DNA damage sensors ATM, ATR, DNA-PKcs, and PARP-1 are dispensable for human immunodeficiency virus type 1 integration

Abstract

Integration of a DNA copy of the viral RNA genome is a crucial step in the life cycle of human immunodeficiency virus type 1 (HIV-1) and other retroviruses. While the virally encoded integrase is key to this process, cellular factors yet to be characterized are suspected to participate in its completion. DNA damage sensors such as ATM (ataxia-telangiectasia mutated), ATR (ATM- and Rad3-related), DNA-PK (DNA-dependent protein kinase), and PARP-1 [poly(ADP-ribose) polymerase 1] play central roles in responses to various forms of DNA injury and as such could facilitate HIV integration. To test this hypothesis, we examined the susceptibility to infection with wild-type HIV-1 and to transduction with a vesicular stomatitis virus G protein (VSV-G)-pseudotyped HIV-1-derived lentiviral vector of human cells stably expressing small interfering RNAs against ATM, ATR, and PARP-1. We found that integration normally occurred in these knockdown cells. Similarly, the VSV-G-pseudotyped HIV-1-based vector could effectively transduce ATM and PARP-1 knockout mouse cells as well as human cells deficient for DNA-PK. Finally, treatment of target cells with the ATM and ATR inhibitors caffeine and wortmannin was without effect in these infectivity assays. We conclude that the DNA repair enzymes ATM, ATR, DNA-PKcs, and PARP-1 are not essential for HIV-1 integration.

FIG. 1.

Both ATM and ATR are dispensable for HIV-1 integration. (a) Inhibition of ATM and/or ATR expression by shRNA-producing lentiviral vectors. Western blotting of cellular lysates with anti-ATM, anti-ATR, and anti-Chk2 antibodies in ATM- (ATMi), ATR- (ATRi), and double-knockdown (DKD) P4.2 cells as well as in P4.2 cells transduced with a control (C) lentiviral vector results are shown. (b) Transduction efficiency of VSV-G-pseudotyped GFP-expressing HIV-1-derived lentiviral vector in control, ATM, ATR, and DKD cells. Two days after transduction at an MOI of 0.1, transduction efficiency was determined by FACS. For this and other figures, experiments were done in duplicate and columns represent the mean percentage of transduced cells, with mean fluorescence intensity (MFI) indicated below. (c) Single-round wild-type HIV-1 infectivity in the same cells, using an MOI of 0.005. The LTR-LacZ-containing cells produce β-galactosidase upon Tat production from integrated proviruses, which is revealed by 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside staining (performed 2 days after infection). (d) Transduction susceptibility of ATM wild-type (+/+) or ATM-knockout (−/−) MEFs, determined by FACS 2 days after exposure to GFP-expressing HIV-derived vector at the indicated MOI.

FIG. 2.

Effect of caffeine and wortmannin on HIV-1 infection. (a) Single-round HIV-1 infectivity in P4.2 cells treated with 4 mM caffeine (dissolved in water) or 4 μM wortmannin (dissolved in dimethyl sulfoxide). Drugs were added 1 h prior to infection, and 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside staining was performed 2 days later. (b) Transduction efficiency of VSV-G-pseudotyped GFP-expressing HIV-1 vector under similar conditions.

FIG. 3.

Transduction efficiency of GFP-expressing VSV-G-pseudotyped HIV-1-derived vector in human DNA-PKcs wild-type (+/+) M059K or DNA-PKcs-deficient (−/−) M059J cells.

FIG. 4.

(a) Transduction efficiency of HIV-derived GFP-expressing lentiviral vector in PARP-1 wild-type MEF (+/+) and PARP-1 knockout MEF (−/−) cells, using the indicated MOIs. (b) Similar experiment in PARP-1 wild-type (+/+) and PARP-1 knockout (−/−) ES cells.

FIG. 5.

(a) Inhibition of PARP-1 expression by small interfering RNAs. Results shown are from Western blotting of lysates from P4.2 cells transduced with control (C) or anti-PARP-1 shRNA-expressing lentiviral vectors, using antibodies specific for PARP-1 or α-tubulin. (b and c) The same cells were then used to score their susceptibility to infection with wild-type HIV-1 (b) and transduction with a GFP-expressing VSV-G-pseudotyped HIV-1-derived vector (c).