Integration of viral DNA into host DNA is an essential step in the replication cycle of retroviruses and retrotransposons (4
). The first two steps in this process, viral DNA processing and joining, are catalyzed by the retroviral protein integrase and require specific sequences at the ends of the viral DNA. In the first step, nucleotides (usually two) are removed from the 3′ ends of the viral DNA, and in the second step, these newly created ends are joined to staggered phosphates in the complementary strands of the host cell DNA, creating an integration intermediate with gaps in the DNA sequence. Complete, stable integration of the viral DNA depends on repair of these gaps: filling in the missing nucleotides, removing the short flap on the 5′ ends of the viral DNA, ligation to the newly synthesized 3′ end of host DNA and, likely, reconstitution of appropriate chromatin structure and composition, all by mechanisms not yet fully understood.
At each step, the retroviral DNA integration process may be assisted by host cellular proteins, including those that participate in DNA repair. Mechanistically, retroviral DNA integration resembles RAG1/2-mediated V(D)J recombination, the process by which immunoglobulin genes are assembled (5
). Successful completion of V(D)J recombination depends on proteins that make up the nonhomologous end joining (NHEJ) DNA repair pathway, including the DNA-dependent protein kinase (DNA-PK), which consists of a large, phosphatidylinositol 3-kinase-related catalytic subunit (DNA-PKCS
) and a DNA binding heterodimer Ku70/Ku80. We have shown that DNA-PKCS
mouse pre-B cells undergo apoptotic cell death in response to infection by avian sarcoma virus (ASV) or human immunodeficiency virus type 1 (HIV-1) retroviral vectors and that this cell death requires an active viral integrase (9
). We also showed that the ability of an ASV vector to stably transduce selectable markers is reduced by 80 to 90% in scid
fibroblasts and other adherent cells that bear mutations in three components of the NHEJ DNA repair pathway: DNA-PKCS
, Ku80, and Xrcc4 (9
). Therefore, we have proposed that the NHEJ DNA repair pathway is required for efficient completion of the retroviral DNA integration process and that failure in repair of the integration intermediate triggers growth arrest or cell death. Others have shown that yeast Ku80 and Ku70 proteins are required for Ty1 retrotransposition in Saccharomyces cerevisiae
) and that HIV-1 replication and integration are reduced in Ku80-deficient human cells (16
). In addition, we have reported that two DNA-PKCS
-related kinases, ATM and ATR, also participate in the retroviral DNA integration process (7
). However, whereas ATM is required only for the residual integration observed in the DNA-PK-deficient cells, ATR appears to be required even in the presence of DNA-PKCS
). These results suggest that integrase-mediated joining of viral to host DNA is sensed as DNA damage by the cell and that the viral DNA integration process has evolved to exploit the cellular DNA damage response and double-strand break DNA repair pathways.
Another group of investigators (1
) has reported that DNA-PK is not required for efficient integration by lentiviral vectors. These researchers found that infection by HIV-1-based vectors induced death of DNA-PKCS
-deficient mouse embryo fibroblasts (MEFs) and other NHEJ-deficient cell lines; however, they did not observe deficiencies in stable transduction when cells were infected at a low multiplicity. In addition, a second group (20
) reported that retroviral infection induces death in a human lymphoid cell line lacking another NHEJ protein, ligase IV. In these experiments, cell death was also induced by a vector carrying an integration-defective integrase; furthermore, an inhibitor of reverse transcriptase suppressed cell death. Therefore, these researchers suggested that the presence of unintegrated viral DNA, rather than an unrepaired integration intermediate, is the causative factor in retrovirus-induced death of NHEJ-deficient cells.
In the experiments presented herein, we measured the transduction efficiency of HIV-1-based vectors with DNA-PKCS-deficient scid MEFs and also examined the viability of a ligase IV-deficient lymphoid cell line after infection with such vectors. Our experiments reveal a striking deficiency in stable transduction of scid MEFs and an Xrcc4-deficient cell line with these lentiviral vectors compared to control cells. We also demonstrate that the transduction deficiency is overcome in scid cells in which a functional DNA-PKCS gene had been introduced. Reduced HIV-1 vector-mediated transduction of the ligase IV-deficient lymphoid cells was also observed, but we found that the vector did not induce cell killing if it carried an inactive integrase. Several possible explanations for the discrepancies noted above are addressed. Data provided in this study lend further support for our proposal that components of the NHEJ DNA repair pathway are required for efficient completion of the retroviral DNA integration process and the survival of cultured cells in which a chromosome has been damaged by integrase-mediated insertion of viral DNA.