134   A Sensitive Integration Assay Using HIV-1 Preintegration Complexes (PICs), Concatameric Target DNA, and PCR Quantification of Integration Junctions.

A. Brooun*, D. Richman, and R. Kornbluth.
Univ. of California, San Diego and VA San Diego Healthcare System, La Jolla, CA.

Background:Previously, studies on PICs have been impeded by the lack of a sensitive and facile assay of their ability to integrate into target DNA in vitro .

Methods:Cytoplasmic detergent extracts containing PICs were prepared from SupT1 cells acutely infected by HIV-1 LAI. 10ml of PIC extract was added to a concatameric target DNA consisting of 32 head-to-tail repeats of a 105-bp sequence. Samples were deproteinized with proteinase K, then 8 exonuclease was added to deplete unreacted target DNA (which interferes with the subsequent PCR step). Real-time PCR for integration junctions used primers in the 3´ LTR, the target DNA, and a TaqMan probe for the 3´. (Alternatively, an ultrasensitive, end- point dilution assay was constructed using detection by heminested PCR and gel electrophoresis.)

Results:As a target DNA, a 32-mer concatamer (~3,300 bp) detected 2-6× more PICs than did an equal amount of monomeric units (105 bp), indicating that PICs prefer high-m.w. target DNAs. The TaqMan assay was used to measure PIC concentration in HIV-1-infected cells, to guide the purification of PICs, and to quantify the inhibition of PIC integration by known inhibitors of HIV-1 integrase. Using inactive, salt-stripped PICs, the assay was also used to detect host cell factors that restore PIC activity. Interestingly, extracts from CD3/CD28- stimulated, primary CD4⁺T cells activated these salt-stripped PICs, whereas extracts from resting CD4⁺T cells did not.

Conclusions:A sensitive and facile assay of PIC integration in vitro has been constructed which can be used for studies on HIV replication, the regulation of host cell factors needed for integration, and the development of new antiviral agents.