Abstract
The last decade has contributed to our understanding of the three-dimensional structure of the human immunodeficiency virus, type 1 (HIV-1) integrase (IN) and to the description of how the enzyme catalyzes the viral DNA integration into the host DNA. Recognition of the viral DNA termini by IN is sequence-specific, and that of the host DNA does not require particular sequence, although in physicochemical studies IN fails to discriminate between the two interactions. Here, such discrimination was allowed thanks to a model system using designed oligonucleotides and peptides as binding structures. Spectroscopic (circular dichroism, NMR, and fluorescence anisotropy) techniques and biochemical (enzymatic and filter binding) assays clearly indicated that the amphipathic helix alpha4, located at the catalytic domain surface, is responsible for the specific high affinity binding of the enzyme to viral DNA. Analogues of the alpha4 peptide having increased helicity and still bearing the biologically relevant lysines 156 and 159 on the DNA binding face, and oligonucleotides conserving an intact attachment site, are required to achieve high affinity complexes (Kd of 1.5 nm). Data corroborate previous in vivo results obtained with mutated viruses.
Highlights
The last decade has contributed to our understanding of the three-dimensional structure of the human immunodeficiency virus, type 1 (HIV-1) integrase (IN) and to the description of how the enzyme catalyzes the viral DNA integration into the host DNA
20 years into the human immunodeficiency virus (HIV)1/AIDS epidemic, an estimated 40 million people worldwide are currently living with the virus, and some 20 million people have already died [1, 2]
Peptide ␣4 reproduces the natural helix ␣4 sequence of the catalytic domain; K156 derives from ␣4 through selected helix stabilizing mutations, E156 is obtained from K156 through replacement of Lys-156 with Glu, and E159 results from substitution of Lys-159 with Glu
Summary
20 years into the human immunodeficiency virus (HIV)1/AIDS epidemic, an estimated 40 million people worldwide are currently living with the virus, and some 20 million people have already died [1, 2]. Treatment of AIDS still requires the development of effective inhibitors of HIV replication [5,6,7]. Those targeted to reverse transcriptase and protease have demonstrated their efficiency in antiviral therapy. The free terminal 3Ј-hydroxyl groups attack the targethost DNA, and the viral cDNA is integrated by a transesterification reaction into the cell genome [14]. The C-terminal domain, less conserved, contains an SH3 fold [28, 30]. The catalytic domain contains five  strands surrounded by six ␣ helices, numbered from one to six, as well as a highly conserved catalytic D, DX35E motif embedded in a protein RNase H fold (19 –22). Peptides deriving from helix ␣4 behave as competitive inhibitors of IN, and this is true for monospecific antibodies raised against a peptide containing ␣4 [43,44,45,46,47]
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