Interaction of HIV-1 Integrase with DNA Repair Protein hRad18
2002; Elsevier BV; Volume: 277; Issue: 30 Linguagem: Inglês
10.1074/jbc.m203061200
ISSN1083-351X
AutoresLubbertus C. F. Mulder, Lisa A. Chakrabarti, Mark A. Muesing,
Tópico(s)Biochemical and Molecular Research
ResumoWe have previously shown that human immunodeficiency virus-1 (HIV-1) integrase is an unstable protein and a substrate for the N-end rule degradation pathway. This degradation pathway shares its ubiquitin-conjugating enzyme, Rad6, with the post-replication/translesion DNA repair pathway. Because DNA repair is thought to play an essential role in HIV-1 integration, we investigated whether other molecules of this DNA repair pathway could interact with integrase. We observed that co-expression of human Rad18 induced the accumulation of an otherwise unstable form of HIV-1 integrase. This accumulation occurred even though hRAD18 possesses a RING finger domain, a structure that is generally associated with E3 ubiquitin ligase function and protein degradation. Evidence for an interaction between integrase and hRad18 was obtained through reciprocal co-immunoprecipitation. Moreover we found that a 162-residue region of hRad18 (amino acids 65–226) was sufficient for both integrase stabilization and interaction. Finally, we observed that HIV-1 integrase co-localized with hRad18 in nuclear structures in a subpopulation of co-transfected cells. Taken together, these findings identify hRad18 as a novel interacting partner of HIV-1 integrase and suggest a role for post-replication/translesion DNA repair in the retroviral integration process. We have previously shown that human immunodeficiency virus-1 (HIV-1) integrase is an unstable protein and a substrate for the N-end rule degradation pathway. This degradation pathway shares its ubiquitin-conjugating enzyme, Rad6, with the post-replication/translesion DNA repair pathway. Because DNA repair is thought to play an essential role in HIV-1 integration, we investigated whether other molecules of this DNA repair pathway could interact with integrase. We observed that co-expression of human Rad18 induced the accumulation of an otherwise unstable form of HIV-1 integrase. This accumulation occurred even though hRAD18 possesses a RING finger domain, a structure that is generally associated with E3 ubiquitin ligase function and protein degradation. Evidence for an interaction between integrase and hRad18 was obtained through reciprocal co-immunoprecipitation. Moreover we found that a 162-residue region of hRad18 (amino acids 65–226) was sufficient for both integrase stabilization and interaction. Finally, we observed that HIV-1 integrase co-localized with hRad18 in nuclear structures in a subpopulation of co-transfected cells. Taken together, these findings identify hRad18 as a novel interacting partner of HIV-1 integrase and suggest a role for post-replication/translesion DNA repair in the retroviral integration process. integrase post-replication DNA repair human immunodeficiency virus-1 non-homologous end joining base excision repair monoclonal antibody human embryonic kidney piperazine-N,N′-bis(2-ethanesulfonic acid) internal ribosome entry sequence ubiquitin constitutive transport element polymerase enhanced green fluorescent protein Integrase (IN)1 is essential for the completion of the retroviral life cycle. This viral protein performs different tasks that include cooperation in reverse transcription (1Wu X. Liu H. Xiao H. Conway J.A. Hehl E. Kalpana G.V. Prasad V. Kappes J.C. J. Virol. 1999; 73: 2126-2135Crossref PubMed Google Scholar), nuclear import of the pre-integration complex 2D. R. Kaufman and M. A. Muesing, submitted manuscript. 2D. R. Kaufman and M. A. Muesing, submitted manuscript. (2Gallay P. Hope T. Chin D. Trono D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9825-9830Crossref PubMed Scopus (413) Google Scholar, 3Bouyac-Bertoia M. Dvorin J.D. Fouchier R.A. Jenkins Y. Meyer B.E., Wu, L.I. Emerman M. Malim M.H. Mol. Cell. 2001; 7: 1025-1035Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar), HIV-1 particle production (4Bukovsky A. Gottlinger H. J. Virol. 1996; 70: 6820-6825Crossref PubMed Google Scholar), and integration of the viral DNA into the host genome (5LaFemina R.L. Schneider C.L. Robbins H.L. Callahan P.L. LeGrow K. Roth E. Schleif W.A. Emini E.A. J. Virol. 1992; 66: 7414-7419Crossref PubMed Google Scholar, 6Wiskerchen M. Muesing M.A. J. Virol. 1995; 69: 376-386Crossref PubMed Google Scholar). In order to mediate these very diverse processes, integrase is believed to associate with a number of cellular factors and to exploit their functions. One such instance is the filling of the gaps left after the ligation of 3′-ends of the viral DNA to the 5′-ends of the staggered cleavage in cellular DNA. The factors involved are thought to be proteins specialized in the maintenance of the genome integrity. It has recently been reported that a particular DNA repair mechanism, the non-homologous end joining (NHEJ) repair pathway, has a major impact on integration frequency (7Daniel R. Katz R.A. Skalka A.M. Science. 1999; 284: 644-647Crossref PubMed Scopus (220) Google Scholar) and that some of its components interact with the pre-integration complex (8Li L. Olvera J.M. Yoder K.E. Mitchell R.S. Butler S.L. Lieber M. Martin S.L. Bushman F.D. EMBO J. 2001; 20: 3272-3281Crossref PubMed Scopus (288) Google Scholar). NHEJ is thought to act on the repair of the gaps (7Daniel R. Katz R.A. Skalka A.M. Science. 1999; 284: 644-647Crossref PubMed Scopus (220) Google Scholar, 9Daniel R. Katz R.A. Merkel G. Hittle J.C. Yen T.J. Skalka A.M. Mol. Cell. Biol. 2001; 21: 1164-1172Crossref PubMed Scopus (70) Google Scholar, 10Daniel R. Litwin S. Katz R.A. Skalka A.M. J. Virol. 2001; 75: 3121-3128Crossref PubMed Scopus (18) Google Scholar), although other evidence suggests that it plays a role in the formation of non-integrated HIV-1 DNA circles (8Li L. Olvera J.M. Yoder K.E. Mitchell R.S. Butler S.L. Lieber M. Martin S.L. Bushman F.D. EMBO J. 2001; 20: 3272-3281Crossref PubMed Scopus (288) Google Scholar). Regardless, failure to accomplish either of these processes results in apoptosis, which is observed in NHEJ-deficient cells upon infection with HIV-1 (7Daniel R. Katz R.A. Skalka A.M. Science. 1999; 284: 644-647Crossref PubMed Scopus (220) Google Scholar, 8Li L. Olvera J.M. Yoder K.E. Mitchell R.S. Butler S.L. Lieber M. Martin S.L. Bushman F.D. EMBO J. 2001; 20: 3272-3281Crossref PubMed Scopus (288) Google Scholar). Components of another DNA repair pathway, the base excision repair (BER), have been successfully tested in vitro for their ability to repair retroviral integration-dependent gaps (11Yoder K.E. Bushman F.D. J. Virol. 2000; 74: 11191-11200Crossref PubMed Scopus (174) Google Scholar). Poly(ADP-ribose) polymerase-1 (PARP-1), an enzyme activated by DNA strand breaks, has also been linked to the retroviral integration process (12Gaken J.A. Tavassoli M. Gan S.U. Vallian S. Giddings I. Darling D.C. Galea-Lauri J. Thomas M.G. Abedi H. Schreiber V. Menissier-de Murcia J. Collins M.K. Shall S. Farzaneh F. J. Virol. 1996; 70: 3992-4000Crossref PubMed Google Scholar, 13Ha H.C. Juluri K. Zhou Y. Leung S. Hermankova M. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3364-3368Crossref PubMed Scopus (89) Google Scholar). Its proposed role in chromatin decondensation would facilitate access of the repair machinery to the integration site (13Ha H.C. Juluri K. Zhou Y. Leung S. Hermankova M. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3364-3368Crossref PubMed Scopus (89) Google Scholar).We recently reported that integrase is a substrate for the N-end rule (14Mulder L.C. Muesing M.A. J. Biol. Chem. 2000; 275: 29749-29753Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) a distinct ubiquitin-proteasome pathway that defines the half-life of target proteins according to their N-terminal residue (15Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12142-12149Crossref PubMed Scopus (715) Google Scholar). Authentic HIV-1 integrase is the proteolytic product of the Gag-Pol polyprotein and specifies an N-terminal phenylalanine, which renders it particularly prone to N-end rule degradation, and therefore makes it very unstable. In general, degradation of proteins by the proteasome proceeds after the substrate has been covalently modified by linkage of a ubiquitin chain. This type of protein tagging is sequentially performed by enzymes or clusters of enzymes broadly named E1, E2, and E3 (16Voges D. Zwickl P. Baumeister W. Annu. Rev. Biochem. 1999; 68: 1015-1068Crossref PubMed Scopus (1581) Google Scholar). In yeast, where the N-end rule was first described (17Bachmair A. Finley D. Varshavsky A. Science. 1986; 234: 179-186Crossref PubMed Scopus (1359) Google Scholar), the E2 molecule is Rad6 (18Sung P. Berleth E. Pickart C. Prakash S. Prakash L. EMBO J. 1991; 10: 2187-2193Crossref PubMed Scopus (83) Google Scholar). Rad6 is involved in a number of apparently diverse processes, such as sporulation, retrotransposition, N-end rule protein degradation, and DNA repair (19Broomfield S. Hryciw T. Xiao W. Mutat. Res. 2001; 486: 167-184Crossref PubMed Scopus (204) Google Scholar). Rad6 is able to bind directly to the E3 molecule of the N-end rule, Ubr-1 (20Madura K. Dohmen R.J. Varshavsky A. J. Biol. Chem. 1993; 268: 12046-12054Abstract Full Text PDF PubMed Google Scholar), and also associates with Rad18 when involved in DNA repair (21Bailly V. Lamb J. Sung P. Prakash S. Prakash L. Genes Dev. 1994; 8: 811-820Crossref PubMed Scopus (277) Google Scholar). The DNA repair pathway in which Rad6 and Rad18 participate is known as DNA post-replication (PRR)/translesion repair (19Broomfield S. Hryciw T. Xiao W. Mutat. Res. 2001; 486: 167-184Crossref PubMed Scopus (204) Google Scholar, 22Lawrence C. Bioessays. 1994; 16: 253-258Crossref PubMed Scopus (147) Google Scholar). One of the primary roles of the PRR/translesion pathway is to overcome structural restraints such as DNA adducts or UV-induced thymine-dimers that prevent the chromosomal DNA replication from being completed (19Broomfield S. Hryciw T. Xiao W. Mutat. Res. 2001; 486: 167-184Crossref PubMed Scopus (204) Google Scholar). The precise mechanism of action of the Rad6-Rad18 complex has not yet been elucidated. However, it is widely assumed that the Rad6-Rad18 heterodimer converges to the site of blocked DNA synthesis by means of the single strand DNA binding ability of Rad18 (23Bailly V. Lauder S. Prakash S. Prakash L. J. Biol. Chem. 1997; 272: 23360-23365Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 24Bailly V. Prakash S. Prakash L. Mol. Cell. Biol. 1997; 17: 4536-4543Crossref PubMed Scopus (96) Google Scholar). Subsequently, the stalled replicative machinery is removed, possibly by Rad6-dependent ubiquitin/proteasome-mediated degradation, and is replaced by protein complexes that include molecules such as Mms2-Ubc13 and Rad5 or repair polymerases such as Polη or Polζ (19Broomfield S. Hryciw T. Xiao W. Mutat. Res. 2001; 486: 167-184Crossref PubMed Scopus (204) Google Scholar).The putative involvement of Rad6 in the degradation of integrase and the similarity between the repair of the retroviral gaps with the proposed post-replication/translesion DNA repair model prompted us to investigate whether PRR/translesion molecules are implicated in integrase function. We demonstrate here that hRad18 interacts with HIV-1 integrase and that it protects integrase from an accelerated degradation. Moreover, hRad18 causes a re-localization of integrase in a subset of transfected cells so that both proteins co-localize in the same sub-nuclear structures.RESULTSAn in silico amino acid sequence analysis (28Schultz J. Milpetz F. Bork P. Ponting C.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5857-5864Crossref PubMed Scopus (2983) Google Scholar) revealed that hRad18 contains a specific type of zinc finger motif known as RING finger. This domain has been proposed to be a distinguishing feature of molecules with E3 ubiquitin ligase activity (29Kamura T. Koepp D.M. Conrad M.N. Skowyra D. Moreland R.J. Iliopoulos O. Lane W.S. Kaelin W.G., Jr. Elledge S.J. Conaway R.C. Harper J.W. Conaway J.W. Science. 1999; 284: 657-661Crossref PubMed Scopus (662) Google Scholar, 30Lorick K.L. Jensen J.P. Fang S. Ong A.M. Hatakeyama S. Weissman A.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11364-11369Crossref PubMed Scopus (937) Google Scholar, 31Joazeiro C.A. Wing S.S. Huang H. Leverson J.D. Hunter T. Liu Y.C. Science. 1999; 286: 309-312Crossref PubMed Scopus (909) Google Scholar, 32Jackson P.K. Eldridge A.G. Freed E. Furstenthal L. Hsu J.Y. Kaiser B.K. Reimann J.D. Trends Cell Biol. 2000; 10: 429-439Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar). The RING finger domain is also required for the degradative activity of Ubr1, the N-end rule E3 ligase in yeast (33Xie Y. Varshavsky A. EMBO J. 1999; 18: 6832-6844Crossref PubMed Scopus (142) Google Scholar).Unexpectedly, Western blot analysis showed that three different forms of integrase, two inherently unstable (Phe-IN and Arg-IN) (14Mulder L.C. Muesing M.A. J. Biol. Chem. 2000; 275: 29749-29753Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), as well as a stable form (Met-IN) significantly accumulated in the presence of co-transfected hRad18 (Fig.1B). This indicated that hRad18 did not increase degradation of integrase. Rather, this experiment showed that hRad18-mediated stabilization of integrase was largely independent from the N-end rule, since all three forms of integrase were similarly affected. No variation in the amounts of EGFP were detected in the presence or absence of hRad18 (data not shown) ruling out a transcriptional effect on the integrase expression vectors as the cause of the accumulation.To determine whether the hRad18-induced accumulation of integrase might depend on an interaction between the two molecules, an unstable form of integrase (Phe-IN) was transfected into HEK 293T cells together with FLAG-hRad18 or with an empty vector as control. Following lysis and sonication protein complexes were precipitated either with an anti-integrase or with an anti-FLAG antibody and analyzed by reciprocal immunoblotting. Integrase could be precipitated with an anti-FLAG antibody and hRad18 with an anti-integrase antibody (Fig.2), which demonstrated an interaction between the two proteins.Figure 2Co-immunoprecipitation (I.P.) of human Rad18 and HIV-1 integrase. The plasmid encoding FLAG-hRad18 was transfected either with authentic integrase (Phe-IN) or with an empty vector (pEGFP*IRES-ATG); "−." Cell lysates were incubated with anti-FLAG or anti-integrase antibodies. The lysates were precipitated and separated by polyacrylamide gel electrophoresis. Blots with anti-FLAG precipitates were probed with anti-integrase mAb12, and conversely, blots with anti-integrase precipitates were probed with anti-FLAG mAb M2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Human Rad18 has been localized to the nucleus (34Tateishi S. Sakuraba Y. Masuyama S. Inoue H. Yamaizumi M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7927-7932Crossref PubMed Scopus (124) Google Scholar), which is compatible with its DNA repair function. Indeed, when hRad18 was transfected in HEK 293T cells it was strictly confined to the nuclear compartment (Fig. 3A). However, hRad18 distribution was not always homogeneous, and we consistently observed that it displayed a structured nuclear pattern in a subpopulation of cells. These different distributions conceivably depend on the different physiologic states of the cells.Figure 3Intracellular distribution of hRad18 and its co-localization with HIV-1 integrase. A, cells transfected with FLAG-hRad18 were fixed and permeabilized. FLAG-hRad18 was detected with anti-FLAG mAb M2 and goat anti-mouse AlexaFluor 594 (red). DNA was stained with Hoechst 33258 (blue). B, cells transfected with FLAG-hRad18 and Met-IN were first permeabilized and then fixed. Integrase was detected with anti-integrase mAb 6G5 and goat anti-mouse AlexaFluor 647 (red), whereas hRad18 was detected with anti-FLAG rabbit polyclonal antibody and goat anti-rabbit fluorescein isothiocyanate-conjugated antibody (green). DNA was stained with Hoechst 33258 (blue).View Large Image Figure ViewerDownload Hi-res image Download (PPT)When expressed alone, integrase usually exhibited a fine punctate nuclear pattern in 293T cells. However, further immunofluorescence experiments revealed that in a subset of cells co-transfected with hRad18, integrase re-localized within the nucleus to larger structures that coincided with the distribution of hRad18 (Fig. 3B). The permeabilization step used before fixation allowed us to visualize only the complexes that were detergent-resistant and thus more likely to have a relevant role in vivo.To determine which region of hRad18 was responsible for stabilization of integrase, we performed a deletion mutagenesis analysis. Human Rad18 has at least three putative functional domains, a RING finger, a zinc finger, and a SAP domain (Fig.4A). We constructed a total of eight deletion mutants (Fig. 4B), each of them harboring an N-terminal FLAG tag for antibody recognition. Experiments were carried out by co-transfecting each of the deletion mutants of hRad18 with the physiologic, unstable form of integrase (Phe-IN). Cell lysates were analyzed by Western blotting for stabilization of integrase and by co-immunoprecipitation to detect binding. All of the hRad18 mutants, except for mutant 1-76, were expressed at levels comparable to that of wild-type hRad18 (Fig. 4B, lower panel). Fig.4B shows that a number of the mutants were able to stabilize integrase to various degrees (upper panel), but that only one of these mutants (65-226) exhibited a detectable level of binding after our immunoprecipitation protocol (middle panel). The portion of hRad18 encoded by this mutant encompasses the region C-terminal of the RING finger and the putative zinc finger. Interestingly, longer exposure times revealed a minor accumulation of integrase also in two mutants lacking the zinc finger motif, namely 239-495 and to a lesser degree 285-495 (data not shown). This stabilization, though less in magnitude, could be evidence for a second region of interaction with integrase positioned in the C terminus of hRad18. This observation could in part explain why the interaction between integrase and mutant 65-226 is not as effective as with full-length hRad18.Weaker interactions due to steric interference with improperly folded domains could be the reason why integrase did not co-precipitate with mutants encoding fragments larger than 65–226. Taken together these results show that a relatively small region within hRad18 encompassing amino acids 65–226 is sufficient for both accumulation of and binding to integrase.DISCUSSIONRepair of host chromosomal DNA at the insertion site is pivotal for productive retroviral integration. In this work we tested the hypothesis that components of the DNA postreplication/translesion repair pathway could play a role in HIV-1 integrase biology. We describe the interaction between HIV-1 integrase and hRad 18, whoseSaccharomyces cerevisae homologue is an essential member of the PRR/translesion pathway. This interaction results in an increased stabilization of integrase, which in its natural form is a particularly unstable protein (14Mulder L.C. Muesing M.A. J. Biol. Chem. 2000; 275: 29749-29753Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). In addition to the accumulation of integrase, we observed through reciprocal co-immunoprecipitation an association of hRad18 with integrase. These results suggest that the stability of integrase is tightly regulated by its interaction with hRad18.A parallel can be drawn between the possible role of hRad18 in HIV-1 integration and that of the protein MuB in the transposition of bacteriophage Mu. Indeed, Mu transposition is a prototype model for retroviral integration. The Mu bacteriophage requires multiple transpositions into the bacterial chromosome to replicate. This complex reaction is orchestrated by its transposase, MuA, in concert with both host and viral factors. After recombination is completed, the nucleoprotein complex (transpososome) is remodeled by the bacterial chaperone molecule ClpX (35Levchenko I. Luo L. Baker T.A. Genes Dev. 1995; 9: 2399-2408Crossref PubMed Scopus (245) Google Scholar, 36Kruklitis R. Welty D.J. Nakai H. EMBO J. 1996; 15: 935-944Crossref PubMed Scopus (104) Google Scholar) that specifically recognizes MuA and mediates its unfolding and eventually its destabilization (37Burton B.M. Williams T.L. Baker T.A. Mol. Cell. 2001; 8: 449-454Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The susceptibility of HIV-1 integrase to rapid degradation by the ubiquitin/proteasome pathway N-end rule (14Mulder L.C. Muesing M.A. J. Biol. Chem. 2000; 275: 29749-29753Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) and a recent report indicating that in vitro an excess of integrase inhibits closure of the DNA single strand intervals (11Yoder K.E. Bushman F.D. J. Virol. 2000; 74: 11191-11200Crossref PubMed Scopus (174) Google Scholar) seems to suggest that indeed the integration complex (intasome) needs to be disassembled for integration to be completed. The phage protein MuB has been found to exert part of its function before the destabilization of the transpososome. MuB has been reported to stabilize the transposition complex by binding the transposase MuA in a region that overlaps with the binding site recognized by ClpX for disassembly and remodeling (38Levchenko I. Yamauchi M. Baker T.A. Genes Dev. 1997; 11: 1561-1572Crossref PubMed Scopus (102) Google Scholar). Therefore, it is suggested that MuB has a modulatory activity, allowing the remodeling of the transpososome to proceed exclusively during the transition to the replicative stage (38Levchenko I. Yamauchi M. Baker T.A. Genes Dev. 1997; 11: 1561-1572Crossref PubMed Scopus (102) Google Scholar). By analogy we propose that hRad18 may regulate the stability of integrase to guarantee a productive insertion of the viral genome into the host DNA.A ubiquitin ligase function of hRad18 favoring the degradation of integrase cannot be excluded, and one could interpret the stabilization effect as a titration of the endogenous factors necessary for an efficient proteolytic degradation due to over-expression of hRad18 itself. However, the possibility that hRad18 titrates the human homologues of Rad6 is unlikely since it has been reported that the RING finger of hRad18 is necessary for hRad6A/B-hRad18 binding (34Tateishi S. Sakuraba Y. Masuyama S. Inoue H. Yamaizumi M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7927-7932Crossref PubMed Scopus (124) Google Scholar). Our deletion analysis showed that the RING finger motif does not play an essential role in the interaction between hRad18 and HIV-1 integrase, ruling out a direct competition between HIV-1 integrase and hRad6A/B for hRad18 binding. Indeed, the 162-amino acid peptide that encompasses a putative zinc finger seems to be sufficient to both stabilize and associate with integrase.The re-localization of integrase and its co-localization with hRad18 in a subset of cells suggests an additional function for this association. Human Rad18 contains a putative SAP-box (39Aravind L. Koonin E.V. Trends Biochem. Sci. 2000; 25: 112-114Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar), a domain recently recognized to mediate the binding of certain proteins to specific A/T-rich DNA regions known as the scaffold attachment regions (SAR) (40Kipp M. Gohring F. Ostendorp T. van Drunen C.M. van Driel R. Przybylski M. Fackelmayer F.O. Mol. Cell. Biol. 2000; 20: 7480-7489Crossref PubMed Scopus (160) Google Scholar). Interestingly, PARP-1, Ku antigens, and HMG-I/Y, which are involved in retroviral integration (7Daniel R. Katz R.A. Skalka A.M. Science. 1999; 284: 644-647Crossref PubMed Scopus (220) Google Scholar, 12Gaken J.A. Tavassoli M. Gan S.U. Vallian S. Giddings I. Darling D.C. Galea-Lauri J. Thomas M.G. Abedi H. Schreiber V. Menissier-de Murcia J. Collins M.K. Shall S. Farzaneh F. J. Virol. 1996; 70: 3992-4000Crossref PubMed Google Scholar, 13Ha H.C. Juluri K. Zhou Y. Leung S. Hermankova M. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3364-3368Crossref PubMed Scopus (89) Google Scholar, 41Farnet C.M. Bushman F.D. Cell. 1997; 88: 483-492Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar), have all been found to be SAR-binding proteins (42Galande S. Kohwi-Shigematsu T. J. Biol. Chem. 1999; 274: 20521-20528Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 43Zhao K. Kas E. Gonzalez E. Laemmli U.K. EMBO J. 1993; 12: 3237-3247Crossref PubMed Scopus (258) Google Scholar, 44Strick R. Laemmli U.K. Cell. 1995; 83: 1137-1148Abstract Full Text PDF PubMed Scopus (143) Google Scholar). An intriguing possibility is that the molecules relevant for HIV-1 integration cluster together, perhaps in the vicinity of SARs, achieving in this way the coordination required for these complex reactions.A role for the molecules of the PRR/translesion pathway in retroviral integration does not rule out other DNA repair mechanisms such as NHEJ or BER (7Daniel R. Katz R.A. Skalka A.M. Science. 1999; 284: 644-647Crossref PubMed Scopus (220) Google Scholar, 11Yoder K.E. Bushman F.D. J. Virol. 2000; 74: 11191-11200Crossref PubMed Scopus (174) Google Scholar). Examples for an overlap between DNA repair pathways that can function on the same damaged site have been recently reported in yeast. For instance, the activity of the Srs2 helicase is the main switch for the interaction between RAD6-RAD18 and the RAD52 epistasis (19Broomfield S. Hryciw T. Xiao W. Mutat. Res. 2001; 486: 167-184Crossref PubMed Scopus (204) Google Scholar). Thus, it is conceivable that different DNA repair pathways complement each other to complete the sequence of events that leads to a productive HIV-1 integration. Integrase (IN)1 is essential for the completion of the retroviral life cycle. This viral protein performs different tasks that include cooperation in reverse transcription (1Wu X. Liu H. Xiao H. Conway J.A. Hehl E. Kalpana G.V. Prasad V. Kappes J.C. J. Virol. 1999; 73: 2126-2135Crossref PubMed Google Scholar), nuclear import of the pre-integration complex 2D. R. Kaufman and M. A. Muesing, submitted manuscript. 2D. R. Kaufman and M. A. Muesing, submitted manuscript. (2Gallay P. Hope T. Chin D. Trono D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9825-9830Crossref PubMed Scopus (413) Google Scholar, 3Bouyac-Bertoia M. Dvorin J.D. Fouchier R.A. Jenkins Y. Meyer B.E., Wu, L.I. Emerman M. Malim M.H. Mol. Cell. 2001; 7: 1025-1035Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar), HIV-1 particle production (4Bukovsky A. Gottlinger H. J. Virol. 1996; 70: 6820-6825Crossref PubMed Google Scholar), and integration of the viral DNA into the host genome (5LaFemina R.L. Schneider C.L. Robbins H.L. Callahan P.L. LeGrow K. Roth E. Schleif W.A. Emini E.A. J. Virol. 1992; 66: 7414-7419Crossref PubMed Google Scholar, 6Wiskerchen M. Muesing M.A. J. Virol. 1995; 69: 376-386Crossref PubMed Google Scholar). In order to mediate these very diverse processes, integrase is believed to associate with a number of cellular factors and to exploit their functions. One such instance is the filling of the gaps left after the ligation of 3′-ends of the viral DNA to the 5′-ends of the staggered cleavage in cellular DNA. The factors involved are thought to be proteins specialized in the maintenance of the genome integrity. It has recently been reported that a particular DNA repair mechanism, the non-homologous end joining (NHEJ) repair pathway, has a major impact on integration frequency (7Daniel R. Katz R.A. Skalka A.M. Science. 1999; 284: 644-647Crossref PubMed Scopus (220) Google Scholar) and that some of its components interact with the pre-integration complex (8Li L. Olvera J.M. Yoder K.E. Mitchell R.S. Butler S.L. Lieber M. Martin S.L. Bushman F.D. EMBO J. 2001; 20: 3272-3281Crossref PubMed Scopus (288) Google Scholar). NHEJ is thought to act on the repair of the gaps (7Daniel R. Katz R.A. Skalka A.M. Science. 1999; 284: 644-647Crossref PubMed Scopus (220) Google Scholar, 9Daniel R. Katz R.A. Merkel G. Hittle J.C. Yen T.J. Skalka A.M. Mol. Cell. Biol. 2001; 21: 1164-1172Crossref PubMed Scopus (70) Google Scholar, 10Daniel R. Litwin S. Katz R.A. Skalka A.M. J. Virol. 2001; 75: 3121-3128Crossref PubMed Scopus (18) Google Scholar), although other evidence suggests that it plays a role in the formation of non-integrated HIV-1 DNA circles (8Li L. Olvera J.M. Yoder K.E. Mitchell R.S. Butler S.L. Lieber M. Martin S.L. Bushman F.D. EMBO J. 2001; 20: 3272-3281Crossref PubMed Scopus (288) Google Scholar). Regardless, failure to accomplish either of these processes results in apoptosis, which is observed in NHEJ-deficient cells upon infection with HIV-1 (7Daniel R. Katz R.A. Skalka A.M. Science. 1999; 284: 644-647Crossref PubMed Scopus (220) Google Scholar, 8Li L. Olvera J.M. Yoder K.E. Mitchell R.S. Butler S.L. Lieber M. Martin S.L. Bushman F.D. EMBO J. 2001; 20: 3272-3281Crossref PubMed Scopus (288) Google Scholar). Components of another DNA repair pathway, the base excision repair (BER), have been successfully tested in vitro for their ability to repair retroviral integration-dependent gaps (11Yoder K.E. Bushman F.D. J. Virol. 2000; 74: 11191-11200Crossref PubMed Scopus (174) Google Scholar). Poly(ADP-ribose) polymerase-1 (PARP-1), an enzyme activated by DNA strand breaks, has also been linked to the retroviral integration process (12Gaken J.A.
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