HIV Integrase, a Brief Overview from Chemistry to Therapeutics (2001)

human immunodeficiency virus avian sarcoma virus Retroviruses are a large and diverse family of RNA viruses that synthesize a DNA copy of their RNA genome after infection of the host cell. Integration of this viral DNA into host DNA is an essential step in the replication cycle of HIV1 and other retroviruses (reviewed in Refs. 1Brown P.O. Coffin J.M. Hughes S.H. Varmus H.E. Retroviruses.in: Hughes J.M. Varmus S.H. H.E. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1997: 161-203Google Scholar, 2Asante-Appiah E. Skalka A.M. Antiviral Res. 1997; 36: 139-156Crossref PubMed Scopus (146) Google Scholar, 3Hindmarsh P. Leis J. Microbiol. Mol. Biol. Rev. 1999; 63: 836-843Crossref PubMed Google Scholar). The integrated viral DNA is transcribed to make the RNA genome of progeny virions and the template for translation of viral proteins. Following assembly, virions bud from the cell surface and subsequently infect previously uninfected cells, thus completing the replication cycle. An infecting retrovirus introduces a large nucleoprotein complex into the cytoplasm of the host cell. This complex, which is derived from the core of the infecting virion, contains two copies of the viral RNA together with a number of viral proteins, including reverse transcriptase and integrase. Reverse transcription of the viral RNA occurs within the complex to make a double-stranded DNA copy of the viral genome, the viral DNA substrate for integration. The viral DNA remains associated with both viral and cellular proteins in a nucleoprotein complex termed the preintegration complex. One constituent of the preintegration complex is the viral integrase protein, the key player in the integration of the viral DNA into the host genome. The other components of the preintegration complex that are transported to the nucleus along with the viral DNA and integrase, and their possible functions, have not been firmly established and are not discussed here. The critical DNA cutting and joining events that integrate the viral DNA are carried out by the integrase protein itself. Here we review our current knowledge of the molecular mechanism of this reaction and discuss some of the key issues that are yet to be understood. Biochemical studies have elucidated the basic chemical mechanism of integration, even though the organization of the active complex of integrase with its DNA substrates remains to be determined. We will focus on HIV integrase, but the key properties of this enzyme appear to be shared among the entire retroviral integrase family. In the first step of the integration process, two nucleotides are removed from each 3′-end of the viral DNA, a reaction termed 3′-end processing. Cleavage occurs to the 3′-side of a CA dinucleotide that is conserved among retroviruses, retrotransposons, and many DNA transposons, both in prokaryotes and eukaryotes. This reaction exposes the terminal 3′-hydroxyl group that is to be joined to target DNA (Fig.1 B). In the second step, DNA strand transfer, a pair of processed viral DNA ends is inserted into the target DNA (Fig. 1 C). In the case of HIV, the sites of integration on the two target DNA strands are separated by 5 base pairs. Repair of this integration intermediate (Fig. 1 D) results in a direct duplication of 5 base pairs flanking the integrated viral DNA (not shown). The repair step requires removal of the two unpaired nucleotides at the 5′-ends of the viral DNA, filling in the single gaps, and finally ligation. Integrase is responsible for 3′-processing and DNA strand transfer, but the latter repair steps are likely to be carried out by cellular enzymes (4Brin E. Yi J.Z. Skalka A.M. Leis J. J. Biol. Chem. 2000; 275: 39287-39295Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 5Yoder K.E. Bushman F.D. J. of Virol. 2000; 74: 11191-11200Crossref PubMed Scopus (177) Google Scholar). There is little specificity for the sites of integration in host DNA, and insertion can occur at essentially any location. The DNA cutting and joining steps of 3′-end processing and DNA strand transfer closely parallel the reactions used by many transposons to move to new sites in the genome. The 3′-processing and DNA strand transfer reactions can be carried outin vitro with purified integrase, a duplex oligonucleotide that mimics one end of the viral DNA, and a divalent metal ion. This simplified in vitro system has proved to be invaluable for dissecting the biochemical mechanism of DNA integration. Stereochemical analysis of the reaction pathway has demonstrated that both 3′-processing and DNA strand transfer occur by a one-step transesterification mechanism (6Engelman A. Mizuuchi K. Craigie R. Cell. 1991; 67: 1211-1221Abstract Full Text PDF PubMed Scopus (543) Google Scholar), the same result that was previously obtained for the corresponding reactions mediated by the related Mu transposase protein (7Mizuuchi K. Adzuma K. Cell. 1991; 66: 129-140Abstract Full Text PDF PubMed Scopus (138) Google Scholar). In the 3′-processing reaction, water serves as the nucleophile for cleavage at the ends of the viral DNA. We may envisage that DNA strand transfer occurs by a chemically similar mechanism, except that integrase positions the 3′-hydroxyl groups at the ends of the viral DNA to simultaneously cleave the target DNA and make covalent connections between the viral and target DNA. Although 3′-processing and DNA strand transfer are very similar reactions at the chemical level, the way the active site region of integrase engages DNA substrate must differ between processing and DNA strand transfer; for the latter reaction the active sites must accommodate target DNA in addition to the viral DNA ends. HIV-1 integrase is comprised of three domains (Fig.2) based on the susceptibility of the linker regions to proteolysis (8Engelman A. Craigie R. J. Virol. 1992; 66: 6361-6369Crossref PubMed Google Scholar), functional studies (8Engelman A. Craigie R. J. Virol. 1992; 66: 6361-6369Crossref PubMed Google Scholar, 9Engelman A. Bushman F.D. Craigie R. EMBO J. 1993; 12: 3269-3275Crossref PubMed Scopus (319) Google Scholar, 10van Gent D.C. Vink C. Groeneger A.A. Plasterk R.H. EMBO J. 1993; 12: 3261-3267Crossref PubMed Scopus (213) Google Scholar), and the structures of the domains (Fig. 3), which have been individually determined by x-ray crystallography or NMR.Figure 3Structures of the three domains of HIV-1 integrase shown as ribbon diagrams (48Carson M. J. Mol. Graph. 1987; 5: 103-106Crossref Scopus (514) Google Scholar).A, the catalytic core domain; B, the N-terminal domain;C, the C-terminal domain. Protein Data Bank codes are 1BIS,1WJC, and 1IHV, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The catalytic core domain contains the invariant triad of acidic residues, the D,D-35-E motif (8Engelman A. Craigie R. J. Virol. 1992; 66: 6361-6369Crossref PubMed Google Scholar, 11Kulkosky J. Jones K.S. Katz R.A. Mack J.P. Skalka A.M. Mol. Cell. Biol. 1992; 12: 2331-2338Crossref PubMed Scopus (523) Google Scholar, 12Polard P. Chandler M. Mol. Microbiol. 1995; 15: 13-23Crossref PubMed Scopus (195) Google Scholar, 13Rowland S.J. Dyke K.G. Mol. Microbiol. 1990; 4: 961-975Crossref PubMed Scopus (182) Google Scholar), comprising residues Asp64, Asp116, and Glu152 in the case of HIV-1 integrase. Mutagenesis of these residues and their counterparts in related retroviral integrases abolishes or severely diminishes all catalytic activities in parallel (8Engelman A. Craigie R. J. Virol. 1992; 66: 6361-6369Crossref PubMed Google Scholar, 11Kulkosky J. Jones K.S. Katz R.A. Mack J.P. Skalka A.M. Mol. Cell. Biol. 1992; 12: 2331-2338Crossref PubMed Scopus (523) Google Scholar, 14Leavitt A.D. Shiue L. Varmus H.E. J. Biol. Chem. 1993; 268: 2113-2119Abstract Full Text PDF PubMed Google Scholar, 15van Gent D.C. Groeneger A.A. Plasterk R.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9598-9602Crossref PubMed Scopus (182) Google Scholar). By analogy with models of catalysis by DNA polymerases (16Beese L.S. Steitz T.A. EMBO J. 1991; 10: 25-33Crossref PubMed Scopus (924) Google Scholar, 17Freemont P.S. Friedman J.M. Beese L.S. Sanderson M.R. Steitz T.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8924-8928Crossref PubMed Scopus (330) Google Scholar, 18Steitz T.A. Steitz J.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6498-6502Crossref PubMed Scopus (1046) Google Scholar), it has been proposed that coordination of divalent metal ion to these residues plays a key role in catalysis (11Kulkosky J. Jones K.S. Katz R.A. Mack J.P. Skalka A.M. Mol. Cell. Biol. 1992; 12: 2331-2338Crossref PubMed Scopus (523) Google Scholar). The structures of catalytic domain of HIV-1 integrase (19Dyda F. Hickman A.B. Jenkins T.M. Engelman A. Craigie R. Davies D.R. Science. 1994; 266: 1981-1986Crossref PubMed Scopus (725) Google Scholar, 20Goldgur Y. Dyda F. Hickman A.B. Jenkins T.M. Craigie R. Davies D.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9150-9154Crossref PubMed Scopus (371) Google Scholar) and the corresponding domain ASV integrase (21Bujacz G. Jaskolski M. Alexandratos J. Wlodawer A. Merkel G. Katz R.A. Skalka A.M. J. Mol. Biol. 1995; 253: 333-346Crossref PubMed Scopus (198) Google Scholar, 22Bujacz G. Jaskolski M. Alexandratos J. Wlodawer A. Merkel G. Katz R.A. Skalka A.M. Structure. 1996; 4: 89-96Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar) have been determined by x-ray crystallography. The catalytic residues Asp64, Asp116, and Glu152of HIV-1 integrase and their counterparts in the ASV structures are in close proximity, coordinate divalent metal ion, and define the active site. However, the residues comprising the active site region exhibit considerable flexibility, suggesting that binding of DNA substrate is required to impose the precise configuration of residues that is required for catalysis. The structures of the HIV-1 and ASV integrase core domains are very similar to each other and to the catalytic domain of Mu transposase (23Rice P. Mizuuchi K. Cell. 1995; 82: 209-220Abstract Full Text PDF PubMed Scopus (191) Google Scholar), reinforcing the parallelism of retroviral DNA integration and transposition. These structures revealed that retroviral integrases and their transposase cousins belong to a superfamily of polynucleotidyltransferases that share the same overall fold as Escherichia coli RNase H and have similar active sites (24Yang W. Steitz T.A. Structure. 1995; 3: 131-134Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 25Rice P. Craigie R. Davies D.R. Curr. Opin. Struct. Biol. 1996; 6: 76-83Crossref PubMed Scopus (187) Google Scholar). The HIV-1 catalytic domain is dimeric in solution (26Jenkins T.M. Hickman A.B. Dyda F. Ghirlando R. Davies D.R. Craigie R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6057-6061Crossref PubMed Scopus (141) Google Scholar) and in the crystal structures (Fig. 3 A). The extensive surface area of the dimer interface suggests that it is biologically relevant. Yet, the spacing between the active sites in the nearly spherical dimer is not compatible with the spacing between the sites of insertion on the two strands of target DNA. The sites of insertion on each strand of target DNA are separated by 5 base pairs, corresponding to about 15 Å for helical B-form DNA. The functional unit of integrase should therefore have a pair of active sites separated by a similar spacing. However, in the crystal structures (Fig. 3 A), the active sites in the dimer are separated by more than 30 Å measured as a straight line through the protein and by an even greater distance measured around the circumference of the dimer. Assuming that the dimer interface is maintained in the functional integrase multimer, at least a tetramer of integrase must be required for the complete integration reaction. The N-terminal domain of HIV-1 integrase contains a conserved pair of His and Cys residues, a motif similar to the zinc-coordinating residues of zinc fingers. Although this domain does indeed bind zinc (27Burke C.J. Sanyal G. Bruner M.W. Ryan J.A. LaFemina R.L. Robbins H.L. Zeft A.S. Middaugh C.R. Cordingley M.G. J. Biol. Chem. 1992; 267: 9639-9644Abstract Full Text PDF PubMed Google Scholar, 28Zheng R. Jenkins T.M. Craigie R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13659-13664Crossref PubMed Scopus (309) Google Scholar), its structure (Fig. 3 B) (29Lodi P.J. Ernst J.A. Kuszewski J. Hickman A.B. Engelman A. Craigie R. Clore G.M. Gronenborn A.M. Biochemistry. 1995; 34: 9826-9833Crossref PubMed Scopus (271) Google Scholar) is totally different from that of zinc fingers. It consists of a bundle of three α-helices (Fig. 3 C) (29Lodi P.J. Ernst J.A. Kuszewski J. Hickman A.B. Engelman A. Craigie R. Clore G.M. Gronenborn A.M. Biochemistry. 1995; 34: 9826-9833Crossref PubMed Scopus (271) Google Scholar, 30Eijkelenboom A.P. Lutzke R.A. Boelens R. Plasterk R.H. Kaptein R. Hard K. Nat. Struct. Biol. 1995; 2: 807-810Crossref PubMed Scopus (219) Google Scholar). It has an SH3 fold, although there is no known functional relationship with the SH3 domains of other proteins. Although the core domain of integrase is clearly responsible for catalysis, the functional roles of the other two domains are less clear. The C-terminal domain binds DNA nonspecifically. Because the sites of integration into target DNA are relatively nonspecific, it has been suggested that this domain may interact with target DNA. However, experiments with chimeric integrases (31Katzman M. Sudol M. J. Virol. 1995; 69: 5687-5696Crossref PubMed Google Scholar, 32Shibagaki Y. Chow S.A. J. Biol. Chem. 1997; 272: 8361-8369Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) assign recognition of the target site to the core domain, and cross-linking studies (33Esposito D. Craigie R. EMBO J. 1998; 17: 5832-5843Crossref PubMed Scopus (263) Google Scholar, 34Jenkins T.M. Esposito D. Engelman A. Craigie R. EMBO J. 1997; 16: 6849-6859Crossref PubMed Scopus (220) Google Scholar, 35Heuer T.S. Brown P.O. Biochemistry. 1997; 36: 10655-10665Crossref PubMed Scopus (134) Google Scholar, 36Heuer T.S. Brown P.O. Biochemistry. 1998; 37: 6667-6678Crossref PubMed Scopus (155) Google Scholar) suggest that the C-terminal domain interacts with a subterminal region just inside the very ends of the viral DNA end. The C-terminal domain of retroviral integrases may therefore play a similar role to that of the site-specific DNA binding domain of transposases, which also recognize a subterminal sequence at the ends of the transposon DNA. The function of the N-terminal domain of integrase is at present unknown. Although the structures of all three domains of integrase have been individually determined, their spatial arrangement in the active complex with DNA substrate is unknown. Three retroviral integrase structures of the core together with the C-terminal domain have been recently reported in the absence of bound DNA (37Chen Z.G. Yan Y.W. Munshi S. Li Y. Zugay-Murphy J. Xu B. Witmer M. Felock P. Wolfe A. Sardana V. Emini E.A. Hazuda D. Kuo L.C. J. Mol. Biol. 2000; 296: 521-533Crossref PubMed Scopus (102) Google Scholar, 38Chen J.C.H. Krucinski J. Miercke L.J.W. Finer-Moore J.S. Tang A.H. Leavitt A.D. Stroud R.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8233-8238Crossref PubMed Scopus (383) Google Scholar, 39Yang Z.N. Mueser T.C. Bushman F.D. Hyde C.C. J. Mol. Biol. 2000; 296: 535-548Crossref PubMed Scopus (112) Google Scholar). The spatial relationship between the core and C-terminal domains is different in each of these structures indicating considerable flexibility in the linkage between the two domains. It is likely that binding of viral DNA substrate imposes the proper configuration of domains for the reaction to occur. Although integrase exists as monomers, dimers, and tetramers at high ionic strength, formation of large aggregates under reaction conditions has frustrated attempts to directly determine the organization of the active unit. Efforts to crystallize integrase together with DNA are further challenged by the nonspecific nature of the interaction between integrase and the viral DNA ends. Many transposases, unlike their retroviral integrase counterparts, bind specifically to the transposon ends and form discrete nucleoprotein complexes that are amenable to direct structural and functional analysis. In the case of Mu transposase, the active unit of transposase is tetramer. Within the tetramer, only two of the four active sites directly participate in the DNA cleavage and joining reactions. A dimer of transposase carries out these reactions in the case of Tn5 (40Davies D.R. Goryshin I.Y. Reznikoff W.S. Rayment I. Science. 2000; 289: 77-85Crossref PubMed Scopus (321) Google Scholar) and Tn10. 2D. Haniford, personal communication. Thus, a dimer would seem to constitute the fundamental unit for this class of reactions, and the requirements for higher order multimers in some systems probably reflect differences in regulatory systems rather than a fundamental mechanism. The recent determination of the crystal structure of the Tn5 transposase dimer in complex with DNA substrate (40Davies D.R. Goryshin I.Y. Reznikoff W.S. Rayment I. Science. 2000; 289: 77-85Crossref PubMed Scopus (321) Google Scholar) provides a platform for modeling the interactions of DNA with the catalytic domain of integrase. Because the integrase catalytic domain is itself a dimer with an extensive interface, a similar architecture to that of the Tn5 complex would require a pair of dimers. Then, as in the case of Mu transposase, two of the active sites in the resulting tetramer would not directly participate in catalysis. A puzzling phenomenon is the almost exclusive integration of a single viral DNA substrate into one strand of the DNA target in reactions with purified HIV integrase. In the cell and in vitro with preintegration complexes, integration is coupled so that a pair of viral DNA ends is integrated with a spacing of 5 base pairs separating the sites of insertion on the two strands of target DNA. Although inclusion of additional protein factors in the reaction has been reported to stimulate coupled integration (41Carteau S. Gorelick R.J. Bushman F.D. J. Virol. 1999; 73: 6670-6679Crossref PubMed Google Scholar, 42Hindmarsh P. Leis J. Adv. Virus Res. 1999; 52: 397-410Crossref PubMed Google Scholar), single-end integration events still predominate. It appears that a yet to be understood assembly pathway, and possibly additional factors, are required to reconstitute the integration reaction with the full fidelity observed with preintegration complexes in vitro and in vivo. An alternative possibility is that aggregation of purified integrase disfavors assembly of the correct complexes with two DNA ends poised for integration; such aggregation may normally be prevented by interaction with other components of the preintegration complex. Indeed, it has been shown that improving the solubility of the closely related Tn552 transposase greatly enhances the efficiency of double-endversus single-end strand transfer (43Leschziner A.E. Griffin T.J. Grindley N.D.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7345-7350Crossref PubMed Scopus (20) Google Scholar). Perhaps the strongest suggestion that additional host proteins may not be required is the finding that disrupted HIV-1 virions, which contain few host proteins, support efficient double-end strand transfer of exogenous viral substrate DNA (44Goodarzi G. Im G.J. Brackmann K. Grandgenett D. J. Virol. 1995; 69: 6090-6097Crossref PubMed Google Scholar). The development of effective of HIV replication to reverse transcriptase and has demonstrated the of for the of to integrase would be a to reverse transcriptase and However, no have yet been that on integrase. The has been the absence of to as the for Although many have been reported to integrase, of these and many other enzymes as A has also been that many copies of integrase the cell with the infecting virus and only two are integrase be an enzyme to of a class of a that HIV replication in cell by integrase Felock P. Witmer M. Wolfe A. K. J.A. A. L. W. C. Science. 2000; PubMed Scopus Google Scholar) this and the of integrase as an The structure of a related with the active site of integrase has also recently been determined Y. Craigie R. Davies D.R. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). However, the higher of these for integrase in the of viral DNA A.S. Felock P. Wolfe A. Witmer M. J. M. S. Hazuda Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google Scholar) and for the DNA strand transfer reaction suggest that the binding in the absence and of DNA substrate are not It will likely be to determine the structure of these and other in complex with an integrase active site with DNA to the interaction in and a platform for the of basic knowledge in the mechanism of retroviral integration to in to the for with of the ribbon diagrams and and Adzuma for on the