The purified integration protein (IN) of avian myeloblastosis virus is shown to nick double-stranded oligodeoxynucleotide substrates that mimic the ends of the linear form of viral DNA. In the presence of Mg2+, nicks are created 2 nucleotides from the 3' OH ends of both the U5 plus strand and the U3 minus strand. Similar cleavage is observed in the presence of Mn2' but only when the extent of the reaction is limited. Neither

Drug resistance conferred by specific human immunodeficiency virus type 1 (HIV-1) pol gene mutations has been associated with clinical progression in HIV-infected patients receiving anti-retroviral therapy. This study examined drug susceptibilities and pol mutations of HIV-1 strains from patients treated for 1 year with zidovudine, didanosine (ddI), or zidovudine and ddI. Ten (42%) of 24 patients receiving combination therapy versus 8/26 (31%) receiving only zidovudine had HIV-1 strains with phenotypic zidovudine resistance or a zidovudine resistance pol mutation at codon 215 (P = .6). In contrast, a ddI resistance mutation at codon 74 was less common among patients receiving combination therapy (2/24) than among those receiving ddI only (17/26; P < .001). Two patients receiving combination therapy developed resistance to zidovudine and ddI; they had HIV strains with amino acid mutations at codons 62, 75, 77, 116, and 151. Combination therapy with zidovudine and ddI selects for zidovudine-resistant HIV-1 strains lacking a ddI resistance mutation and for multidrug-resistant strains containing novel pol mutations.

A secondary structure in the 5' noncoding region of avian retrovirus RNA, called the U5-leader stem, was shown previously to have a role in initiation of reverse transcription (D. Cobrinik, L. Soskey, and J. Leis, J. Virol. 62:3622-3630, 1988). We now show that an additional RNA secondary structure near the U5 terminus, called the U5-IR stem, is also important for reverse transcription. Mutations that disrupt the U5-IR stem cause a replication defect associated with both a decrease in synthesis of viral DNA in infected cells and a decrease in initiation of reverse transcription in melittin-permeabilized virions. Structure-compensating base substitutions in the U5-IR restore reverse transcription efficiency. In viral DNA, U5-IR sequences are included in the U5 term'inal region that functions as a viral integration donor site. When base substitutions are introduced into these sequences, a reduced efficiency of integration in vitro and in vivo is observed. These observations indicate that U5-IR sequences have a structural role in reverse transcription of viral RNA and a sequence-specific role in the integration of viral DNA.

Human immunodeficiency virus type 1 (HIV-1) and visna virus integrases were purified from a bacterial expression system and assayed on oligonucleotide substrates derived from each terminus of human immunodeficiency virus type 1 and visna virus linear DNA. Three differences between the proteins were identified, including levels of specific 3-end processing, patterns of strand transfer, and target site preferences. To map domains of integrase (IN) responsible for viral DNA specificity and target site selection, we constructed and purified chimeric proteins in which the N-terminal, central, and C-terminal regions of these lentiviral integrases were exchanged. All six chimeric proteins were active for disintegration, demonstrating that the active site in the central region of each chimera maintained a functional conformation. Analysis of endonucleolytic processing activity indicated that the N terminus of IN does not contribute to viral DNA specificity; this function must reside in the central region or C terminus of IN. In the viral DNA integration assay, chimeric proteins gave novel patterns of strand transfer products which did not match that of either wild-type IN. Thus, target site selection with a viral DNA terminus as nucleophile could not be mapped to regions of IN defined by these boundaries and may involve interactions between regions. In contrast, when target site preferences were monitored with a new assay in which glycerol stimulates IN-mediated cleavage of nonviral DNA, chimeras clearly segregated between the two wild-type patterns. Target site selection for this nonspecific alcoholysis activity mapped to the central region of IN. This report represents the first detailed description of functional chimeras between any two retroviral integrases.

No abstract

To identify parts of retroviral integrase that interact with cellular DNA, we tested patient-derived human immunodeficiency virus type 1 (HIV-1) integrases for alterations in the choice of nonviral target DNA sites. This strategy took advantage of the genetic diversity of HIV-1, which provided 75 integrase variants that differed by a small number of amino acids. Moreover, our hypothesis that biological pressures on the choice of nonviral sites would be minimal was validated when most of the proteins that catalyzed DNA joining exhibited altered target site preferences. Comparison of the sequences of proteins with the same preferences then guided mutagenesis of a laboratory integrase. The results showed that single amino acid substitutions at one particular residue yielded the same target site patterns as naturally occurring integrases that included these substitutions. Similar results were found with DNA joining reactions conducted with Mn 2؉ or with Mg 2؉and were confirmed with a nonspecific alcoholysis assay. Other amino acid changes at this position also affected target site preferences. Thus, this novel approach has identified a residue in the central domain of HIV-1 integrase that interacts with or influences interactions with cellular DNA. The data also support a model in which integrase has distinct sites for viral and cellular DNA.

Integrase can insert retroviral DNA into almost any site in cellular DNA; however, target site preferences are noted in vitro and in vivo. We recently demonstrated that amino acid 119, in the ␣2 helix of the central domain of the human immunodeficiency virus type 1 integrase, affected the choice of nonviral target DNA sites. We have now extended these findings to the integrases of a nonprimate lentivirus and a more distantly related alpharetrovirus. We found that substitutions at the analogous positions in visna virus integrase and Rous sarcoma virus integrase changed the target site preferences in five assays that monitor insertion into nonviral DNA. Thus, the importance of this protein residue in the selection of nonviral target DNA sites is likely to be a general property of retroviral integrases. Moreover, this amino acid might be part of the cellular DNA binding site on integrase proteins.

Purified retroviral integrase (IN) from avian sarcoma-4eukosis viruses can appropriately process the termini of linear viral DNA, cleave host DNA in a sequenceindependent manner, and catalyze integrative recombination; an exogenous source of energy is not required for these reactions. Using DNA substrates containing radioactive phosphate groups, we demonstrate that IN becomes covalently joined to the new 5' phosphate ends of DNA produced at sites of cleavage. Most of the phosphodiester linkages between IN and DNA involve serine, but some involve threonine. Computer-assisted alignment of 80 retroviral and retrotransposon IN sequences identified one serine that is conserved in all of these proteins and three less-conserved threonine residues. These results identify candidate active-site residues and provide support for the participation of a covalent IN-DNA intermediate in retroviral integration.Integration of a double-stranded DNA copy of the retroviral genome into host-cell chromosomal DNA requires cis-acting sequences at the ends of viral DNA and results in loss of 2 terminal viral base pairs (bp) that follow the invariant C-A dinucleotide. Although there is no sequence specificity for host integration sites, integration produces a characteristic duplication offlanking host base pairs (1, 2). These alterations of viral and host DNA suggest that the process of integration requires at least two nuclease activities with different specificities-i.e., sequence-specific cleavage of viral DNA that removes precisely two nucleotides from each terminus and sequence-independent staggered cleavage of host DNA at the site of insertion of the provirus. The pol gene-encoded integrase (IN) protein, shown by genetic experiments to be required for integration (1, 2), can perform both types of endonucleolytic cleavages necessary for retroviral integration (3-5) and can catalyze joining of viral long terminal repeat (LTR) sequences to target DNA in vitro (6-8). Because results from cell-free assays showed that a source of exogenous energy is not required forjoining viral to host DNA (6, 7, 9, 10), we examined our previously described oligodeoxynucleotide assay system for evidence ofa protein-DNA intermediate that might conserve energy from a DNA-cleavage event and utilize it for a DNA-joining reaction. We report here that avian sarcoma-leukosis virus IN forms a covalent complex involving serine, and to a lesser extent threonine, linked to 5' phosphate ends produced at sites of DNA cleavage.MATERIALS AND METHODS Purification of IN. IN was purified to homogeneity from avian myeloblastosis virus (AMV) and from Rous sarcoma virus (RSV) sequences expressed in bacteria as described (3, 4). AMV IN was able to nick 23 pmol of substrate DNA per