A mutation in integrase can compensate for mutations in the simian immunodeficiency virus att site.

Sequences at the left terminus of U3 in the left long terminal repeat (LTR) and at the right terminus of U5 in the right LTR are important for integration of retroviral DNA. In the infectious pathogenic molecular clone of simian immunodeficiency virus strain mac239 (SIVmac239), 10 of the 12 terminal base pairs form an imperfect inverted repeat structure (5' TGGAAGGGATTT 3' [nucleotides 1 to 12] and 3' ACGATCCCTAAA 5' [nucleotides 10279 to 10268]). Nineteen different mutant forms of SIVmac239 proviral DNA with changes at one or more of the positions in each of the 12-terminal-base-pair regions were constructed. Viral replication was severely or completely compromised with nine of these mutants. Revertants appeared 40 to 50 days after transfection in two independent experiments with mutant 7, which contained changes of AGG to TAC at positions 5 to 7 in U3 and TCC to GAA at positions 10275 to 10273 in U5. Virus produced at these times from mutant 7 transfection replicated upon reinfection with only a slight delay when compared to the wild type. Sequence analysis of the LTR and integrase regions from infected cultures revealed two predominant changes: G to A at position 10275 in U5 and Glu to Lys at position 136 in integrase. Derivatives of clone 7 in which these changes were introduced individually and together were constructed by site-specific mutagenesis. Each change individually restored replication capacity only partially. However, the combination of both mutations restored replicative capacity to that of the original revertants. These results indicate that changes in integrase can compensate for mutations in the terminal nucleotides of the SIV LTR. The results further indicate that resistance to integrase inhibitors may include both integrase and LTR mutations.     

Mutations in the U5 sequences adjacent to the primer binding site do not affect tRNA cleavage by rous sarcoma virus RNase H but do cause aberrant integrations in vivo

In most retroviruses, the first nucleotide added to the tRNA primer becomes the right end of the U5 region in the right long terminal repeat (LTR); the removal of this tRNA primer by RNase H defines the right end of the linear double-stranded DNA. Most retroviruses have two nucleotides between the 5' end of the primer binding site (PBS) and the CA dinucleotide that will become the end of the integrated provirus. However, human immunodeficiency virus type 1 (HIV-1) has only one nucleotide at this position, and HIV-2 has three nucleotides. We changed the two nucleotides (TT) between the PBS and the CA dinucleotide of the Rous sarcoma virus (RSV)-derived vector RSVP(A)Z to match the HIV-1 sequence (G) and the HIV-2 sequence (GGT), and we changed the CA dinucleotide to TC. In all three mutants, RNase H removes the entire tRNA primer. Sequence analysis of RSVP(HIV2) proviruses suggests that RSV integrase can remove three nucleotides from the U5 LTR terminus of the linear viral DNA during integration, although this mutation significantly reduced virus titer, suggesting that removing three nucleotides is inefficient. However, the results obtained with RSVP(HIV1) and RSVP(CATC) show that RSV integrase can process and integrate the normal U3 LTR terminus of a linear DNA independently of an aberrant U5 LTR terminus. The aberrant end can then be joined to the host DNA by unusual processes that do not involve the conserved CA dinucleotide. These unusual events generate either large duplications or, less frequently, deletions in the host genomic DNA instead of the normal 5- to 6-base duplications.     

Substrate specificity of recombinant human immunodeficiency virus integrase protein.

Recombinant human immunodeficiency virus type 1 (HIV-1) integrase (IN) produced in Escherichia coli efficiently cleaves two nucleotides from the 3' end of synthetic oligonucleotide substrates which mimic the termini of HIV-1 proviral DNA. Efficient cleavage was restricted to HIV-1 substrates and did not occur with substrates derived from other retroviruses. Mutagenesis of the U5 long terminal repeat (LTR) terminus revealed only moderate effects of mutations outside the terminal four bases of the U5 LTR and highlighted the critical nature of the conserved CA dinucleotide motif shared by all retroviral termini. Integration of the endonuclease cleavage products occurs subsequent to cleavage, and evidence that the cleavage and integration reactions may be uncoupled is presented. Competition cleavage reactions demonstrated that IN-mediated processing of an LTR substrate could be inhibited by competition with LTR and non-LTR oligonucleotides.     

An integration-defective U5 deletion mutant of human immunodeficiency virus type 1 reverts by eliminating additional long terminal repeat sequences.

Nonoverlapping deletions that eliminated the 5' (HIV-1US/603del), middle (HIV-1U5/206del), and 3' (HIV-1U5/604del) thirds of the U5 region of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) were studied for their effects on virus replication (transient transfection of HeLa cells) and infectivity (T-cell lines and peripheral blood mononuclear cells). All three mutants exhibited a wild-type phenotype in directing the production and release of virus particles from transfected HeLa cells. In infectivity assays, HIV-1U5/206del was usually indistinguishable from wild-type virus whereas HIV-1U%/603del was unable to infect human peripheral blood mononuclear cells or MT4 and CEM cells. Investigations of HIV-1U5/603del particles revealed a packaging defect resulting in a 10-fold reduction of encapsidated genomic RNA. The HIV-1U5/604del mutant either was noninfectious or exhibited delayed infection kinetics, depending on the cell type and multiplicity of infection. Quantitative competitive PCR indicated that HIV-1U5/604del synthesized normal amounts of viral DNA in newly infected cells. During the course of a long-term infectivity assay, a revertant of the HIV-1U5/604del mutant that displayed rapid infection kinetics emerged. Nucleotide sequence analysis indicated that the original 26-nucleotide deletion present in HIV-1U5/604del had been extended an additional 19 nucleotides in the revertant virus. Characterization of the HIV-1U5/604del mutant LTR in in vitro integration reactions revealed defective 3' processing and strand transfer activities that were partially restored when the revertant LTR substrate was used, suggesting that the reversion corrected a similar defect in the mutant virus.     

Molecular cloning of viral DNA from leukemogenic Gross passage A murine leukemia virus and nucleotide sequence of its long terminal repeat.

The viral DNA genome of the leukemogenic Gross passage A virus was cloned in phage Charon 21A as an infectious molecule. The virus recovered by transfection with this infectious DNA was ecotropic, N-tropic, fibrotropic, and XC+. It was leukemogenic when reinjected into newborn SIM mice, indicating that ecotropic murine leukemia virus (MuLV) from an AKR mouse thymoma can harbor leukemogenic sequences. Its restriction map was similar to that of nonleukemogenic AKR MuLV, its putative parent, but differed at the 3' end and in the long terminal repeat (LTR). The nucleotide sequence of the Gross A virus LTR was identical to the AKR MuLV LTR sequence (Van Beveren et al., J. Virol. 41:542-556, 1982) in U5, R, and part of U3. All differences between both LTRs were found in U3. Only one copy of the U3 tandem direct repeat was conserved in the Gross A virus LTR, and it was rearranged by the insertion of a 36-base-pair sequence and by five point mutations. Only one additional point mutation common to several oncogenic MuLVs was present in U3. These structural changes in the U3 LTR and at the 3' end of the genome may be related to the leukemogenicity of this virus.     

Influence of substrate structure on disintegration activity of Moloney murine leukemia virus integrase.

The disintegration activity of Moloney murine leukemia virus (M-MuLV) integrase (IN) was investigated through structural and sequence modifications of a Y substrate that resembles an integration intermediate. The Y substrates, constructed from individual oligonucleotides, contain a single viral long terminal repeat (LTR) joined to a nicked target DNA. Truncation of the double-stranded LTR sequences distal to the conserved 5'-CA-3' dinucleotide progressively diminished disintegration activity. M-MuLV IN was also able to catalyze disintegration of a heterologous double-stranded LTR sequence. Significantly, the activity of M-MuLV IN on single-stranded LTR Y substrates was more dependent on the sequence and length of the LTR strand than that reported for human immunodeficiency virus type 1 (HIV-1) IN. Modifications introduced at the Y-substrate junction demonstrated that the 3'-hydroxyl group at the terminus of the target strand was necessary for efficient joining of the target DNA strands. The presence of a 2'-hydroxyl group at the 3' end of the target strand, as well as a single-nucleotide gap at the LTR-target junction, reduced disintegration activity. The absence of hydroxyl groups on the terminal nucleotide abolished joining of the target strands. The results presented here suggest that M-MuLV IN disintegration activity is dependent on substantially different LTR sequence requirements than those reported for HIV-1 IN and may be mediated primarily through a structural recognition event.     

Efficient replication, integration, and packaging of retroviral vectors with modified long terminal repeats containing the packaging signal.

Retroviral vectors were modified to contain packaging (psi) signals of varying lengths (nucleotides 211-355, 211-565, or 211-1039 of MoMuLV RNA) between the U3-r and U5 sequences of their 5' long terminal repeat (LTR). For the vector MoTN-PR3, containing the full length 211-1039 nucleotide-long psi signal within the 5' LTR, replication, integration, and packaging were almost as efficient as for the original unmodified vector. This result confirmed that the 211-1039 nucleotide-long sequence from the MoMuLV RNA is sufficient and necessary to allow efficient packaging of RNAs. In addition, an important site was revealed where insertion of foreign DNA sequences of up to 829 nucleotides can be made within the 5' LTR, between U3-r and U5 sequences, without affecting viral replication, integration, or packaging.     

Pre-organized structure of viral DNA at the binding-processing site of HIV-1 integrase

The integration of the human immunodeficiency virus type 1 DNA into the host cell genome is catalysed by the viral integrase (IN). The reaction consists of a 3'-processing [dinucleotide released from each 3' end of the viral long terminal repeat (LTR)] followed by a strand transfer (insertion of the viral genome into the human chromosome). A 17 base pair oligonucleotide d(GGAAAATCTCTAGCAGT), d(ACTGCTAGAGATTTTCC) reproducing the U5-LTR extremity of viral DNA that contains the IN attachment site was analysed by NMR using the classical NOEs and scalar coupling constants in conjunction with a small set of residual dipolar coupling constants (RDCs) measured at the 13C/15N natural abundance. The combination of these two types of parameters in calculations significantly improved the DNA structure determination. The well-known features of A-tracts were clearly identified by RDCs in the first part of the molecule. The binding/cleavage site at the viral DNA end is distinguishable by a loss of regular base stacking and a distorted minor groove that can aid its specific recognition by IN.     

Activity of recombinant HIV-1 integrase on mini-HIV DNA

Integration of the human immunodeficiency virus type 1 (HIV-1) cDNA into the genome of a human cell is an essential step in the viral replication cycle. Understanding of the integration process has been facilitated by the development of in vitro assays using specific oligonucleotides and recombinant integrase. However, understanding of the biology of retroviral integration will require in vitro and in vivo model systems using long DNA substrates that mimic the HIV cDNA. We have now studied the activity of recombinant HIV-1 integrase on a linear 4.7 kb double-stranded DNA, containing flanking regions of approximately 200 bp that represent the intact ends of the HIV-1 long terminal repeat (LTR) sequences (mini-HIV). The strand transfer products of the integration reaction can be directly visualized after separation in agarose gels by ethidium bromide staining. The most prominent reaction product resulted from integration of one LTR end into another LTR end (U5 into U5 and U5 into U3). Sequence analysis of the reaction products showed them to be products of legitimate integration preceded by correct processing of the viral LTR ends. Hotspots for integration were detected. Electron microscopy revealed the presence of a range of reaction products resulting from single or multiple integration events. The binding of HIV-1 integrase to mini-HIV DNA was visualized. Oligomers of integrase seem to induce DNA looping whereby the enzyme often appears to be bound to the DNA substrate that adopts the structure of a three-site synapsis that is reminiscent of the Mu phage transposase complex.