector, was consistently inferior to subtype-B Tat in a variety of biological assays. Furthermore, using ELISA, we evaluated the anti-Tat antibody titers in a large number of primary clinical samples (n = 200) collected from all four southern Indian states. Our analysis of the Indian populations demonstrated that Tat is non-immunodominant and that a large variation exists in the antigen-specific antibody titers. CONCLUSION: Our report not only describes a simple protein purification strategy for Tat but also demonstrates important structural and functional differences between subtype-B and -C Tat proteins. Furthermore, this is the first report of protein purification and characterization of subtype-C Tat.

Identification of unique B/C recombinant strains of HIV-1 in the southern state of Karnataka, India.

We characterized the molecular nature of a large number of primary HIV-1 isolates in the four southern states of India. In addition to confirming a predominance of subtype C infection, for the first time we identified three B/C recombinant viruses in a subset of 115 samples. Unexpectedly, env sequences of two of the three B/C recombinants phylogenetically clustered with subtype B strains of the USA. The determination of the real incidence of the recombinant viruses is of importance.