Molecular and genetic characterization of natural HIV-1 Tat Exon-1 variants from North India and their functional implications.
BACKGROUND
Designing an ideal vaccine against HIV-1 has been difficult due to enormous genetic variability as a result of high replication rate and lack of proofreading activity of reverse transcriptase leading to emergence of genetic variants and recombinants. Tat transactivates HIV-1 LTR, resulting in a remarkable increase in viral gene expression, and plays a vital role in pathogenesis. The aim of this study was to characterize the genetic variations of Tat exon-1 from HIV-1 infected patients from North India.


METHODS
Genomic DNA was isolated from PBMCs and Tat exon-1 was PCR amplified with specific primers followed by cloning, sequencing and sequence analyses using bioinformatic tools for predicting HIV-1 subtypes, recombination events, conservation of domains and phosphorylation sites, and LTR transactivation by luciferase assay.


RESULTS
Phylogenetic analysis of Tat exon-1 variants (n = 120) revealed sequence similarity with South African Tat C sequences and distinct geographical relationships were observed for B/C recombinants. Bootscan analysis of our variants showed 90% homology to Tat C and 10% to B/C recombinants with a precise breakpoint. Natural substitutions were observed with high allelic frequencies which may be beneficial for virus. High amino acid conservation was observed in Tat among Anti Retroviral Therapy (ART) recipients. Barring few changes, most of the functional domains, predicted motifs and phosphorylation sites were well conserved in most of Tat variants. dN/dS analysis revealed purifying selection, implying the importance of functional conservation of Tat exon-1. Our Indian Tat C variants and B/C recombinants showed differential LTR transactivation.


CONCLUSIONS
The possible role of Tat exon-1 variants in shaping the current HIV-1 epidemic in North India was highlighted. Natural substitutions across conserved functional domains were observed and provided evidence for the emergence of B/C recombinants within the ORF of Tat exon-1. These events are likely to have implications for viral pathogenesis and vaccine formulations.
Virology Laboratory, National Institute of Immunology, New Delhi, India ; Department of Microbiology, University College of Medical Sciences and Guru Teg Bahadur Hospital, Delhi, India.
Larance
L
Ronsard
Department of Microbiology, University College of Medical Sciences and Guru Teg Bahadur Hospital, Delhi, India.
Sneh
S
Lata
Virology Laboratory, National Institute of Immunology, New Delhi, India ; Department of Microbiology, University College of Medical Sciences and Guru Teg Bahadur Hospital, Delhi, India.
Jyotsna
J
Singh
Department of Microbiology, University College of Medical Sciences and Guru Teg Bahadur Hospital, Delhi, India.
Vishnampettai G
VG
Ramachandran
Department of Microbiology, University College of Medical Sciences and Guru Teg Bahadur Hospital, Delhi, India.
Shukla
S
Das
Virology Laboratory, National Institute of Immunology, New Delhi, India.
Akhil C
AC
Banerjea
D000293:Adolescent; D000328:Adult; D000595:Amino Acid Sequence; D002648:Child; D002675:Child, Preschool; D004252:DNA Mutational Analysis; D005091:Exons; D005260:Female; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D058889:Molecular Typing; D058873:Pandemics; D010766:Phosphorylation; D010802:Phylogeny; D058974:Phylogeography; D011499:Protein Processing, Post-Translational; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D055815:Young Adult; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics*
D054322:tat Gene Products, Human Immunodeficiency Virus