Human immunodeficiency virus reverse transcriptase expressed in transformed yeast cells. Biochemical properties and interactions with bovine tRNALys.
Marie-Line Sallafranque-Andreola,Dominique Robert,Philip J. Barr,Michel Fournier,Simon Litvak,Leila Sarih-Cottin,Laura Tarrago-Litvak +6 more
TL;DR: A complex between the human retroviral enzyme and the bovine t RNALys was shown, using a direct approach, by glycerol gradient centrifugation, as well as by the protective and specific effect of the tRNALys against enzyme inactivation by thermal denaturation and trypsin digestion.
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Abstract: Human immunodeficiency virus (HIV) reverse transcriptase has been purified from yeast transformed by an autoreplicating plasmid containing the retroviral DNA polymerase gene The previously described purification procedure for the yeast-expressed reverse transcriptase [Barr, P J, Power, M D, Chun Ting Lee-Ng, Gibson, H & Luciw, P (1987) Bio/Technology 5, 486–489] has been substantially modified, leading to an increased yield and a higher degree of purity Several biochemical properties of the enzyme are described (template specificity, effect of DNA synthesis inhibitors); interestingly, HIV reverse transcriptase is highly resistant to N-ethylmaleimide A complex between the human retroviral enzyme and the bovine tRNALys was shown, using a direct approach, by glycerol gradient centrifugation, as well as by the protective and specific effect of the tRNALys against enzyme inactivation by thermal denaturation and trypsin digestion A competitive type of inhibition of HIV reverse transcriptase by tRNALys, but not by tRNAVal, is observed when viral RNA or activated DNA are used as templates
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RNA pseudoknots that inhibit human immunodeficiency virus type 1 reverse transcriptase
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Construction of a type 1 human immunodeficiency virus that maintains a primer binding site complementary to tRNA(His).
TL;DR: This study is the first report of a stable human immunodeficiency virus type 1 which utilizes an alternative tRNA primer and suggests that interactions between the primer tRNA anticodon loop and viral sequences upstream of the PBS contribute to the specificity of the t RNA primer used in reverse transcription.
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Human immunodeficiency virus type 1 can use different tRNAs as primers for reverse transcription but selectively maintains a primer binding site complementary to tRNA(3Lys).
TL;DR: Results suggest that factors other than complementarity between the PBS and the primer tRNA contribute to the selectivity of tRNA3(Lys) to initiate HIV-1 reverse transcription.
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