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Abstract
On the Role of b8-aE Loop of Human Immunodefficiency Virus TYPE-1 Reverse Transcriptase (HIV-1RT) Replication
Sharma B.1
Introduction: HIV-1 RT is known to catalyze the synthesis of viral cDNA which gets integrated into the human genome. It has RNA / DNA dependent DNA polymerase as well as RNase H activities. The highly conserved amino acid residues on the LPQG motif of the â8-áE loop in the larger subunit (p66) of heterodimeric (p66/p51) HIV-1 RT are critical for RT function as well as to confer nucleoside drug resistance and fidelity to the enzyme. In order to ascertain the role of some amino acid residues, we decided to study Trp153 and Lys154 by the site-directed mutagenesis and their mutant derivatives were biochemically characterized.
Methods: Site directed mutagenesis was carried out by PCR and the muation positive clones were expressed in suitable media. The proteins were extracted from the cells and purified to homogeneity using Ni2+ chelated metalloaffinity column chromatographic technique.
Results: . 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 containing opposite charge or different size exhibited drastic alterations in the polymerase activity. All the mutants of Trp153 and of Lys154 with like size/charge substitution were found to be showing error prone DNA synthesis. The mutants of Lys154 containing opposite charge were more stringent in recruiting the correct versus incorrect nucleotides and displayed greater fidelity in DNA synthesis. The similar results were also obtained with rNTPs incorporation. These results indicated the involvement of Trp153 and Lys154 in conferring fidelity of DNA synthesis catalyzed by HIV-1 RT. The implications of these findings are discussed in the context of the binary and ternary crystal structures of HIV-1 RT.
Conclusions: The conservative mutations at Trp153 and Lys154 positions showed contrary responses to the antiHIV-1 compound (3TC), the former being sensitive whereas the later was resistant to 3TCTP.
The 3rd IAS Conference on HIV Pathogenesis and Treatment
Abstract no.
TuPe4.2B01
Suggested Citation
" Sharma B.
On the Role of b8-aE Loop of Human Immunodefficiency Virus TYPE-1 Reverse Transcriptase (HIV-1RT) Replication.
Poster Exhibition:
The 3rd IAS Conference on HIV Pathogenesis and Treatment:
Abstract no.
TuPe4.2B01"
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