Suppression of retroviral infection by the RAD52 DNA repair protein

Abstract

Reverse transcription of retroviral RNA into linear double-stranded DNA and its integration into the host cell genome are essential steps in the retroviral life cycle. The nonhomologous end-joining (NHEJ) DNA repair pathway has been implicated in protecting cells from retrovirus-induced apoptosis caused by strand breaks in host cell DNA or unintegrated linear viral DNA. In eukaryotes, both the NHEJ and homologous recombination (HR) pathways play important roles in repairing DNA double-strand breaks. Here we show that the HR repair protein RAD52 modulates the outcome of recombinant HIV-l vector infection by markedly reducing the efficiency of productive integration events. Increased retroviral integration is the first major phenotype described for a RAD52 deficiency in mammalian cells. Mutations in other HR proteins (XRCC2, XRCC3 and BRCA2) do not markedly affect retroviral transduction rates, suggesting that the HR repair pathway per se does not influence retroviral infection. Instead, the mechanism of attenuation of retroviral infection by RAD52 appears to be based upon competition between the RAD52 protein and active integration complexes for the retroviral cDNA genome.

Figure 1

RAD52 (but not HR in general) is involved in modulating retroviral infection. (A) HIV-1 transduction assay results for XRCC2 (IRS1)- and XRCC3 (IRS1-SF)-defective hamster cell lines infected with HIV-1 luciferase retrovirus stocks. (B) HIV-1 luciferase transduction assays of BRCA2-deficient (Capan-1) human pancreatic cells. (C) HIV-1 luciferase transduction assay results for RAD52-deficient mouse ES cells. (D) HIV-1 luciferase transduction assay results for HeLa cells transfected with the siRNAs directed against RAD52. The assay itself is described in more detail in Supplementary information.

Figure 2

Overexpression of RAD52 impairs retroviral integration. (A) Parental HeLa cells (HeLa) and clones stably transfected with vector DNA only (IRES-1 and -2) or stably overexpressing HA-RAD52 (RAD52-1 through -5) infected with HIV-1 luciferase retroviral stocks. HIV-1 luciferase transduction assay results are shown in the left graph and immunoblot analysis of the HeLa cell clones are on the right. Luciferase transduction assay results are expressed as luciferase activity relative to parental HeLa cells. Western blots of HeLa cell clones were performed using antibodies raised against the HA tag epitope, RAD52, Ku70, Ku80 and β-actin (loading control). (B) Quantitative real-time PCR analysis of late RT HIV-1 DNA isolated from Rad52^−/− ES cells and HeLa cell clones overexpressing HA-RAD52 at 21 days after infection. The standard curve for quantification was generated from HeLa cell DNA isolated 24 h after infection (left panel). The amount of late RT HIV-1 DNA in each cell line (pg per 50 ng genomic DNA), determined from the standard curve, is shown in the right panel. UD: undetectable.

Figure 3

The DNA-binding domain of RAD52 is required for inhibition of retroviral infection. (A) Schematic of HA-tagged RAD52 and deletion mutants showing functional domains (DNA: DNA-binding domain; RAD52: RAD52 self-association domain; RPA: RPA-binding domain; RAD51: RAD51-binding domain; NLS: nuclear localisation signal, with amino-acid residues numbered). (B) 293 cells transiently transfected with full-length (FL) RAD52 and RAD52 deletion mutant expression plasmids subsequently infected with HIV-1 luciferase retroviral stocks. HIV-1 luciferase transduction results (left) and immunoblot analysis of cell lysates (right) are shown. Results are expressed as luciferase activity relative to untransfected 293 cells. Immunoblots were performed using anti-HA tag antibodies before re-probing for β-actin (loading control). (C) Chromatin immunoprecipitation (ChIP) analysis of 293 cells transiently transfected with FL-RAD52 or RAD52 deletion mutant expression plasmids and infected with HIV-1 luciferase retroviral stocks. Levels of HIV-1 DNA physically associated with HA-RAD52 were determined by immunoprecipitation with anti-HA antibodies and PCR performed using primers against HIV-1 LTR sequences (upper panel) or genomic GAPDH sequences (lower panel). Nonspecific control immunoprecipitations were performed using either no antibody (−) or an IgG1 isotype control anti-FLAG tag antibody. For reference, 0.5% of the total amount of HIV-1 LTR DNA formed during a typical infection is also shown (INPUT).

Figure 4

RAD52 can compete with Ku for binding to HIV-1. (A) 293 cells transiently transfected with an increasing amount of HA-RAD52 expression plasmid, then infected with HIV-1 luciferase retroviral stocks. HIV-1 luciferase transduction assay results are shown (left) along with immunoblot analysis of cell lysates (right). Results are expressed as luciferase activity relative to untransfected 293 cells. Immunoblots were performed using anti-HA tag antibodies before re-probing for β-actin (loading control). (B) Competitive PCR–ChIP analysis of 293 cells transiently transfected with increasing amounts of HA-RAD52 expression plasmid and infected with HIV-1 luciferase retroviral stocks. Results show HIV-1 LTR PCRs for ChIPs using antibodies against the HA-tag, Ku80 and nonspecific controls (no antibody (−) and IgG1 isotype control anti-FLAG tag antibody). PCR amplifications were normalised to the amount of HIV-1 LTR DNA used per ChIP (INPUT DNA). (C) Immunoblot analysis of input extracts used to perform the ChIP assays. The amount of HA-RAD52 or Ku80 protein used per ChIP analysis was determined by immunoblotting using anti-HA tag or anti-Ku80 antibodies.

Figure 5

2-LTR circle DNA formation but not apoptosis is modulated by RAD52 expression. Semiquantitative PCR analysis of DNA extracted from HIV-1-infected cells with different levels of RAD52 expression. (A) 2-LTR PCR analysis of wild-type (Rad52^+/+; 1B10) and Rad52^−/− mouse ES cells. (B) 2-LTR PCR analysis of 293 cells transfected with either full-length HA-RAD52 (RAD52 FL) or DNA-binding deletion mutant HA-RAD52 (RAD52 Δ43–177) expression plasmids. All PCR quantification results are expressed as a normalised ratio of 2-LTR DNA:GAPDH control DNA. HeLa and two RAD52-overexpressing stable cells clones (RAD52-3 and RAD52-5) were infected with HIV-1 and apoptosis was quantified by annexin-V staining and flow cytometry. Live/dead cell discrimination was also performed by counter-staining with propidium iodide (PI). (C) The percentage of cells undergoing the early stages of apoptosis at increasing time points after infection with HIV-1 luciferase retrovirus stocks. (D) The percentage of both dead and apoptotic cells (all high annexin-V-stained cells) at increasing time points after HIV-1 luciferase retrovirus infection.