Integration complexes derived from HIV vectors for rapid assays in vitro

Abstract

Of three enzymes encoded by HIV-reverse transcriptase, protease, and integrase-only the first two have been exploited clinically as inhibitor targets. Efforts to develop inhibitors of purified integrase protein have yielded many compounds, but none with clinical utility. A different source of integration activity for studies in vitro is provided by replication intermediates isolated from HIV-infected cells. These preintegration complexes (PICs) can direct integration of the endogenously synthesized viral cDNA into an added target DNA in vitro. Despite their authentic activities, assays of PICs have not been widely used due to technical obstacles, particularly the requirement for handling large amounts of infectious HIV. Here, we describe greatly improved methods for producing PICs using HIV-based vectors that are capable of establishing an integrated provirus but not a spreading infection. We also report the development of a PIC integration assay using DNA-coated microtiter plates, which speeds assays of PIC integration in vitro. We used this method to screen a library of chemicals related to known integrase inhibitors and found a new compound, quinalizarin sulfate, that displayed enhanced activity against PICs.

Identification of RFPL3 Protein as a Novel E3 Ubiquitin Ligase Modulating the Integration Activity of Human Immunodeficiency Virus, Type 1 Preintegration Complex Using a Microtiter Plate-based Assay

“…5 mg/ml of proteinase K and 5% SDS were added to stop the reaction, with incubation at 37°C for another hour. Wells were then washed five times with buffer A at 65°C (with the third wash done at 68°C for 20 min) and five times with buffer K. Integrated DNA products were detached from the plate by incubating with 30 l of 0.04 N NaOH at 50°C for 10 min before neutralizing with 30 l of 0.04 N HCl and 50 mM HEPES (pH 7.5) (17). Quantitative PCR (qPCR) for the detection of an eluted HIV-1 DNA product was performed using a primer pair (AA55 and M667) amplifying the R-U5 region of the LTR and a specific fluorogenic probe (HIV-FAM) as described previously (24).…”

“…Preparation of the Target DNA-coated Microtiter PlateImmobilization of target DNA to a 96-well plate was performed as described previously (17), with slight modifications. 1 g of pUC19 plasmid (2.7 kb) that had been linearized with EcoRI was suspended in 75 l of 20 mM 1-methyl-imidazole (pH 7.0) (Sigma) and 200 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Sigma) and added to a Covalink amine-coated plate (Corning) that had been prewashed three times with 20 mM 1-methyl-imidazole (pH 7.0).…”

“…The development of a rapid and efficient HIV-1 PIC integration assay using DNA-coated microtiter plates was first described in a study by Hansen et al (17) in which a library of chemical compounds with structures related to known IN inhibitors had been screened for the ability to inhibit PIC integration activity in vitro. For the purpose of our study, the microtiter plate-based PIC integration assay was adapted for a screen with recombinant proteins to identify potential cellular modulators of the HIV-1 PIC.…”

“…On the other hand, VRK1 has been shown A PIC sample (25 l) was incubated in the well of a microtiter plate in which 2.7-kb target DNA was covalently immobilized at 37°C for 30 min. The reaction was then inactivated by treatment with proteinase K and SDS, and, after washing, integrated DNA were released using NaOH to be quantified via qPCR using LTR-specific primers (17). Inactivated PICs were prepared by pretreatment with proteinase K/SDS before being subjected to the microtiter plate-based PIC assay to check the background level of viral DNA.…”

“…To this end, we employed a microtiter plate-based in vitro assay reported previously for the HIV-1 PIC that allows for a rapid measure of PIC integration activity on a 96-well plate, thus enabling the handling of large scale sample sets (17). In addition, as a novel aspect of our study, the microtiter plate-based PIC integration assay was combined with a library of human proteins that had been produced using wheat germ cell-free technology to screen for new cellular modulators of HIV-1 PIC integration activity in vitro (18).…”