Integrase of Mason-Pfizer monkey virus

The gene encoding an integrase of Mason-Pfizer monkey virus (M-PMV) is located at the 3'-end of the pol open reading frame. The M-PMV integrase has not been previously isolated and characterized. We have now cloned, expressed, isolated, and characterized M-PMV integrase and compared its activities and primary structure with those of HIV-1 and other retroviral integrases. M-PMV integrase prefers untranslated 3'-region-derived long-terminal repeat sequences in both the 3'-processing and the strand transfer activity assays. While the 3'-processing reaction catalyzed by M-PMV integrase was significantly increased in the presence of Mn(2+) and Co(2+) and was readily detectable in the presence of Mg(2+) and Ni(2+) cations, the strand transfer activity was strictly dependent only on Mn(2+). M-PMV integrase displays more relaxed substrate specificity than HIV-1 integrase, catalyzing the cleavage and the strand transfer of M-PMV and HIV-1 long-terminal repeat-derived substrates with similar efficiency. The structure-based sequence alignment of M-PMV, HIV-1, SIV, and ASV integrases predicted critical amino acids and motifs of M-PMV integrase for metal binding, interaction with nucleic acids, dimerization, protein structure maintenance and function, as well as for binding of human immunodeficiency virus type 1 and Rous avian sarcoma virus integrase inhibitors 5-CI-TEP, DHPTPB and Y-3.     

Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases.

Our comparison of deduced amino acid sequences for retroviral/retrotransposon integrase (IN) proteins of several organisms, including Drosophila melanogaster and Schizosaccharomyces pombe, reveals strong conservation of a constellation of amino acids characterized by two invariant aspartate (D) residues and a glutamate (E) residue, which we refer to as the D,D(35)E region. The same constellation is found in the transposases of a number of bacterial insertion sequences. The conservation of this region suggests that the component residues are involved in DNA recognition, cutting, and joining, since these properties are shared among these proteins of divergent origin. We introduced amino acid substitutions in invariant residues and selected conserved and nonconserved residues throughout the D,D(35)E region of Rous sarcoma virus IN and in human immunodeficiency virus IN and assessed their effect upon the activities of the purified, mutant proteins in vitro. Changes of the invariant and conserved residues typically produce similar impairment of both viral long terminal repeat (LTR) oligonucleotide cleavage referred to as the processing reaction and the subsequent joining of the processed LTR-based oligonucleotides to DNA targets. The severity of the defects depended upon the site and the nature of the amino acid substitution(s). All substitutions of the invariant acidic D and E residues in both Rous sarcoma virus and human immunodeficiency virus IN dramatically reduced LTR oligonucleotide processing and joining to a few percent or less of wild type, suggesting that they are essential components of the active site for both reactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional identification of nucleotides conferring substrate specificity to retroviral integrase reactions.

The long terminal repeats (LTRs) that flank the retroviral DNA genome play a distinct role in the integration process by acting as specific substrates for the integrase (IN). The role of LTR sequences in providing substrate recognition and specificity to integration reactions was investigated for INs from human immunodeficiency virus type 1 (HIV-1), Moloney murine leukemia virus (M-MuLV), human T-cell leukemia virus type 1 (HTLV-1), and human T-cell leukemia virus type 2 (HTLV-2). Overall, these INs required specific LTR sequences for optimal catalysis of 3'-processing reactions, as opposed to strand transfer and disintegration reactions. It is of particular note that in strand transfer reactions the sites of integration were similar among the four INs. In the 3'-processing reaction, sequence specificity for each IN was traced to the three nucleotides proximal to the conserved CA. Reactions catalyzed by M-MuLV IN were additionally influenced by upstream regions. The nucleotide requirements for optimal catalysis differed for each IN. HIV-1 IN showed a broad range of substrate specificities, while HTLV-1 IN and HTLV-2 IN had more defined sequence requirements. M-MuLV IN exhibited greater activity with the heterologous LTR substrates than with its own wild-type substrate. This finding was further substantiated by the high levels of activity catalyzed by the IN on modified M-MuLV LTRs. This work suggests that unlike the other INs examined, M-MuLV IN has evolved with an IN-LTR interaction that is suboptimal.     

Effect of serine and tyrosine phosphorylation on retroviral proteinase substrates.

Vimentin, a cellular substrate of HIV type 1 (HIV-1) proteinase, contains a protein kinase C (PKC) phosphorylation site at one of its cleavage sites. Peptides representing this site were synthesized in P2 Ser-phosphorylated and nonphosphorylated forms. While the nonphosphorylated peptide was a fairly good substrate of the enzyme, phosphorylation prevented hydrolysis. Phosphorylation of human recombinant vimentin by PKC prevented its processing within the head domain, where the phosphorylation occurred. Oligopeptides representing naturally occurring cleavage sites at the C-terminus of the Rous sarcoma virus integrase were assayed as substrates of the avian proteinase. Unlike the nonphosphorylated peptides, a Ser-phosphorylated peptide was not hydrolyzed by the enzyme at the Ser-Pro bond, suggesting the role of previously established phosphorylation in processing at this site. Ser-phosphorylated and Tyr-phosphorylated forms of model substrates were also tested as substrates of the HIV-1 and the avian retroviral proteinases. In contrast to the moderate effect of P4 Ser phosphorylation, phosphorylation of P1 Tyr prevented substrate hydrolysis by HIV-1 proteinase. Substrate phosphorylation had substantially smaller effects on the hydrolysis by the avian retroviral proteinase. As the active retroviral proteinase as well as various protein kinases are incorporated into mature virions, substrate phosphorylation resulting in attenuation or prevention of proteolytic processing may have important consequences in the regulation of the retroviral life cycle as well as in virus-host cell interactions.     

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.     

Defining the DNA Substrate Binding Sites on HIV-1 Integrase

A tetramer model for human immunodeficiency virus type 1 (HIV-1) integrase (IN) with DNA representing long terminal repeat (LTR) termini was previously assembled to predict the IN residues that interact with the LTR termini; these predictions were experimentally verified for nine amino acid residues [Chen, A., Weber, I. T., Harrison, R. W. & Leis, J. (2006). Identification of amino acids in HIV-1 and avian sarcoma virus integrase subsites required for specific recognition of the long terminal repeat ends. J. Biol. Chem., 281, 4173-4182]. In a similar strategy, the unique amino acids found in avian sarcoma virus IN, rather than HIV-1 or Mason-Pfizer monkey virus IN, were substituted into the structurally related positions of HIV-1 IN. Substitutions of six additional residues (Q44, L68, E69, D229, S230, and D253) showed changes in the 3′ processing specificity of the enzyme, verifying their predicted interaction with the LTR DNA. The newly identified residues extend interactions along a 16-bp length of the LTR termini and are consistent with known LTR DNA/HIV-1 IN cross-links. The tetramer model for HIV-1 IN with LTR termini was modified to include two IN binding domains for lens-epithelium-derived growth factor/p75. The target DNA was predicted to bind in a surface trench perpendicular to the plane of the LTR DNA binding sites of HIV-1 IN and extending alongside lens-epithelium-derived growth factor. This hypothesis is supported by the in vitro activity phenotype of HIV-1 IN mutant, with a K219S substitution showing loss in strand transfer activity while maintaining 3′ processing on an HIV-1 substrate. Mutations at seven other residues reported in the literature have the same phenotype, and all eight residues align along the length of the putative target DNA binding trench.     

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.     

Transfer of active oncogenes and promoters into the mouse cell genome

Different cell DNA's (normal NIH 3T3 DNA; human osteosarcoma cell DNA; human malignant glioma cell DNA with amplified c-Ha-ras) were cotransfected onto NIH 3T3 cells with cloned long terminal repeat (LTR) sequences of Rous sarcoma virus. LTR RSV and normal NIH 3T3 DNA c-fos oncogen expression was detected in tumors induced in nude mice. In the same system human tumour cell DNA with amplified c-Ha-ras gene was used, that to the integration and amplification of LTP sequences with simultaneous maintenance of c-Ha-ras amplification. Nude mouse tumour DNA with integrated LTR sequences was active in successive rounds of transfection.     

Helper-independent retrovirus vectors with Rous-associated virus type O long terminal repeats.

We have constructed nonpermuted replication-competent avian retrovirus vectors that derive from Rous sarcoma virus (S. H. Hughes, J. J. Greenhouse, C. J. Petropoulos, and P. Sutrave, J. Virol. 61:3004-3012, 1987). We describe here the construction and properties of corresponding vectors in which the long terminal repeats (LTRs) of the parental virus have been replaced by the LTRs of the endogenous chicken virus Rous-associated virus type O. The Rous-associated virus type O LTR vectors replicated approximately 1/10 as well as the parental vectors and expressed a test gene, chloramphenicol acetyltransferase, approximately 1/30 to 1/50 as well.     

Avian retroviral long terminal repeats bind CCAAT/enhancer-binding protein.

DNA-protein interactions involving enhancer and promoter sequences within the U3 regions of several avian retroviral long terminal repeats (LTRs) were studied by DNase I footprinting. The rat CCAAT/enhancer-binding protein, C/EBP, bound to all four viral LTRs examined. The Rous sarcoma virus binding site corresponded closely to the 5' limit of the LTR enhancer; nucleotides -225 to -188 were protected as a pair of adjacent binding domains. The Fujinami sarcoma virus LTR bound C/EBP at a single site at nucleotides -213 to -195. C/EBP also bound to the promoter region of the enhancerless Rous-associated virus-0 LTR at nucleotides -77 to -57. The avian myeloblastosis virus LTR bound C/EBP at three sites: nucleotides -262 to -246, -154 to -134, and -55 to -39. We have previously observed binding of C/EBP to an enhancer in the gag gene of avian retroviruses. A heat-treated nuclear extract from chicken liver bound to all of the same retroviral sequences as did C/EBP. Alignment of the avian retroviral binding sequences with the published binding sites for C/EBP in two CCAAT boxes and in the simian virus 40, polyoma, and murine sarcoma virus enhancers suggested TTGNNGCTAATG as a consensus sequence for binding of C/EBP. When two bases of this consensus sequence were altered by site-specific mutagenesis of the Rous sarcoma virus LTR, binding of the heat-stable chicken protein was eliminated.     

v-src mutations outside the carboxyl-coding region are not sufficient to fully activate transformation by pp60c-src in NIH 3T3 cells.

Previous studies have shown that carboxyl-terminal mutation of pp60c-src can activate its transforming ability. Conflicting results have been reported for the transforming ability of pp60c-src mutants having only mutations outside its carboxyl-terminal region. To clarify the effects of such mutations, we tested the activities of chimeric v(amino)- and c(carboxyl)-src (v/c-src) proteins at different dosages in NIH 3T3 cells. The focus-forming activity of Rous sarcoma virus long terminal repeat (LTR)-src expression plasmids was significantly reduced when the v-src 3' coding region was replaced with the corresponding c-src region. This difference was masked when the Rous sarcoma virus LTR was replaced with the Moloney murine leukemia virus LTR, which induced approximately 20-fold more protein expression, but even focus-selected lines expressing v/c-src proteins were unable to form large colonies in soft agarose or tumors in NFS mice. This suggests that pp60c-src is not equally sensitive to mutations in its different domains and that there are at least two distinguishable levels of regulation, the dominant one being associated with its carboxyl terminus. v/c-src chimeric proteins expressed with either LTR had high in vitro specific kinase activity equal to that of pp60v-src but, in contrast, were phosphorylated at both Tyr-527 and Tyr-416. Total cell protein phosphotyrosine was enhanced in cells incompletely transformed by v/c-src proteins to the same extent as in v-src-transformed cells, suggesting that the carboxyl-terminal region may affect substrate specificity in a manner that is important for transformation.     

Alternate polypurine tracts affect rous sarcoma virus integration in vivo

When the endogenous polypurine tract (PPT) of the Rous sarcoma virus (RSV)-derived vector RSVP(A)Z was replaced with alternate retroviral PPTs, the fraction of unintegrated viral DNA with the normal consensus ends significantly decreased and the retention of part of the PPT significantly increased. If the terminus of the U3 long terminal repeat (LTR) is aberrant, RSV integrase can correctly process and integrate the normal U5 LTR into the host genome. However, the canonical CA is not involved in joining the aberrant U3 LTR to the host DNA, generating either large duplications or deletions of the host sequences instead of the normal 5- or 6-bp duplication.     

Interactions of HIV-1 DNA heterocyclic bases with viral DNA

Human immunodeficiency virus type 1 integrase is one of three viral enzymes, and it realizes a key process of the viral replication cycle, i.e. viral DNA integration into infected cell genome. Integrase recognizes nucleotide sequences located at the ends of the viral DNA U3 and U5 LTRs and catalyzes 3'-processing and strand transfer reactions. To study the interactions between integrase and viral DNA at present work, we used modified integrase substrates mimicking the terminal U5 LTR sequence and containing non-nucleoside insertions in one or/and both strands. It is shown that the substrate modifications have no influence on the integrase binding rate, while the heterocyclic bases removal in the 5th and 6th substrate positions and in the 3rd position of the substrate processed strand distinctly inhibits the integrase catalytic activity. This fact demonstrates these bases significance for the active enzyme/substrate complex formation. On the contrary, modification of the 3rd position within substrate non-processed strand stimulates 3'-processing. Since heterocyclic base elimination results in disruption of the DNA complementary and staking interactions, this result shows that DNA double helix destabilization close to the cleaved bond promotes the 3'-processing.     

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.     

Nucleophile selection for the endonuclease activities of human, ovine, and avian retroviral integrases

Retroviral integrases catalyze four endonuclease reactions (processing, joining, disintegration, and nonspecific alcoholysis) that differ in specificity for the attacking nucleophile and target DNA sites. To assess how the two substrates of this enzyme affect each other, we performed quantitative analyses, in three retroviral systems, of the two reactions that use a variety of nucleophiles. The integrase proteins of human immuno- deficiency virus type 1, visna virus, and Rous sarcoma virus exhibited distinct preferences for water or other nucleophiles during site-specific processing of viral DNA and during nonspecific alcoholysis of nonviral DNA. Although exogenous alcohols competed with water as the nucleophile for processing, the alcohols stimulated nicking of nonviral DNA. Moreover, different nucleophiles were preferred when the various integrases acted on different DNA targets. In contrast, the nicking patterns were independent of whether integrase was catalyzing hydrolysis or alcoholysis and were not influenced by the particular exogenous alcohol. Thus, although the target DNA influenced the choice of nucleophile, the nucleophile did not affect the choice of target sites. These results indicate that interaction with target DNA is the critical step before catalysis and suggest that integrase does not reach an active conformation until target DNA has bound to the enzyme.     

Cellular co-factors of HIV-1 integration

To achieve productive infection, the reverse transcribed cDNA of human immunodeficiency virus type 1 (HIV-1) is inserted in the host cell genome. The main protein responsible for this reaction is the viral integrase. However, studies indicate that the virus is assisted by cellular proteins, or co-factors, to achieve integration into the infected cell. The barrier-to-autointegration factor (BAF) might prevent autointegration. Its ability to bridge DNA and the finding that the nuclear lamina-associated polypeptide-2alpha interacts with BAF suggest a role in nuclear structure organization. Integrase interactor 1 was found to directly interact with HIV-1 integrase and to activate its DNA-joining activity, and the high mobility group chromosomal protein A1 might approximate both long terminal repeat (LTR) ends and facilitate integrase binding by unwinding the LTR termini. Furthermore, the lens-epithelium-derived growth factor (LEDGF; also known as p75) seems to tether HIV-1 integrase to the chromosomes. Although a direct role in integration has only been demonstrated for LEDGF/p75, to date, each validated cellular co-factor for HIV-1 integration could constitute a promising new target for antiviral therapy.     

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.     

Coordinated disintegration reactions mediated by Moloney murine leukemia virus integrase.

The protein-DNA and protein-protein interactions important for function of the integrase (IN) protein of Moloney murine leukemia virus (M-MuLV) were investigated by using a coordinated-disintegration assay. A panel of M-MuLV IN mutants and substrate alterations highlighted distinctions between the intermolecular and intramolecular reactions of coordinated disintegration. Mispairing of the crossbone single-strand region and altered long terminal repeat (LTR) positioning affected the intermolecular, but not the intramolecular, reactions of coordinated disintegration. Partial components of the crossbone substrate were coordinated by M-MuLV IN, indicating a reliance on both LTR and target DNA determinants for substrate assembly. The intramolecular reaction was dependent on the presence of either the HHCC domain or a crossbone LTR 5' single-stranded tail. An M-MuLV IN mutant without the HHCC domain (Ndelta105) catalyzed reduced levels of double disintegration but not single disintegration. A separately purified HHCC domain protein (Cdelta232) stimulated double disintegration mediated by Ndelta105, suggesting a role of the N-terminal HHCC domain in stable IN-IN and IN-DNA interactions. Significantly, crossbone substrates lacking the LTR 5' tails were not recognized by the fingerless Ndelta105 protein. Collectively, these data suggest similar roles of the HHCC domain and 5' LTR tail in substrate recognition and modulation of IN activity.     

Crystal structure of an active two-domain derivative of Rous sarcoma virus integrase

Integration of retroviral cDNA is a necessary step in viral replication. The virally encoded integrase protein and DNA sequences at the ends of the linear viral cDNA are required for this reaction. Previous studies revealed that truncated forms of Rous sarcoma virus integrase containing two of the three protein domains can carry out integration reactions in vitro. Here, we describe the crystal structure at 2.5 A resolution of a fragment of the integrase of Rous sarcoma virus (residues 49-286) containing both the conserved catalytic domain and a modulatory DNA-binding domain (C domain). The catalytic domains form a symmetric dimer, but the C domains associate asymmetrically with each other and together adopt a canted conformation relative to the catalytic domains. A binding path for the viral cDNA is evident spanning both domain surfaces, allowing modeling of the larger integration complexes that are known to be active in vivo. The modeling suggests that formation of an integrase tetramer (a dimer of dimers) is necessary and sufficient for joining both viral cDNA ends at neighboring sites in the target DNA. The observed asymmetric arrangement of C domains suggests that they could form a rotationally symmetric tetramer that may be important for bridging integrase complexes at each cDNA end.