Abstract
We investigated which features of the substrate specificity of human immunodeficiency virus type 1 (HIV-1) integrase could be assigned to the central domain of the 288-residue HIV-1 integrase protein, composed of amino acids 50-212. This domain contains the active site and shares structural homology with a large family of polynucleotidyl transferases. Using model substrates with defined alterations in critical features we found that this domain alone is sufficient for recognition of: 1) the phylogenetically conserved CA/TG base pairs near the viral DNA end; 2) the 5'-terminal dinucleotide that is left unpaired after end processing; and 3) target DNA flanking the site of joining. Future efforts aimed at identifying specific amino acids involved in recognition of these key substrate features can now be targeted at this domain.
Highlights
Integration of a double-stranded DNA copy of the retroviral genome into a host cell chromosome is essential for viral replication
The Catalytic Core Domain Recognizes the Gross Structural Features of the Viral DNA End—Y-mer disintegration substrates with altered viral DNA portions were constructed to investigate the sensitivity of the HIV-1 catalytic core domain to gross structural alterations in the viral DNA
The Catalytic Core Domain Can Recognize the Conserved CA/TG Dinucleotide Pair at the Viral DNA End—To determine whether the core domain could recognize the conserved CA/TG bps of the viral DNA end, we measured the catalytic activity of the core domain on Y-mer disintegration substrates with base substitutions in the phylogenetically conserved A/T bp (Fig. 2A) or C/G bp (Fig. 2B)
Summary
A deletion construct containing amino acids 50 –212 of HIV-1 integrase in the expression strain BL21 was a gift from Tim Jenkins. The pellet was resuspended in 50 ml of 20 mM Tris-HCl, pH 8.0, 1 M NaCl, 2 mM -mercaptoethanol (TNM) containing 5 mM imidazole, processed 30 times in a Dounce homogenizer This high salt extract was stirred at 4 °C for 1 h, centrifuged at 28,000 rpm in an SW28 rotor for 1 h at 4 °C. The thrombin-digested material was loaded onto a 1-ml DEAE-Sepharose (Pharmacia Biotech Inc.) column This column was washed with 10 column volumes of buffer (HCDG) containing 50 mM HEPES, pH 7.5, 10 mM CHAPS, 2 mM dithiothreitol, 10% glycerol with 100 mM NaCl. One ml each of HCDG containing 250 mM NaCl, 500 mM NaCl, 750 mM NaCl, or 1 M NaCl was added sequentially, and 1-ml fractions were collected. The additional oligonucleotide sequences used to make the U3 viral DNA end Y-mer disintegration substrates were as follows. Sequences of oligonucleotides used to test the dependence of the core domain on target DNA were as follows. 40-mer, dby, Standard Dumbbell—5Ј-ACTGCTAGTTCTAGCAGGCCCTTGGGCCGGCGCTTGCGCC-3Ј. 48-mer, dby4ϩ2—5Ј-ACTGCTAGTTCTAGCAGGCCCCATTTGGGGCCGGCGCTTTTAAGCGCC-3Ј. 56-mer, dby4ϩ4 —5Ј-ACTGCTAGTTCTAGCAGGCCCCAGGTTGGTGGGGCCGGCGCTTGCTTGCAGCGCC-3Ј. 64-mer, dby4ϩ6 —5Ј-ACTGCTAGTTCTAGCAGGGCCCCAGGTCTTGACCTGGGGCCGGCGCTTGCGTTTACGCAAGCGCC-3Ј. 80-mer, dby4ϩYT—5Ј-ACTGCTAGTTCTAGCAGGCTGCAGGTCGACTTGTCGACCTGCAGCCCAAGCTTGCGTTGCTGTTCAGCAACGCAAGCTTG-3Ј. 66-mer, dby4ϩYV—5Ј-ACTGCTAGAGATTTTCCACATTTATGTGGAAAATCTCTAGCAGGCCCTTGGGCCGGCGCTTGCGCC-3Ј. 52-mer, dby4ϩLL—5Ј-ACTGCTAGTTCTAGCAGGCTGCAGGTCGACTTGTCGACCTGCAGCCCGTTCG-3Ј. 52-mer, dby4ϩLR—5Ј-ACTGCTAGTTCTAGCAGCTTGCCAAGCTTGCGTTGCTTGCAACGCAAGCTTG-3Ј
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