Virus Research 147 (2010) 195–201 Contents lists available at ScienceDirect Virus Research journal homepage: www.elsevier.com/locate/virusres Molecular characterization of tat gene and long terminal repeat region of human immunodeficiency virus type-1 detected among the injecting drug users (IDUs) of Manipur, India: Identification of BC recombinants Ranajoy Mullick, Satarupa Sengupta, Kamalesh Sarkar, Sekhar Chakrabarti ∗ HIV/AIDS Research Laboratory, Divison of Virology, National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road, Scheme-XM, Beliaghata, Kolkata 700010, West Bengal, India article info Article history: Received 28 July 2009 Received in revised form 27 October 2009 Accepted 30 October 2009 Available online 6 November 2009 Keywords: HIV-1 tat gene LTR India Recombinants abstract The tat gene of human immunodeficiency virus type-1 (HIV-1) is responsible for the initiation and elon- gation of viral transcription through the LTR (long terminal repeat) transactivation process. Our study included structural and functional analyses of the tat gene and LTR region of 35 injecting drug users (IDUs) from Manipur (a north-eastern state in India and a potential source of HIV-1 recombinants) in order to search for the recombinants and variation in the transactivation process if any due to recom- bination. Analysis showed prevalence of subtype C with few BC recombinants for the tat gene showing identical recombination breakpoints. Phylogenetic analysis of the LTR region of those IDU strains showed strong resemblance to Indian subtype C forming a completely separate cluster from the other global C LTR sequences. The TAR element (transactivator response region) in all the LTR sequences was fairly con- served. Further study of the transactivation rate of the C and BC tat for the Manipur C LTR showed almost equal transactivity in both the cases. This is the first report of characterisation of tat gene and LTR region of HIV-1 samples among IDUs from north-eastern India. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Human immunodeficiency virus type-1 (HIV-1) gene expression and replication are highly dependent on the interaction between the viral (Joseph et al., 2003) and host cellular factors (Brigati et al., 2003). Tat protein encoded by the tat gene of HIV-1 is an essen- tial element for viral gene expression (Muesing et al., 1987; Jeang et al., 1999) in the early phase of viral life cycle from the multiple spliced viral transcripts (Schwartz et al., 1990). It transactivates the HIV-1 long terminal repeat (LTR) promoter by binding to a nascent RNA stem-loop structure known as the transactivator response region (TAR) (Laspia et al., 1989; Berkhout et al., 1989) followed by subsequent assembly of positive transcription elongation factor b (pTEFb) (Karn, 1999). Tat is responsible for both initiation and elon- gation of transcription (Muesing et al., 1987; Schwartz et al., 1990). The tat gene contains certain highly conserved domains in order to regulate its activities. The majority of these functional domains lie within the exon1 region. Therefore structural and functional anal- yses of the tat exon1 region of HIV-1 are important to understand the transcriptional regulation process of different subtype specific viruses. ∗ Corresponding author. Tel.: +91 33 2370 1176; fax: +91 33 2370 5066. E-mail address: drsekharchakrabarti@yahoo.co.in (S. Chakrabarti). The main objective of our study was to characterize the tat and LTR regions of HIV-1 among the IDU populations of Manipur, a north-eastern state in India both at the structural and func- tional levels in search for recombinants. Manipur is a potential hub for HIV-1 recombinants as studied earlier based on gag and env subgenomic regions (Bhanja et al., 2005). Earlier study showed BF intragenic recombination of HIV-1 tat among the Argentinean sam- ples showing higher rate of transactivation (Turk et al., 2006), but recombination among the tat gene from India was not reported earlier. Our study revealed interesting findings on both tat and LTR genomic regions of HIV-1 among IDU populations from this part of India. 2. Materials and methods 2.1. Sample collection, PBMC DNA isolation and HMA study Blood samples were collected in EDTA coated vacutainer tubes (Beckton Dickinson) and peripheral blood mononuclear cells (PBMCs) were separated from whole blood by Ficoll–Hypaque gra- dient centrifugation (Boyam, 1968). DNA was extracted by using the QIAamp DNA Blood Mini Kit 250 (QIAGEN, Germany) accord- ing to the manufacturer’s protocol. Subtyping analysis of all the samples were done by gag and env heteroduplex mobility assays (Heyndrickx et al., 2000) followed by sequencing analysis. 0168-1702/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2009.10.024