Multiple Integrase Functions Are Required to Form the Native Structure of the Human Immunodeficiency Virus Type I Intasome

Abstract

Mu-mediated polymerase chain reaction footprinting was used to investigate the protein-DNA structure of human immunodeficiency virus type I (HIV-I) preintegration complexes. Preintegration complexes were partially purified from cells after using an established coculture infection technique as well as a novel technique using cell-free supernatant from transfected cells as the source of virus. Footprinting revealed that bound proteins protected the terminal 200–250 base pairs of each viral end from nuclease attack. Bound proteins also caused strong transpositional enhancements near each end of HIV-I. In contrast, regions of viral DNA internal to the ends did not show evidence of strong protein binding. The end regions of preintegrative HIV-I apparently form a unique nucleoprotein structure, which we term the intasome to distinguish it from the greater preintegration complex. Our novel system also allowed us to analyze the structure and function of preintegration complexes isolated from cells infected with integrase mutant viruses. Complexes were derived from viruses defective for either integrase catalysis, integrase binding to the viral DNA substrate, or an unknown function in the carboxyl-terminal domain of the integrase protein. None of these mutant complexes supported detectable integration activity. Despite the presence of the mutant integrase proteins in purified samples, none of these nucleoprotein complexes displayed the native intasome structure detected in wild-type preintegration complexes. We conclude that multiple integrase functions are required to form the native structure of the HIV-I intasome in infected cells. Mu-mediated polymerase chain reaction footprinting was used to investigate the protein-DNA structure of human immunodeficiency virus type I (HIV-I) preintegration complexes. Preintegration complexes were partially purified from cells after using an established coculture infection technique as well as a novel technique using cell-free supernatant from transfected cells as the source of virus. Footprinting revealed that bound proteins protected the terminal 200–250 base pairs of each viral end from nuclease attack. Bound proteins also caused strong transpositional enhancements near each end of HIV-I. In contrast, regions of viral DNA internal to the ends did not show evidence of strong protein binding. The end regions of preintegrative HIV-I apparently form a unique nucleoprotein structure, which we term the intasome to distinguish it from the greater preintegration complex. Our novel system also allowed us to analyze the structure and function of preintegration complexes isolated from cells infected with integrase mutant viruses. Complexes were derived from viruses defective for either integrase catalysis, integrase binding to the viral DNA substrate, or an unknown function in the carboxyl-terminal domain of the integrase protein. None of these mutant complexes supported detectable integration activity. Despite the presence of the mutant integrase proteins in purified samples, none of these nucleoprotein complexes displayed the native intasome structure detected in wild-type preintegration complexes. We conclude that multiple integrase functions are required to form the native structure of the HIV-I intasome in infected cells. A pivotal step in the retroviral life cycle is forming the provirus, an integrated cDNA copy of the viral RNA genome. The key viral players in integration are the trans-acting integrase (IN) 1The abbreviations used are: IN, integrase; PIC, preintegration complex; PCR, polymerase chain reaction; MM-PCR, Mu-mediated polymerase chain reaction; HIV-I, human immunodeficiency virus type I; LTR, long terminal repeat; MoMLV, Moloney murine leukemia virus 1The abbreviations used are: IN, integrase; PIC, preintegration complex; PCR, polymerase chain reaction; MM-PCR, Mu-mediated polymerase chain reaction; HIV-I, human immunodeficiency virus type I; LTR, long terminal repeat; MoMLV, Moloney murine leukemia virus protein and thecis-acting DNA attachment site. Integration proceeds through three steps, the first two of which are known to require IN function. The linear ends of the cDNA are initially processed adjacent to phylogenetically conserved CA dinucleotides, resulting in a pair of recessed 3′ ends. After nuclear localization, the exposed 3′-hydroxyls are joined to the 5′-phosphates of a double-stranded staggered cut in chromosomal DNA. The final step is DNA repair, wherein the single-stranded gaps at the sites of joining are sealed, resulting in the sequence duplication of the double-stranded cut flanking the integrated provirus (for a review, see Ref. 1Brown P.O. Coffin J.M. Hughes S.H. Varmus H.E. Retroviruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1997: 161-203Google Scholar). In infected cells, integration is mediated by large nucleoprotein complexes known as preintegration complexes (PICs), which are derived from the cores of infecting virions (2Bowerman B. Brown P.O. Bishop J.M. Varmus H.E. Genes Dev. 1989; 3: 469-478Crossref PubMed Scopus (282) Google Scholar). PICs isolated from infected cells can integrate their endogenous cDNA into an exogenously added target DNA in vitro (2Bowerman B. Brown P.O. Bishop J.M. Varmus H.E. Genes Dev. 1989; 3: 469-478Crossref PubMed Scopus (282) Google Scholar, 3Brown P.O. Bowerman B. Varmus H.E. Bishop J.M. Cell. 1987; 49: 347-356Abstract Full Text PDF PubMed Scopus (324) Google Scholar, 4Fujiwara T. Mizuuchi K. Cell. 1988; 54: 497-504Abstract Full Text PDF PubMed Scopus (256) Google Scholar, 5Farnet C.M. Haseltine W.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4164-4168Crossref PubMed Scopus (201) Google Scholar, 6Ellison V. Abrams H. Roe T. Lifson J. Brown P. J. Virol. 1990; 64: 2711-2715Crossref PubMed Google Scholar, 7Lee Y.M.H. Coffin J.M. J. Virol. 1990; 64: 5958-5965Crossref PubMed Google Scholar). Additionally, recombinant IN proteins purified after expression in bacteria can cut and join oligonucleotide attachment site DNA substrates (reviewed in Ref. 1Brown P.O. Coffin J.M. Hughes S.H. Varmus H.E. Retroviruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1997: 161-203Google Scholar). These latter in vitro assays have been invaluable for deciphering the structure and function of retroviral IN proteins. IN can be divided into three distinct functional domains: the amino-terminal, catalytic core, and carboxyl-terminal domains (reviewed in Ref. 8Andrake M.D. Skalka A.M. J. Biol. Chem. 1996; 271: 19633-19636Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). The central domain contains the highly conserved D,D(35)E amino acid motif that comprises the IN active site (9Dyda F. Hickman A.B. Jenkins T.M. Engelman A. Craigie R. Davies D.R. Science. 1994; 266: 1981-1986Crossref PubMed Scopus (709) Google Scholar, 10Bujacz G. Jaskolski M. Alexandratos J. Wlodower A. Merkel G. Katz R.A. Skalka A.M. J. Mol. Biol. 1995; 253: 336-346Crossref Scopus (193) Google Scholar). Although simplified in vitro assays using purified IN proteins have provided essential knowledge toward understanding the overall integration process, these systems only partially mimic integration in vivo, because the predominant recombination products result from the insertion of only one viral DNA end into just one strand of target DNA. Virus replication requires integration of both DNA ends into both strands of target DNA; the single-ended activity typical of some in vitro systems would not yield a productive viral infection. Although altering the source of purified IN protein from bacterial to viral and/or modifying reaction conditions can increase the frequency of two-ended integration products (11Vora A.C. McCord M. Fitzgerald M.L. Inman R.B. Grandgenett D.P. Nucleic Acids Res. 1994; 22: 4454-4461Crossref PubMed Scopus (54) Google Scholar, 12Goodarzi G. Im G.-J. Brackmann K. Grandgenett D.P. J. Virol. 1995; 69: 6090-6097Crossref PubMed Google Scholar, 13Aiyar A. Hindmarsh P. Skalka A.M. Leis J. J. Virol. 1996; 70: 3571-3580Crossref PubMed Google Scholar), these systems still do not recapitulate the efficiency of two-ended integration activity displayed by PICs isolated from infected cells (14Miller M.D. Farnet C.M. Bushman F.D. J. Virol. 1997; 71: 5382-5390Crossref PubMed Google Scholar). The discrepancy in reaction products catalyzed by PICs as compared with purified IN proteins suggests that efficient two-ended integration activity might require higher-order protein-protein and/or protein-DNA interactions specific to nucleoprotein complexes derived from infected cells. To begin to address this, we have used in vitroMu-mediated polymerase chain reaction (MM-PCR) footprinting to analyze the protein-DNA structure of human immunodeficiency virus type I (HIV-I) PICs partially purified from infected cells. We have established an efficient HIV-I infection system initiated from transfected cell supernatant that yields active PICs. This has allowed us to analyze the structure and function of HIV-I PICs derived from a number of IN mutant viruses. Our results indicate that multiple IN functions are required to form the native protein-DNA structure of the HIV-I intasome. MOLT IIIB (5Farnet C.M. Haseltine W.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4164-4168Crossref PubMed Scopus (201) Google Scholar), SupT1 (15Smith S.D. Shatsky M. Cohen P.S. Warnke R. Link M.P. Glader B.E. Cancer Res. 1984; 44: 5657-5660PubMed Google Scholar), C8166 (16Salahuddin S.Z. Markham P.D. Wong-Staal F. Franchini G. Kalyanaraman V.S. Gallo R.C. Virology. 1983; 129: 51-64Crossref PubMed Scopus (256) Google Scholar), MT-4 (17Miyoshi I. Taguchi H. Kubonishi I. Yoshimoto S. Ohrauki Y. Shiraishi Y. Akagi T. Jpn. J. Cancer Res. 1982; 28: 219-228Google Scholar), Jurkat (18Weiss A. Wiskocil R.L. Stobo J.D. J. Immunol. 1984; 133: 123-128PubMed Google Scholar), and CEM-12D7 (19Ross E.K. Buckler-White A.J. Rabson A. Englund G. Martin M.A. J. Virol. 1991; 65: 4350-4358Crossref PubMed Google Scholar) T-cell lines were maintained in RPMI 1640 medium containing 10% fetal calf serum. 293T (20Pear W.S. Nolan G.P. Scott M.L. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8392-8396Crossref PubMed Scopus (2291) Google Scholar) and HeLa-CD4 (21Wu 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) cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Two different infection systems were used to isolate HIV-I PICs. In one system, chronically infected MOLT IIIB cells were cocultured with uninfected SupT1 cells, essentially as described previously (5Farnet C.M. Haseltine W.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4164-4168Crossref PubMed Scopus (201) Google Scholar). HIV-I production (HTLV-IIIB strain) was stimulated 2- to 3-fold by pretreating MOLT IIIB cells (4 × 105 cells/ml) for 24 h with phorbol 12-myristate 13-acetate (10 μg/ml). In the other infection system, cell-free HIV-I (NL4-3 strain) produced from transfected 293T cells was used to infect C8166 cells (see below). Wild-type pNL4-3 (22Adachi A. Gendelman H.E. Koenig S. Folks T. Willey R. Rabson A. Martin M.A. J. Virol. 1986; 59: 284-291Crossref PubMed Google Scholar) and IN mutants D116N (23Engelman A. Englund G. Orenstein J.M. Martin M.A. Craigie R. J. Virol. 1995; 69: 2729-2736Crossref PubMed Google Scholar) and K156E/K159E (24Jenkins T.M. Esposito D. Engelman A. Craigie R. EMBO J. 1997; 16: 6849-6859Crossref PubMed Scopus (214) Google Scholar) have been described previously. HIV-I (NL4-3) carrying the substitution of Lys for Gln-62 (Q62K) in IN displays a replication-defective phenotype similar to that of K156E/K159E. 2A. Engelman, manuscript in preparation. The W235E IN change (25Leavitt A.D. Robles G. Alesandro N. Varmus H.E. J. Virol. 1996; 70: 721-728Crossref PubMed Google Scholar) was incorporated into pNL4-3 using overlapping PCR (23Engelman A. Englund G. Orenstein J.M. Martin M.A. Craigie R. J. Virol. 1995; 69: 2729-2736Crossref PubMed Google Scholar). were and were to DNA; was the PCR HIV-I were to both the and Haseltine R. B. J.A. K. M.L. J.A. J. Gallo R.C. Wong-Staal F. Scholar) The and their in are as to as in Ref. J.A. A. Cell. 1990; Full Text PDF PubMed Scopus Google to and were used in the first to the and 3′ of HIV-I. to and to were used in to analyze the terminal and were used in the to analyze the The was used in the first to analyze a of HIV-I internal to the was used in SupT1 cells (4 × were with phorbol 12-myristate MOLT IIIB cells in of MOLT medium supernatant of cells at × cells/ml) at a of After cells were in and and in of by was with A for at To partially of was through a in The was purified a in K. was for h at × in a at were from the of the 293T cells were at × in a 24 h were transfected with DNA using J. T. A Cold Spring Harbor Laboratory, Cold Spring Harbor, Scholar). cell supernatant was through and with I in the presence of for at This step the of from to the of by in cell cells × and HeLa-CD4 cells (4 × in a h were infected for h with of cell with A were by and to final of and After at for DNA was by and with To isolate C8166 cells × were infected for h with of transfected cell was activity was essentially as described previously (5Farnet C.M. Haseltine W.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4164-4168Crossref PubMed Scopus (201) Google Scholar). of or was with of DNA for at were and DNA was by with DNA was through in J. T. A Cold Spring Harbor Laboratory, Cold Spring Harbor, Scholar), to and with a Integration activity was as the of cDNA into using either or C8166 cell to were in at was added to a final of to and proteins were by essentially as described previously D. 1984; PubMed Scopus Google Scholar). were with of by of Two were after of and at × for at the were using of and at for proteins were in and for and through 10% were to and IN was detected using K. Brown P.O. J. Virol. 1996; 70: PubMed Google Scholar) with an HIV-I IN protein was purified after expression in as described previously A. Liu Y. H. M. F. J. Virol. 1997; 71: PubMed Google Scholar). A protein was provided by Mizuuchi of and and Two different DNA footprinting were In DNA was added to to a final of The other was PICs in K. were by the end with A in and After a at the was in the were with an of PICs or DNA and was initiated by to a final of After at were by and to final of and products were by with DNA was in of of which was used in The first each of and a of each and of DNA polymerase were a PCR system with the PCR at for at for at for and at for A final at was was for the which each of and a viral × 105 of viral of each and of DNA polymerase PCR was essentially as described that the was from to the of the of were In HIV-I PICs were isolated from infected cells using one of two different infection In one system, HIV-I (HTLV-IIIB strain) infection was initiated by uninfected SupT1 cells with chronically infected MOLT IIIB cells, essentially as described previously (5Farnet C.M. Haseltine W.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4164-4168Crossref PubMed Scopus (201) Google Scholar). were h after and containing HIV-I PICs was purified by This step of the protein in the cell PICs that supported a of integration activity in the not The was purified a which was and for HIV-I DNA and activity. DNA and integration activity to and The integration activity of the cell from 10% to the preparation. of was to analyze the structure and function of HIV-I PICs derived from IN mutant viruses. we to an infection technique using transfected cell supernatant as the source of cell-free virus. the of different cell lines to after infection with HIV-I (NL4-3 these cell C8166 cells (16Salahuddin S.Z. Markham P.D. Wong-Staal F. Franchini G. Kalyanaraman V.S. Gallo R.C. Virology. 1983; 129: 51-64Crossref PubMed Scopus (256) Google Scholar) supported the of cDNA A revealed of h after infecting C8166 cells not HIV-I cDNA in the of C8166 cells was not these were purified by The were and for HIV-I DNA and activity. cDNA and integration activity to PICs from C8166 cells integrated of their cDNA into the target of to as compared with PICs isolated from the coculture infection footprinting was to analyze the native protein-DNA structure of Moloney murine leukemia virus PICs Mizuuchi K. Craigie R. EMBO J. 1997; 16: PubMed Scopus Google Scholar). In footprinting are the DNA A DNA at the site of as in protein-DNA footprinting bound protein with the of the to cut the The of and DNA joining technique other footprinting is that the DNA a for PCR We technique to analyze the native protein-DNA structure of HIV-I PICs isolated from cells using two different infection different and native were PICs partially purified after the coculture infection were initially PICs supported a of similar to the detected using DNA and revealed different transpositional Bound proteins protected base pairs from each end and In to these large transpositional enhancements were detected near the ends of HIV-I A the and regions were of the viral DNA internal regions of and native PICs supported similar of were detected that proteins with internal regions of HIV-I. PICs partially purified from infected C8166 cells the overall These PICs displayed regions to their integration activity. The internal of the were the base pairs from the end of HIV-I, the base pairs PICs isolated from infected C8166 cells displayed isolated from the coculture infection not IN is essential for retroviral DNA we the of different IN the structure and function of HIV-I PICs. HIV-I IN mutants can be divided into two I and their the virus replication cycle A. Virus Res. 1999; PubMed Google Scholar). I mutants are at the integration mutants and/or virus of PICs can only be derived from I IN mutant viral HIV-I as well as a number of into A. Virus Res. 1999; PubMed Google Scholar). HIV-I IN contains three functional the amino-terminal, catalytic core, and carboxyl-terminal domains M.D. Skalka A.M. J. Biol. Chem. 1996; 271: 19633-19636Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), and each domain contains at one amino acid that is conserved A. Virus Res. 1999; PubMed Google Scholar). The domain contains two conserved and two conserved mutants carrying of one of these the replication phenotype A. Virus Res. 1999; PubMed Google Scholar), of these PICs were not not The catalytic domain contains the conserved active site and of the D,D(35)E motif as well as as (24Jenkins T.M. Esposito D. Engelman A. Craigie R. EMBO J. 1997; 16: 6849-6859Crossref PubMed Scopus (214) Google Scholar, S. M. J. Brown P.O. J. Virol. PubMed Google Scholar) and Gln-62 A. Liu Y. H. M. F. J. Virol. 1997; 71: PubMed Google Scholar, S. M. J. Brown P.O. J. Virol. PubMed Google D. Craigie R. EMBO J. PubMed Scopus Google Scholar) in viral DNA end binding. of the three active site yields the I mutant viral as do of mutant K156E/K159E was used and Gln-62 The substitution of for the conserved carboxyl-terminal domain also yields the I mutant viral phenotype (25Leavitt A.D. Robles G. Alesandro N. Varmus H.E. J. Virol. 1996; 70: 721-728Crossref PubMed Google Scholar). the of the W235E mutant virus is We different I mutant viruses that three different IN which was defective for and each of which was defective for viral DNA end and which was defective for an unknown carboxyl-terminal domain function. of C8166 cells were after infection with either wild-type or I IN mutant viruses. wild-type PICs of the cDNA to the integration none of the mutant viral PICs supported detectable integration activity The were into mutant in to the as did that the nucleoprotein structure of each was not The were to The results of that none of the mutant PICs displayed the of protein-DNA footprinting and transpositional enhancements of the wild-type HIV-I intasome To that the of intasome structure for each of the mutants was not to the of the IN were by using an The results of that each of the mutant PICs IN protein at a to that of wild-type of HIV-I IN mutant mutant PICs partially purified by were by structure of the end of HIV-I. structure of the The in were the native is the as that described in the to and protein of HIV-I mutant were by and of recombinant HIV-I IN protein. The in were isolated after infection with and HIV-I, The in a from cells. The of in are In we the native protein-DNA structure of preintegrative HIV-I as detected by The structure has two large regions base of protein binding were detected at the ends of HIV-I. regions of nuclease were detected near the ends of the virus. The overall structure is similar to the one previously for Mizuuchi K. Craigie R. EMBO J. 1997; 16: PubMed Scopus Google Scholar). similar intasome be to the preintegrative of a of results to the functional of the retroviral intasome. IN was required to form both the Mizuuchi K. Craigie R. EMBO J. 1997; 16: PubMed Scopus Google Scholar) and HIV-I both the Mizuuchi K. Craigie R. EMBO J. 1997; 16: PubMed Scopus Google Scholar) and HIV-I H. Engelman A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) as well as were after bound proteins with of and both structure and function were in by from uninfected cells. insertion into the regions the of PICs to vitro integration Mizuuchi K. Craigie R. EMBO J. 1997; 16: PubMed Scopus Google Scholar). This the functional of the retroviral intasome as detected by The for the transpositional enhancements at the ends of and HIV-I DNA is integration and their of (reviewed in Ref. G. Immunol. 1996; Google Scholar). HIV-I IN protein oligonucleotide attachment DNA substrates into regions of target DNA D. Bushman F.D. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar), that protein binding in native PICs viral end regions and for it is that proteins bound near the ends of retroviral DNA with the IN protein bound to recombinant viral DNA displayed the viral end footprinting and with the endogenous intasome isolated from infected cells Mizuuchi K. Craigie R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), that IN is for the structure as detected by we have been to evidence for the HIV-I intasome structure using purified recombinant IN with either recombinant viral DNA or cDNA substrates purified from infected cells not To the of IN in the of the HIV-I we established a highly efficient infection system initiated with virus. cell lines were for their to after infection and one T-cell C8166 (16Salahuddin S.Z. Markham P.D. Wong-Staal F. Franchini G. Kalyanaraman V.S. Gallo R.C. Virology. 1983; 129: 51-64Crossref PubMed Scopus (256) Google Scholar), was not only to efficient DNA the resulting HIV-I PICs displayed efficient in vitro integration activity PICs isolated after infection with I viral mutants defective for either IN viral DNA end binding and or an unknown carboxyl-terminal function did not detectable of in vitro integration activity Despite containing their mutant IN proteins none of the mutant PICs displayed the protein-DNA and viral end enhancements of the wild-type HIV-I intasome Although was for IN domain mutants K156E/K159E and are to HIV-I attachment site DNA binding (24Jenkins T.M. Esposito D. Engelman A. Craigie R. EMBO J. 1997; 16: 6849-6859Crossref PubMed Scopus (214) Google Scholar, S. M. J. Brown P.O. J. Virol. PubMed Google Scholar, D. Craigie R. EMBO J. PubMed Scopus Google Scholar), was for both the carboxyl-terminal domain mutant (see and the D116N active site IN active site has been in with the HIV-I attachment site S. M. J. Brown P.O. J. Virol. PubMed Google Scholar). In contrast, recombinant D116N mutant proteins have not revealed evidence of DNA binding using either functional S. M. J. Brown P.O. J. Virol. PubMed Google Scholar) or A. Hickman A.B. Craigie R. J. Virol. 1994; PubMed Google Scholar) in vitro we that IN be to HIV-I intasome in infected cells. is a in other DNA recombination that the active A DNA with each step the recombination G. Immunol. 1996; Google Scholar). 3′ of the viral ends by IN the HIV-I nucleoprotein complex. A revealed that the intasome Mizuuchi K. Craigie R. EMBO J. 1997; 16: PubMed Scopus Google Scholar), with the that IN be required for intasome in infected cells. The W235E in infected cells is in because recombinant W235E mutant IN protein displays wild-type of 3′ and DNA strand in in assays A.D. Varmus H.E. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). A of the and has been in binding to the HIV-I attachment site D. Craigie R. EMBO J. PubMed Scopus Google Scholar, Brown P.O. 1997; PubMed Scopus Google Scholar). In contrast, the and to the target DNA of an in vitro DNA Brown P.O. 1997; PubMed Scopus Google Scholar). is that the W235E mutant virus is defective for with chromosomal DNA in infected cells. because the structure of W235E PICs purified from was from the structure of either the D116N or attachment site DNA binding we that the W235E virus is at a step that target DNA in the of infected cells. the of the W235E mutant higher-order protein-protein and/or protein-DNA interactions for activity that are for recombinant IN function in simplified in vitro integration We also to to analyze the structure and function of HIV-I IN and attachment site mutant PICs isolated from infected cells. We M. Mizuuchi for purified A X. and J. Kappes for HeLa-CD4 cells, D. for cells, and D. for of the