HIV-1 integrase strand transfer inhibitors stabilize an integrase-single blunt-ended DNA complex |
| |
Authors: | Bera Sibes Pandey Krishan K Vora Ajaykumar C Grandgenett Duane P |
| |
Affiliation: | Saint Louis University Health Sciences Center, Institute for Molecular Virology, Doisy Research Center, 1100 South Grand Boulevard, St. Louis, MO 63104, USA |
| |
Abstract: | Integration of human immunodeficiency virus cDNA ends by integrase (IN) into host chromosomes involves a concerted integration mechanism. IN juxtaposes two DNA blunt ends to form the synaptic complex, which is the intermediate in the concerted integration pathway. The synaptic complex is inactivated by strand transfer inhibitors (STI) with IC50 values of ∼ 20 nM for inhibition of concerted integration. We detected a new nucleoprotein complex on a native agarose gel that was produced in the presence of > 200 nM STI, termed the IN-single DNA (ISD) complex. Two IN dimers appear to bind in a parallel fashion at the DNA terminus, producing an ∼ 32-bp DNase I protective footprint. In the presence of raltegravir (RAL), MK-2048, and L-841,411, IN incorporated ∼ 20-25% of the input blunt-ended DNA substrate into the stabilized ISD complex. Seven other STI also produced the ISD complex (≤ 5% of input DNA). The formation of the ISD complex was not dependent on 3′OH processing, and the DNA was predominantly blunt ended in the complex. The RAL-resistant IN mutant N155H weakly forms the ISD complex in the presence of RAL at ∼ 25% level of wild-type IN. In contrast, MK-2048 and L-841,411 produced ∼ 3-fold to 5-fold more ISD than RAL with N155H IN, which is susceptible to these two inhibitors. The results suggest that STI are slow-binding inhibitors and that the potency to form and stabilize the ISD complex is not always related to inhibition of concerted integration. Rather, the apparent binding and dissociation properties of each STI influenced the production of the ISD complex. |
| |
Keywords: | IN, integrase STI, strand transfer inhibitors ISD, IN-single DNA RAL, raltegravir HIV, human immunodeficiency virus PIC, preintegration complex LTR, long terminal repeat SC, synaptic complex STC, strand transfer complex PFV, prototype foamy virus EVG, elvitegravir CHS, circular half site SPA, scintillation proximity assay wt, wild type H-SC, higher-order synaptic complex BS3, bis(sulfosuccinimidyl)suberate EDTA, ethylenediaminetetraacetic acid |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|