The HIV-1 Integrase Monomer Induces a Specific Interaction with LTR DNA for Concerted Integration

• Published in: Biochemistry (Easton) 2011-11, Vol.50 (45), p.9788-9796
• Main Authors: Pandey, Krishan K, Bera, Sibes, Grandgenett, Duane P
• Format: Article
• Language: English
• Subjects: Base Sequence; Foamy virus; HIV Integrase - chemistry; HIV Integrase - genetics; HIV Integrase - physiology; HIV Long Terminal Repeat - genetics; HIV Long Terminal Repeat - physiology; HIV-1 - genetics; HIV-1 - physiology; Human immunodeficiency virus 1; Humans; Protein Structure, Quaternary; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Substrate Specificity; Virus Integration - genetics; Virus Integration - physiology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi201247f

The assembly mechanism for the human immunodeficiency virus type 1 (HIV) synaptic complex (SC) capable of concerted integration is unknown. Molecular and structural studies have established that the HIV SC and prototype foamy virus (PFV) intasome contain a tetramer of integrase (IN) that catalyzes concerted integration. HIV IN purified in the presence of 1 mM EDTA and 10 mM MgSO4 was predominately a monomer. IN efficiently promoted concerted integration of micromolar concentrations of 3′-OH recessed and blunt-ended U5 long terminal repeat (LTR) oligonucleotide (ODN) substrates (19–42 bp) into circular target DNA. Varying HIV IN to U5 DNA showed that an IN dimer:DNA end molar ratio of 1 was optimal for concerted integration. Integration activities decreased with an increasing length of the ODN, starting from the recessed 18/20 or 19/21 bp set to the 31/33 and 40/42 bp set. Under these conditions, the average fidelity for the HIV 5 bp host site duplication with recessed and blunt-ended substrates was 56%. Modifications of U5 LTR sequences beyond 21 bp from the terminus on longer DNA (1.6 kb) did not alter the ∼32 bp DNaseI protective footprint, suggesting viral sequences beyond 21 bp were not essential for IN binding. The results suggest IN binds differentially to an 18/20 bp than to a 40/42 bp ODN substrate for concerted integration. The HIV IN monomer may be a suitable candidate for attempting crystallization of an IN–DNA complex in the absence or presence of strand transfer inhibitors.