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Abstract
Human immunodefficiency virus Type-1 reverse transcriptase (HIV-1RT) exhibits RNA polymerae Activity
Sharma B.1
Objectives: To investigate whether HIV-1 RT and some of its mutant derivatives display RNA polymerase like activity.
Methods: The mutant HIV-1RTs were generated by PCR based site directed mutagnesis.The wild type and mutant heterodimeric (p66/p51) HIV-1RT proteins containing Histag at its C-terminus were expressed separately in E.coli(JM109). Cells were harvested, lysed and the enzyme was purified using standard protocol with the help of IMAC chromatography technique. The purified enzyme preparations were dialysed and concentrated by further dilysis against 50% glycerol buffer. Proteins were estimated using Bradford´s dye and quantitated by densitometric analysis of the protein bands obtained after SDS PAGE.The polymerase activity assay was performed using standard methods.
Results: It was observed that wild type HIV-1 RT and some of its mutant derivatives such as W153F, W153Y, W153A and K154R lying on the b8-aE loop in the larger subunit (p66) of heterodimeric (p66/p51) enzyme have the potential to incorporate rNTPs in place of dNTPs.though upto varying degrees, at the 3’end of the DNA primer annealed to a DNA or RNA template, thereby synthesizing a RNA strand. The incorporation of rNTPs in the primer was higher against an RNA template than that of DNA template. The polymerase activities of the mutant derivatives containing substitutions with the like amino acid side chains exhibited almost wild type polymerase activity. However, their substitutions with unlike amino acid side chains with different size exhibited drastic alterations in the polymerase activity. All the mutants of Trp153 and of Lys154 with like size/charge substitution were also found to be showing error prone DNA synthesis. The similar results were also obtained with rNTPs incorporation. The implications of these findings may be explained in the context of the 3D-crystal structures of HIV-1 RT.
Conclusions: The in vitro results indicate that the wild type and some mutants of HIV-1 RT may synthesise RNA.
4th IAS Conference on HIV Pathogenesis, Treatment and Prevention
Abstract no.
WEPEA020
Suggested Citation
" Sharma B.
Human immunodefficiency virus Type-1 reverse transcriptase (HIV-1RT) exhibits RNA polymerae Activity.
Poster exhibition:
4th IAS Conference on HIV Pathogenesis, Treatment and Prevention:
Abstract no.
WEPEA020"
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