Quantitation of HIV DNA integration: effects of differential integration site distributions on Alu-PCR assays.
In many studies of HIV replication, it is useful to quantify the number of HIV proviruses in cells against a background of unintegrated forms of the HIV DNA. A popular method for doing so involves quantitative PCR using one primer complementary to the HIV long terminal repeat (LTR), and a second primer complementary to a cellular Alu repeat, so that PCR product only forms from templates where a provirus is integrated in the human genome near an Alu repeat. However, several recent studies have identified conditions that alter distributions of HIV integration sites relative to genes. Because Alu repeats are enriched in gene rich regions, this raises the question of whether altered integration site distributions might confound provirus abundance measurements using the Alu-PCR method. Here modified versions of the HIV tethering protein LEDGF/p75 were used to retarget HIV integration outside of transcription units, and show that this has a negligible effect on Alu-PCR quantitation of proviral abundance. Thus altered integration targeting, at least to the degree achieved here, is not a major concern when using the Alu-PCR assay.
University of Pennsylvania Perelman School of Medicine, Department of Microbiology, 3610 Hamilton Walk, Philadelphia, PA 19104-6076, United States.
Troy
T
Brady
Brendan J
BJ
Kelly
Frances
F
Male
Shoshannah
S
Roth
Aubrey
A
Bailey
Nirav
N
Malani
Rik
R
Gijsbers
Una
U
O'Doherty
Frederic D
FD
Bushman
D020087:Alu Elements / Q000235:genetics*; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics*; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics*; D011533:Proviruses / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering; D016662:Virus Integration
D004279:DNA, Viral; D036341:Intercellular Signaling Peptides and Proteins; D034741:RNA, Small Interfering; C119380:lens epithelium-derived growth factor