Host site selection for full-site integration by human immunodeficiency virus type-1 (HIV-1) intergrase (IN) from nonionic detergent-lysed virions was investigated. Linear retrovirus-like DNA (469 bp) possessing 3' OH recessed long terminal repeat termini was efficiently inserted by a bimolecular donor reaction into a supercoiled DNA target (2867 bp), producing the HIV-1 5-bp host site duplication. Sequence data were analyzed from 193 donor-target recombinants obtained from the linear 3.8-kb DNA product. The selection of host target sites appeared randomly distributed and was independent of lysis and assay conditions. The fidelity of the 5-bp duplications in comparison to other size duplications was highest (94%) with high-salt (300 mM NaCl) lysis of the virions and 60 mM NaCl for strand transfer using Mg2+ as the divalent cation. Base sequence analysis demonstrated some biases in the 5-bp duplications at the sites of strand transfer and at the immediate host sequences surrounding the duplications. In addition to the observed duplications, approximately 30% of the recombinants isolated from the linear 3.8-kb DNA product contained specific and repetitive small-size deletions. No deletions smaller that 17 bp were observed and the distance between the deletion sets had a periodicity of approximately 10 bp. The mechanisms involved in how HIV-1 IN produces the 5-bp duplications and the repetitive host site deletions are discussed.

Retroviruses have the capability of inserting their DNA genome into the host DNA by the virus-encoded integrase (IN). IN from different retrovirus species possess three similar structural domains even though their amino acid sequences are significantly different (Fig. 1) (3, 13). The N-terminal domain (ϳ50 residues) contains a zinc binding region (6), promotes IN multimerization (46), and is necessary to catalyze 3Ј OH processing and strand transfer. The 3Ј OH processing activity is responsible for removal of dinucleotides from the catalytic strands on the blunt-ended viral DNA genome. The central or catalytic core domain (ϳ180 residues) contains a conserved triad of amino acids comprising the D,D(35)E motif (28) that is involved in coordinating divalent metal ions for enzymatic activities (2, 13). The core is also involved in target binding for strand transfer (1,21,27). The C-terminal domain (ϳ50 residues) binds to the viral DNA at ϳ7 to 9 bp from the long terminal repeat (LTR) ends (18,26,30), binds to DNA in a nonspecific fashion (3), and also appears to be involved in multimerization of IN (25,30). The reported subunit structure for purified virion IN from avian myeloblastosis virus (AMV) is a dimer (20, 32), while several retrovirus recombinant INs purified from bacteria are observed as monomers, dimers, and tetramers (12,13,14,25).Models describing potential interactions of IN at the viral DNA ends for concerted integration into the host DNA have been proposed (13, 44). The models are based on several approaches, including complementation of IN mutants (6, 17), crystallographic studies of individual or paired domains of IN (5,8,10,42,44), cross-linking studies of IN with DNA substrates (18,19,22,26), and protection of viral DNA ends by IN in preintegration complexes (PICs) (4,7,9,33,43) and in reconstituted nucleoprotein complexes with purified AMV IN with viral DNA substrates (40). The collective theme suggests that IN forms multimers at the viral DNA ends for concerted integration of the two ends into a target, here termed full-site integration.Attempts to compare the mutant retrovirus IN enzymatic activities in vitro to the observed replication and integration capabilities in vivo of retroviruses carrying the same mutations in the IN gene have been difficult for several reasons. First, recombinant wild-type (wt) and mutant IN have either failed to catalyze full-site integration or have done so in a nonefficient manner, thus offering significant insights into only the 3Ј OH processing and half-site integration reactions (3,16,35). Halfsite integration is defined as insertion of one viral DNA end into a target substrate. Second, even though mutations introduced into IN within the context of the viral genome were defective for integration (class I mutants) (16), others produced pleiotropic effects on the replication of retroviruses, indirectly affecting DNA integration in vivo (class II mutants) (3,16).In order to map the functions associated with the different domains in Rous sarcoma virus (RSV) (Prague ...

Integration of linear retrovirus DNA involves the concerted insertion of the viral termini (full-site integration) into the host chromosome. We investigated the interactions that occur between long terminal repeat (LTR) termini bound by avian retrovirus integrase (IN) for full-site integration in vitro. Wild-type (wt) or mutant LTR donors that possess gain-of-function ("G") or loss-of-function ("L") for full-site integration activity were used. G LTR termini are characterized as having significantly higher strand transfer activity than the wt and the L LTR termini. L LTR mutations are classified as partially or extremely defective for strand transfer activity. The L mutations were further classified by their ability to either permit or block the assembly of G or wt LTR termini into nucleoprotein complexes capable of full-site strand transfer. We demonstrated that avian myeloblastosis virus IN bound to G LTR termini increased the incorporation of partially defective L LTR termini into nucleoprotein complexes that were capable of full-site integration. The observed full-site integration activity of these assembled nucleoprotein complexes appeared to be influenced by each individual IN-LTR complex in trans. In contrast, extremely defective L LTR termini exhibited the ability to effectively block the assembly of wt LTR termini into nucleoprotein complexes capable of full-site strand transfer. Data from nonspecific DNA competition experiments suggested that IN had an apparent higher affinity for G LTR donor termini than for partially defective L LTR donor termini as measured by full-site integration activity. However, assembled nucleoprotein complexes containing either two G or two L LTR donors were stable, having a similar half-life of approximately 2 h on ice. The results suggest that LTR termini bound by IN exhibit an allosteric effect to modulate full-site integration in vitro. Similar regulatory controls also appear to exist in vivo between the wt U3 and wt U5 LTR termini in retroviruses as well as purified retrovirus preintegration complexes that promoted full-site integration in vitro.

Concerted integration of retrovirus DNA termini into the host chromosome in vivo requires specific interactions between the cis-acting attachment (att) sites at the viral termini and the viral integrase (IN) in trans.In this study, reconstruction experiments with purified avian myeloblastosis virus (AMV) IN and retroviruslike donor substrates containing wild-type and mutant termini were performed to map the internal att DNA sequence requirements for concerted integration, here termed full-site integration. The avian retrovirus mutations were modeled after internal att site mutations studied at the in vivo level with human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). Systematic overlapping 4-bp deletions starting at nucleotide positions 7, 8, and 9 in the U3 terminus had a decreasing detrimental gradient effect on full-site integration, while more internal 4-bp deletions had little or no effect. This decreasing detrimental gradient effect was measured by the ability of mutant U3 ends to interact with wild-type U3 ends for full-site integration in trans. Modification of the highly conserved C at position 7 on the catalytic strand to either A or T resulted in the same severe decrease in full-site integration as the 4-bp deletion starting at this position. These studies suggest that nucleotide position 7 is crucial for interactions near the active site of IN for integration activity and for communication in trans between ends bound by IN for full-site integration. The ability of AMV IN to interact with internal att sequences to mediate full-site integration in vitro is similar to the internal att site requirements observed with MLV and HIV-1 in vivo and with their preintegration complexes in vitro.The integration of the retrovirus linear DNA genome into the host chromosome is mediated by the retrovirus integrase (IN) (for reviews, see references 4, 17, 24, and 35). The integration process involves the concerted insertion of the viral DNA termini by IN into cellular DNA, here termed full-site integration. In vivo, the first ϳ15 bp of the long terminal repeat (LTR) sequences at the viral DNA termini is required to various degrees for both 3Ј OH trimming of the blunt-ended termini and subsequent insertion of the recessed termini into the host chromosome (3,4,8,10,16,28,32,33). These ϳ15-bp LTR end nucleotides are termed attachment (att) site sequences (4).Several pathways have been used to investigate retrovirus full-site integration at the in vitro level. The most direct route has been by the characterization of preintegration complexes (PIC) isolated from the cytoplasm of newly virus-infected cells (3,5,8,14,25,39). Studies with several retroviruses have suggested that the LTR ends in the PIC are held together by a protein bridge presumably promoted by IN (3, 31, 40), although one or more cellular proteins also may have similar bridging functions (13,26,39,40). Two 3Ј OH recessed termini interact to form a functional PIC for integration in vitro; this entity is termed an intasome (3,8,39,40)...

The U3 and U5 termini of linear retrovirus DNA contain imperfect inverted repeats that are necessary for the concerted insertion of the termini into the host chromosome by viral integrase. Avian myeloblastosis virus integrase can efficiently insert the termini of retroviruslike DNA donor substrates (480 base pairs) by a concerted mechanism (full-site reaction) into circular target DNA in vitro. The specific activities of virion-derived avian myeloblastosis virus integrase and bacterial recombinant Rous sarcoma virus (Prague A strain) integrase (ϳ50 nM or less) appear similar upon catalyzing the full-site reaction with 3-OH recessed wild type or mutant donor substrates. We examined the role of the three nonsymmetrical nucleotides located at the 5th, 8th, and 12th positions in the U3 and U5 15-base pair inverted repeats for their ability to modify the full-site and simultaneously, the half-site strand transfer reactions. Our data suggest that the nucleotide at the 5th position appears to be responsible for the 3-5-fold preference for wild type U3 ends over wild type U5 ends by integrase for concerted integration. Additional mutations at the 5th or 6th position, or both, of U3 or U5 termini significantly increased (ϳ3 fold) the full-site reactions of mutant donors over wild type donors.Upon retrovirus infection, the viral RNA genome is reverse transcribed into a linear blunt ended DNA genome. The retrovirus U3 and U5 DNA termini contain LTR 1 sequences with short imperfect inverted repeats located at the very end of the blunt ended LTRs (1). In vivo, the inverted repeats are necessary for virally encoded integrase to catalyze the removal of a dinucleotide from the 3Ј-OH termini and the subsequent fullsite integration reaction (2). The full-site reaction involves the concerted insertion of the two recessed LTR DNA termini into the host genome. This reaction also results in the formation of a small size host duplication at the site of insertion whose size is virus-specific (1).In vitro, the mechanisms involved in the recognition of the blunt ended LTR termini by integrase for the 3Ј-OH processing reaction and for half-site strand transfer of the recessed LTR termini into target DNA have been investigated (2-10). The half-site reaction involves the insertion of only one LTR terminus into the DNA target. These in vitro analyses using purified integrase from several retrovirus species have established that the imperfect inverted repeat sequences located at the LTR termini are also necessary for catalysis (1,11,12). Besides the essential CA dinucleotide located 2 nucleotides downstream from the blunt ended viral termini, approximately 5-10 nucleotides internally also play varying roles in the 3Ј-OH processing and half-site strand transfer reactions (2, 13).The full-site integration reaction can be catalyzed efficiently using retrovirus-like donor substrates with integrase purified from virions (14, 15). Study of the specific interactions of integrase with the U3 and U5 LTR termini for the full-site integration reaction i...