Juxtaposition of two viral DNA ends in a bimolecular disintegration reaction mediated by multimers of human immunodeficiency virus type 1 or murine leukemia virus integrase.

Integration of retroviral DNA involves a coordinated joining of the two ends of a viral DNA molecule into precisely spaced sites on target DNA. In this study, we designed an assay that requires two separate oligonucleotides to be brought together via interactions between integrase promoters to form a "crossbones" substrate that mimics the integration intermediate. The crossbones substrate contains two viral DNA ends, each joined to one strand of target DNA and separated by a defined length of target DNA. We showed that purified integrases of human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV) could mediate a concerted strand cleavage-ligation between the two half-substrates at one or both viral DNA joining sites (trans disintegration). Another major product, termed fold-back, resulted from an intramolecular attack on the phosphodiester bond at the viral-target DNA junction by the 3'-OH group of the same DNA molecule (cis disintegration). The activity of integrase on the crossbones substrate depended on the presence of viral DNA sequences. For trans disintegration, the optimal length of target DNA between the viral DNA joining sites of the crossbones substrate corresponded to the spacing between the staggered joints formed on two opposite strands of target DNA during retroviral DNA integration in vivo. The activity of integrases on crossbones did not require complementary base pairing between the two half-substrates, indicating that the half-substrates were juxtaposed solely through protein-DNA interactions. The crossbones assay, therefore, measures the ability of integrase to juxtapose two viral DNA ends, an activity which heretofore has been difficult to detect by using purified integrase in conventional assays. Certain mutant integrases that were otherwise inactive with the crossbones substrate could complement one another, indicating that no single protomer in the integrase multimer requires a complete set of functional domains either for catalytic activity or for juxtaposition of the two viral DNA ends by the active multimer.     

Integration of human immunodeficiency virus types 1 and 2 DNA in vitro by cytoplasmic extracts of Moloney murine leukemia virus-infected mouse NIH 3T3 cells.

An essential step in the life cycle of the human immunodeficiency virus (HIV) is integration of a DNA copy of the viral RNA into the genome of the infected cell. We show here that this step can be faithfully accomplished in vitro by the enzymatic machinery of another retrovirus, Moloney murine leukemia virus (MoMLV). Mini-HIV substrates, which are linearized plasmids with long terminal repeat sequences at their ends, were incubated with cytoplasmic extracts of MoMLV-infected NIH 3T3 cells and target DNA. The MoMLV integration apparatus carried out integration of the mini-HIV substrates correctly; the terminal nucleotides of the viral substrate were removed, and a 4-base-pair duplication of the target DNA flanked the inserted viral DNA (C. Shoemaker, S. P. Goff, E. Gilboa, M. Paskind, S. W. Mitra, and D. Baltimore, Proc. Natl. Acad. Sci. USA 77:3932-3936, 1980). Our experiments show that the substrate sequence requirements for integration in vitro were limited to a few nucleotides, as the similarity between HIV and MoMLV long terminal repeat ends is minimal.     

Biochemical Characterization of Novel Retroviral Integrase Proteins

Integrase is an essential retroviral enzyme, catalyzing the stable integration of reverse transcribed DNA into cellular DNA. Several aspects of the integration mechanism, including the length of host DNA sequence duplication flanking the integrated provirus, which can be from 4 to 6 bp, and the nucleotide preferences at the site of integration, are thought to cluster among the different retroviral genera. To date only the spumavirus prototype foamy virus integrase has provided diffractable crystals of integrase-DNA complexes, revealing unprecedented details on the molecular mechanisms of DNA integration. Here, we characterize five previously unstudied integrase proteins, including those derived from the alpharetrovirus lymphoproliferative disease virus (LPDV), betaretroviruses Jaagsiekte sheep retrovirus (JSRV), and mouse mammary tumor virus (MMTV), epsilonretrovirus walleye dermal sarcoma virus (WDSV), and gammaretrovirus reticuloendotheliosis virus strain A (Rev-A) to identify potential novel structural biology candidates. Integrase expressed in bacterial cells was analyzed for solubility, stability during purification, and, once purified, 3′ processing and DNA strand transfer activities in vitro. We show that while we were unable to extract or purify accountable amounts of WDSV, JRSV, or LPDV integrase, purified MMTV and Rev-A integrase each preferentially support the concerted integration of two viral DNA ends into target DNA. The sequencing of concerted Rev-A integration products indicates high fidelity cleavage of target DNA strands separated by 5 bp during integration, which contrasts with the 4 bp duplication generated by a separate gammaretrovirus, the Moloney murine leukemia virus (MLV). By comparing Rev-A in vitro integration sites to those generated by MLV in cells, we concordantly conclude that the spacing of target DNA cleavage is more evolutionarily flexible than are the target DNA base contacts made by integrase during integration. Given their desirable concerted DNA integration profiles, Rev-A and MMTV integrase proteins have been earmarked for structural biology studies.     

Efficiency and Fidelity of Full-Site Integration Reactions Using Recombinant Simian Immunodeficiency Virus Integrase

Full-site integration by recombinant wild-type and mutant simian immunodeficiency virus (SIV) integrase (IN) was investigated with linear retrovirus-like DNA (469 bp) as a donor substrate and circular DNA (2,867 bp) as a target substrate. Under optimized conditions, recombinant SIV IN produced donor-target products consistent with full-site (two donor ends) and half-site (one donor end) reactions with equivalent frequency. Restriction enzyme analysis of the 3.8-kbp full-site reaction products confirmed the concerted insertion of two termini from separate donors into a single target molecule. Donor ends carrying the viral U5 termini were preferred over U3 termini for producing both half-site and full-site products. Bacterial genetic selection was used to isolate individual donor-target recombinants, and the donor-target junctions of the cloned products were characterized by sequencing. Analysis of 149 recombinants demonstrated approximately 84% fidelity for the appropriate simian retrovirus 5-bp host duplication. As seen previously in similar reactions with human immunodeficiency virus type 1 (HIV-1) IN from lysed virions, approximately 8% of the donor-target recombinants generated with recombinant SIV IN incurred specific 17- to 18- or 27- to 29-bp deletions. The efficiency and fidelity of the full-site integration reaction mediated by the purified, recombinant SIV IN is comparable to that of HIV-1 IN from virions. These observations suggest that a purified recombinant lentivirus IN is itself sufficient to recapitulate the full-site integration process.     

Retroviral integration: in vitro host site selection by avian integrase.

Viral integrase catalyzes the integration of the linear viral DNA genome into the chromatin of the infected host cell, an essential step in the life cycle of retroviruses. The reaction produces a characteristic small duplication of host sequences at the site of integration, implying that there is a close juxtaposition of the viral DNA ends during a concerted integration event. We have used an in vitro assay to measure the concerted integration of virus-like plasmid DNA into naked lambda DNA catalyzed by virion purified avian integrase. In contrast to in vivo avian integration, which has strong fidelity for a 6-bp duplication, purified avian integrase in the context of this assay produced a distribution of duplication sizes, with the 6-bp size dominating. The metal cofactor Mg2+ induced increased fidelity for the 6-bp duplication relative to that with Mn2+. The immediate sequence of the host site may also influence duplication size in that we found sites that sustained multiple independent integration events producing the same duplication size. Additionally, for each set of cloned integration sites (5, 6, and 7 bp), a unique but similar symmetrical pattern of G/C and A/T sequence biases was found. Using duplex oligonucleotides as target substrates, we tested the significance of the 6-bp G/C and A/T pattern for site selection. In the context of this assay, which is likely dominated by the integration of only one viral end, the 6-bp pattern was not preferred. Instead, integration was predominantly into the 3' ends of the oligonucleotides. The combined results of the lambda and oligonucleotide assays indicated that although host site selection has properties in common with recognition of the viral DNA termini, the nonrandom sequence preferences seen for host site selection were not identical to the sequence requirements for long terminal repeat recognition.     

Assembly and catalysis of concerted two-end integration events by Moloney murine leukemia virus integrase

Retroviral integration results in the stable and coordinated insertion of the two termini of the linear viral DNA into the host genome. An in vitro concerted two-end integration reaction catalyzed by the Moloney murine leukemia virus (M-MuLV) integrase (IN) was used to investigate the binding and coordination of the two viral DNA ends. Comparison of the two-end integration and strand transfer assays indicates that zinc is required for efficient concerted integration utilizing plasmid DNA as target. Complementation assays using a pair of nonoverlapping integrase domains, consisting of the HHCC domain and the core/C-terminal region, yielded products containing the correct 4-base target site duplication. The efficiency of the coordinated two-end integration varied depending on the order of addition of the individual protein and DNA components in the complementation assay. Two-end integration was most efficient when the long terminal repeat (LTR) was premixed with either the target DNA or the HHCC domain. The preference for two-end integration through preincubation of the HHCC finger with the viral DNA supports the role of this domain in the recognition and/or positioning of the LTR.     

3'-end processing and kinetics of 5'-end joining during retroviral integration in vivo.

Retroviral replication depends on integration of viral DNA into a host cell chromosome. Integration proceeds in three steps: 3'-end processing, the endonucleolytic removal of the two terminal nucleotides from each 3' end of the viral DNA; strand transfer, the joining of the 3' ends of viral DNA to host DNA; and 5'-end joining (or gap repair), the joining of the 5' ends of viral DNA to host DNA. The 5'-end joining step has never been investigated, either for retroviral integration or for any other transposition process. We have developed an assay for 5'-end joining in vivo and have examined the kinetics of 5'-end joining for Moloney murine leukemia virus (MLV). The interval between 3'-end and 5'-end joining is estimated to be less than 1 h. This assay will be a useful tool for examining whether viral or host components mediate 5'-end joining. MLV integrates its DNA only after its host cell has completed mitosis. We show that the extent of 3'-end processing is the same in unsynchronized and aphidicolin-arrested cells. 3'-end processing therefore does not depend on mitosis.     

HIV-1 DNA integration: mechanism of viral DNA cleavage and DNA strand transfer.

Retroviral DNA integration involves a coordinated set of DNA cutting and joining reactions. Linear viral DNA is cleaved at each 3' end to generate the precursor ends for integration. The resulting recessed 3' ends are inserted into target DNA by a subsequent DNA strand transfer reaction. Purified HIV-1 integration protein carries out both of these steps in vitro. Two novel forms of the dinucleotide cleaved from HIV-1 DNA were identified and one, a cyclic dinucleotide, was used to analyze the stereochemical course of viral DNA cleavage. Both viral DNA cleavage and DNA strand transfer display inversion at chiral phosphorothioates during the course of the reaction. These results suggest that both reactions occur by a one-step mechanism without involvement of a covalent protein-DNA intermediate.     

The avian retroviral IN protein is both necessary and sufficient for integrative recombination in vitro

The integration of viral DNA into the host cell chromosome is an essential feature of the retroviral life cycle. The integration reaction requires cis-acting sequences at the ends of linear viral DNA and a trans-acting product of the pol gene, the integration protein (IN). Previously, we demonstrated that avian sarcoma-leukosis virus (ASLV) IN is able to carry out the first step in the integration process in vitro: nicking of the ends of linear viral DNA. In this paper, using two independent assays, we demonstrate that IN, alone, is sufficient to carry out the second step: cleavage and joining to the target DNA. These results demonstrate that the retroviral IN protein is an integrase.     

In vitro activities of purified visna virus integrase.

Although integration generally is considered a critical step in the retrovirus life cycle, it has been reported that visna virus, which causes degenerative neurologic disease in sheep, can productively infect sheep choroid plexus cells without detectable integration. To ascertain whether the integrase (IN) of visna virus is an inherently defective enzyme and to create tools for further study of integration of the phylogenetically related human immunodeficiency virus type 1 (HIV-1), we purified visna virus IN by using a bacterial expression system and applied various in vitro oligonucleotide-based assays to studying this protein. We found that visna virus IN demonstrates the full repertoire of in vitro functions characteristic of retroviral integrases. In particular, visna virus IN exhibits site-specific endonuclease activity following the invariant CA found two nucleotides from the 3' ends of viral DNA (processing activity), joins processed oligonucleotides to various sites on other oligonucleotides (strand transfer or integration activity), and reverses the integration reaction by resolving a complex that mimics one end of viral DNA integrated into host DNA (disintegration activity). In addition, although it has been reported that purified HIV-1 IN cannot specifically nick visna virus DNA ends, purified visna virus IN does specifically process and integrate HIV-1 DNA ends.     

Recombinant human immunodeficiency virus type 1 integrase exhibits a capacity for full-site integration in vitro that is comparable to that of purified preintegration complexes from virus-infected cells

Retrovirus preintegration complexes (PIC) in virus-infected cells contain the linear viral DNA genome (approximately 10 kbp), viral proteins including integrase (IN), and cellular proteins. After transport of the PIC into the nucleus, IN catalyzes the concerted insertion of the two viral DNA ends into the host chromosome. This successful insertion process is termed "full-site integration." Reconstitution of nucleoprotein complexes using recombinant human immunodeficiency virus type 1 (HIV-1) IN and model viral DNA donor substrates (approximately 0.30 to 0.48 kbp in length) that are capable of catalyzing efficient full-site integration has proven difficult. Many of the products are half-site integration reactions where either IN inserts only one end of the viral donor substrate into a circular DNA target or into other donors. In this report, we have purified recombinant HIV-1 IN at pH 6.8 in the presence of MgSO4 that performed full-site integration nearly as efficiently as HIV-1 PIC. The size of the viral DNA substrate was significantly increased to 4.1 kbp, thus allowing for the number of viral DNA ends and the concentrations of IN in the reaction mixtures to be decreased by a factor of approximately 10. In a typical reaction at 37 degrees C, recombinant HIV-1 IN at 5 to 10 nM incorporated 30 to 40% of the input DNA donor into full-site integration products. The synthesis of full-site products continued up to approximately 2 h, comparable to incubation times used with HIV-1 PIC. Approximately 5% of the input donor was incorporated into the circular target producing half-site products with no significant quantities of other integration products produced. DNA sequence analysis of the viral DNA-target junctions derived from wild-type U3 and U5 coupled reactions showed an approximately 70% fidelity for the HIV-1 5-bp host site duplications. Recombinant HIV-1 IN successfully utilized a mutant U5 end containing additional nucleotide extensions for full-site integration demonstrating that IN worked properly under nonideal active substrate conditions. The fidelity of the 5-bp host site duplications was also high with these coupled mutant U5 and wild-type U3 donor ends. These studies suggest that recombinant HIV-1 IN is at least as capable as native IN in virus particles and approaching that observed with HIV-1 PIC for catalyzing full-site integration.     

Efficient concerted integration by recombinant human immunodeficiency virus type 1 integrase without cellular or viral cofactors

Replication of retroviruses requires integration of the linear viral DNA genome into the host chromosomes. Integration requires the viral integrase (IN), located in high-molecular-weight nucleoprotein complexes termed preintegration complexes (PIC). The PIC inserts the two viral DNA termini in a concerted manner into chromosomes in vivo as well as exogenous target DNA in vitro. We reconstituted nucleoprotein complexes capable of efficient concerted (full-site) integration using recombinant wild-type human immunodeficiency virus type I (HIV-1) IN with linear retrovirus-like donor DNA (480 bp). In addition, no cellular or viral protein cofactors are necessary for purified bacterial recombinant HIV-1 IN to mediate efficient full-site integration of two donor termini into supercoiled target DNA. At about 30 nM IN (20 min at 37 degrees C), approximately 15 and 8% of the input donor is incorporated into target DNA, producing half-site (insertion of one viral DNA end per target) and full-site integration products, respectively. Sequencing the donor-target junctions of full-site recombinants confirms that 5-bp host site duplications have occurred with a fidelity of about 70%, similar to the fidelity when using IN derived from nonionic detergent lysates of HIV-1 virions. A key factor allowing recombinant wild-type HIV-1 IN to mediate full-site integration appears to be the avoidance of high IN concentrations in its purification (about 125 microg/ml) and in the integration assay (<50 nM). The results show that recombinant HIV-1 IN may not be significantly defective for full-site integration. The findings further suggest that a high concentration or possibly aggregation of IN is detrimental to the assembly of correct nucleoprotein complexes for full-site integration.     

Comparison of DNA binding and integration half-site selection by avian myeloblastosis virus integrase.

Insertion of the linear retrovirus DNA genome into the host DNA by the virus-encoded integrase (IN) is essential for efficient replication. We devised an efficient virus-like DNA plasmid integration assay which mimics the standard oligonucleotide assay for integration. It permitted us to study, by electron microscopy and sequence analysis, insertion of a single long terminal repeat terminus (LTR half-site) of one plasmid into another linearized plasmid. The reaction was catalyzed by purified avian myeloblastosis virus IN in the presence of Mg2+. The recombinant molecules were easily visualized and quantitated by agarose gel electrophoresis. Agarose gel-purified recombinants could be genetically selected by transformation of ligated recombinants into Escherichia coli HB101 cells. Electron microscopy also permitted the identification and localization of IN-DNA complexes on the virus-like substrate in the absence of the joining reaction. Intramolecular and intermolecular DNA looping by IN was visualized. Although IN preferentially bound to AT-rich regions in the absence of the joining reaction, there was a bias towards GC-rich regions for the joining reaction. Alignment of 70 target site sequences 5' of the LTR half-site insertions with 68 target sites previously identified for the concerted insertion of both LTR termini (LTR full-site reaction) indicated similar GC inflection patterns with both insertional events. Comparison of the data suggested that IN recognized only half of the target sequences necessary for integration with the LTR half-site reaction.     

Concerted integration of retrovirus-like DNA by human immunodeficiency virus type 1 integrase.

The integration of linear retrovirus DNA by the viral integrase (IN) into the host chromosome occurs by a concerted mechanism (full-site reaction). IN purified from avian myeloblastosis virus and using retrovirus-like DNA restriction fragments (487 bp in length) as donors and circular DNA (pGEM-3) as the target can efficiently catalyze that reaction. Nonionic detergent lysates of purified human immunodeficiency virus type 1 (HIV-1) virions were also capable of catalyzing the concerted integration reaction. The donor substrates were restriction fragments (469 bp) containing either U3-U5 (H-2 donor) or U5-U5 (H-5 donor) long terminal repeat sequences at their ends. As was shown previously with bacterially expressed HIV-1 IN, the U5 terminus of H-2 was preferred over the U3 terminus by virion-associated IN. The reactions involving two donors per circular target by HIV-1 IN preferred Mg2+ over Mn2+. Both metal ions were equally effective for the circular half-site reaction involving only one donor molecule. The linear 3.8-kbp recombinant products produced from two donor insertions into pGEM were genetically selected, and the donor-target junctions of individual recombinants were sequenced. A total of 55% of the 87 sequenced recombinants had host site duplications of between 5 and 7 bp, with the HIV-1 5-bp-specific duplication predominating. The other recombinants that migrated at the linear 3.8-kbp position were mainly small deletions that were grouped into four sets of 17, 27, 40, and 47 bp, each having a periodicity mimicking a turn of the DNA helix. Aprotic solvents (dimethyl sulfoxide and 1,4-dioxane) enhanced both the half-site and the linear 3.8-kbp strand transfer reactions which favored low-salt conditions (30 mM NaCl). The order of addition of the donor and target during preincubation with HIV-1 IN on ice did not affect the quantity of linear 3.8-kbp recombinants relative to that of the circular half-site products that were produced; only the quantity of donor-donor versus donor-target recombinants was affected. The presence of Mg2+ in the preincubation mixtures containing donor and target substrates was not necessary for the stability of preintegration complexes on ice or at 22 degrees C. Comparisons of the avian and HIV-1 concerted integration reactions are discussed.     

Structural insights into the retroviral DNA integration apparatus

Retroviral replication depends on successful integration of the viral genetic material into a host cell chromosome. Virally encoded integrase, an enzyme from the DDE(D) nucleotidyltransferase superfamily, is responsible for the key DNA cutting and joining steps associated with this process. Insights into the structural and mechanistic aspects of integration are directly relevant for the development of antiretroviral drugs. Recent breakthroughs have led to biochemical and structural characterization of the principal integration intermediates revealing the tetramer of integrase that catalyzes insertion of both 3' viral DNA ends into a sharply bent target DNA. This review discusses the mechanism of retroviral DNA integration and the mode of action of HIV-1 integrase strand transfer inhibitors in light of the recent visualization of the prototype foamy virus intasome, target DNA capture and strand transfer complexes.     

Retroviral DNA Integration

DNA integration is a unique enzymatic process shared by all retroviruses and retrotransposons. During integration, double-stranded linear viral DNA is inserted into the host genome in a process catalyzed by the virus-encoded integrase (IN). The mechanism involves a series of nucleophillic attacks, the first of which removes the terminal 2 bases from the 3′ ends of the long terminal repeats and of the second which inserts the viral DNA into the host genome. IN specifically recognizes the DNA sequences at the termini of the viral DNA, juxtaposing both ends in an enzyme complex that inserts the viral DNA into a single site in a concerted manner. Small duplications of the host DNA, characteristic of the viral IN, are found at the sites of insertion. At least two host proteins, HMG-I(Y) and BAF, have been shown to increase the efficiency of the integration reaction.     

The HIV-1 integrase monomer induces a specific interaction with LTR DNA for concerted integration

The assembly mechanism for the human immunodeficiency virus type 1 (HIV) synaptic complex (SC) capable of concerted integration is unknown. Molecular and structural studies have established that the HIV SC and prototype foamy virus (PFV) intasome contain a tetramer of integrase (IN) that catalyzes concerted integration. HIV IN purified in the presence of 1 mM EDTA and 10 mM MgSO(4) was predominately a monomer. IN efficiently promoted concerted integration of micromolar concentrations of 3'-OH recessed and blunt-ended U5 long terminal repeat (LTR) oligonucleotide (ODN) substrates (19-42 bp) into circular target DNA. Varying HIV IN to U5 DNA showed that an IN dimer:DNA end molar ratio of 1 was optimal for concerted integration. Integration activities decreased with an increasing length of the ODN, starting from the recessed 18/20 or 19/21 bp set to the 31/33 and 40/42 bp set. Under these conditions, the average fidelity for the HIV 5 bp host site duplication with recessed and blunt-ended substrates was 56%. Modifications of U5 LTR sequences beyond 21 bp from the terminus on longer DNA (1.6 kb) did not alter the ~32 bp DNaseI protective footprint, suggesting viral sequences beyond 21 bp were not essential for IN binding. The results suggest IN binds differentially to an 18/20 bp than to a 40/42 bp ODN substrate for concerted integration. The HIV IN monomer may be a suitable candidate for attempting crystallization of an IN-DNA complex in the absence or presence of strand transfer inhibitors.