Functional oligomeric state of avian sarcoma virus integrase

Retroviral integrase, one of only three enzymes encoded by the virus, catalyzes the essential step of inserting a DNA copy of the viral genome into the host during infection. Using the avian sarcoma virus integrase, we demonstrate that the enzyme functions as a tetramer. In presteady-state active site titrations, four integrase protomers were required for a single catalytic turnover. Volumetric determination of integrase-DNA complexes imaged by atomic force microscopy during the initial turnover additionally revealed substrate-induced assembly of a tetramer. These results suggest that tetramer formation may be a requisite step during catalysis with ramifications for antiviral design strategies targeting the structurally homologous human immunodeficiency virus, type 1 (HIV-1) integrase.     

Role of DNA end distortion in catalysis by avian sarcoma virus integrase.

Retroviral integrase (IN) recognizes linear viral DNA ends and introduces nicks adjacent to a highly conserved CA dinucleotide usually located two base pairs from the 3'-ends of viral DNA (the "processing" reaction). In a second step, the same IN active site catalyzes the insertion of these ends into host DNA (the "joining" reaction). Both DNA sequence and DNA structure contribute to specific recognition of viral DNA ends by IN. Here we used potassium permanganate modification to show that the avian sarcoma virus IN catalytic domain is able to distort viral DNA ends in vitro. This distortion activity is consistent with both unpairing and unstacking of the three terminal base pairs, including the processing site adjacent to the conserved CA. Furthermore, the introduction of mismatch mutations that destabilize the viral DNA ends were found to stimulate the IN processing reaction as well as IN-mediated distortion. End-distortion activity was also observed with mutant or heterologous DNA substrates. However, further analyses showed that using Mn(2+) as a cofactor, processing site specificity of these substrates was also maintained. Our results support a model whereby unpairing and unstacking of the terminal base pairs is a required step in the processing reaction. Furthermore, these results are consistent with our previous observations indicating that unpairing of target DNA promotes the joining reaction.     

A crystal structure of the catalytic core domain of an avian sarcoma and leukemia virus integrase suggests an alternate dimeric assembly

Integrase (IN) is an important therapeutic target in the search for anti-Human Immunodeficiency Virus (HIV) inhibitors. This enzyme is composed of three domains and is hard to crystallize in its full form. First structural results on IN were obtained on the catalytic core domain (CCD) of the avian Rous and Sarcoma Virus strain Schmidt-Ruppin A (RSV-A) and on the CCD of HIV-1 IN. A ribonuclease-H like motif was revealed as well as a dimeric interface stabilized by two pairs of α-helices (α1/α5, α5/α1). These structural features have been validated in other structures of IN CCDs. We have determined the crystal structure of the Rous-associated virus type-1 (RAV-1) IN CCD to 1.8 Å resolution. RAV-1 IN shows a standard activity for integration and its CCD differs in sequence from that of RSV-A by a single accessible residue in position 182 (substitution A182T). Surprisingly, the CCD of RAV-1 IN associates itself with an unexpected dimeric interface characterized by three pairs of α-helices (α3/α5, α1/α1, α5/α3). A182 is not involved in this novel interface, which results from a rigid body rearrangement of the protein at its α1, α3, α5 surface. A new basic groove that is suitable for single-stranded nucleic acid binding is observed at the surface of the dimer. We have subsequently determined the structure of the mutant A182T of RAV-1 IN CCD and obtained a RSV-A IN CCD-like structure with two pairs of buried α-helices at the interface. Our results suggest that the CCD of avian INs can dimerize in more than one state. Such flexibility can further explain the multifunctionality of retroviral INs, which beside integration of dsDNA are implicated in different steps of the retroviral cycle in presence of viral ssRNA.     

Presteady-state analysis of avian sarcoma virus integrase. II. Reverse-polarity substrates identify preferential processing of the U3-U5 pair

The integrase-catalyzed insertion of the retroviral genome into the host chromosome involves two reactions in vivo: 1) the binding and endonucleolytic removal of the terminal dinucleotides of the viral DNA termini and 2) the recombination of the ends with the host DNA. Kukolj and Skalka (Kukolj, G., and Skalka, A. M. (1995) Genes Dev. 9, 2556-2567) have previously shown that tethering of the termini enhances the endonucleolytic activities of integrase. We have used 5'-5' phosphoramidites to design reverse-polarity tethers that allowed us to examine the reactivity of two viral long terminal repeat-derived sequences when concurrently bound to integrase and, additionally, developed presteady-state assays to analyze the initial exponential phase of the reaction, which is a measure of the amount of productive nucleoprotein complexes formed during preincubation of integrase and DNA. Furthermore, the reverse-polarity tether circumvents the integrase-catalyzed splicing reaction (Bao, K., Skalka, A. M., and Wong, I. (2002) J. Biol. Chem. 277, 12089-12098) that obscures accurate analysis of the reactivities of synapsed DNA substrates. Consequently, we were able to establish a lower limit of 0.2 s(-1) for the rate constant of the processing reaction. The analysis showed the physiologically relevant U3/U5 pair of viral ends to be the preferred substrate for integrase with the U3/U3 combination favored over the U5/U5 pair.     

Proper processing of avian sarcoma/leukosis virus capsid proteins is required for infectivity

The formation of the mature carboxyl terminus of CA in avian sarcoma/leukemia virus is the result of a sequence of cleavage events at three PR sites that lie between CA and NC in the Gag polyprotein. The initial cleavage forms the amino terminus of the NC protein and releases an immature CA, named CA1, with a spacer peptide at its carboxyl terminus. Cleavage of either 9 or 12 amino acids from the carboxyl terminus creates two mature CA species, named CA2 and CA3, that can be detected in avian sarcoma/leukemia virus (R. B. Pepinsky, I. A. Papayannopoulos, E. P. Chow, N. K. Krishna, R. C. Craven, and V. M. Vogt, J. Virol. 69:6430-6438, 1995). To study the importance of each of the three CA proteins, we introduced amino acid substitutions into each CA cleavage junction and studied their effects on CA processing as well as virus assembly and infectivity. Preventing cleavage at any of the three sites produced noninfectious virus. In contrast, a mutant in which cleavage at site 1 was enhanced so that particles contained CA2 and CA3 but little detectable CA1 was infectious. These results support the idea that infectivity of the virus is closely linked to proper processing of the carboxyl terminus to form two mature CA proteins.     

Monoclonal antibodies against Rous sarcoma virus integrase protein exert differential effects on integrase function in vitro.

We have prepared and characterized several monoclonal antibodies (MAbs) against the Rous sarcoma virus integrase protein (IN) with the aim of employing these specific reagents as tools for biochemical and biophysical studies. The interaction of IN with the purified MAbs and their Fab fragment derivatives was demonstrated by Western blot (immunoblot), enzyme-linked immunosorbent assay, and size exclusion chromatography. A series of truncated IN proteins was used to determine regions in the protein important for recognition by the antibodies. The MAbs described here recognize epitopes that lie within the catalytic core region of IN (amino acids 50 to 207) and are likely to be conformational. A detailed functional analysis was carried out by investigating the effects of Fab fragments as well as of intact MAbs on the activities of IN in vitro. These studies revealed differential effects which fall into three categories. (i) One of the antibodies completely neutralized the processing as well as the joining activity and also reduced the DNA binding capacity as determined by a nitrocellulose filter binding assay. On the other hand, this MAb did not abolish the cleavage-ligation reaction on a disintegration substrate and the nonspecific cleavage of DNA by IN. The cleavage pattern generated by the IN-MAb complex on various DNA substrates closely resembled that produced by mutant IN proteins which show a deficiency in multimerization. Preincubation of IN with substrate protected the enzyme from inhibition by this antibody. (ii) Two other antibodies showed a general inhibition of all IN activities tested. (iii) In contrast, a fourth MAb stimulated the in vitro joining activity of IN. Size exclusion chromatography demonstrated that IN-Fab complexes from representatives of the three categories of MAbs exhibit different stoichiometric compositions that suggest possible explanations for their contrasting effects and may provide clues to the relationship between the structure and function of IN.     

Identification of amino acids in HIV-1 and avian sarcoma virus integrase subsites required for specific recognition of the long terminal repeat Ends

A tetramer model for HIV-1 integrase (IN) with DNA representing 20 bp of the U3 and U5 long terminal repeats (LTR) termini was assembled using structural and biochemical data and molecular dynamics simulations. It predicted amino acid residues on the enzyme surface that can interact with the LTR termini. A separate structural alignment of HIV-1, simian sarcoma virus (SIV), and avian sarcoma virus (ASV) INs predicted which of these residues were unique. To determine whether these residues were responsible for specific recognition of the LTR termini, the amino acids from ASV IN were substituted into the structurally equivalent positions of HIV-1 IN, and the ability of the chimeras to 3 ' process U5 HIV-1 or ASV duplex oligos was determined. This analysis demonstrated that there are multiple amino acid contacts with the LTRs and that substitution of ASV IN amino acids at many of the analogous positions in HIV-1 IN conferred partial ability to cleave ASV substrates with a concomitant loss in the ability to cleave the homologous HIV-1 substrate. HIV-1 IN residues that changed specificity include Val(72), Ser(153), Lys(160)-Ile(161), Gly(163)-Val(165), and His(171)-Leu(172). Because a chimera that combines several of these substitutions showed a specificity of cleavage of the U5 ASV substrate closer to wild type ASV IN compared with chimeras with individual amino acid substitutions, it appears that the sum of the IN interactions with the LTRs determines the specificity. Finally, residues Ser(153) and Val(72) in HIV-1 IN are among those that change in enzymes that develop resistance to naphthyridine carboxamide- and diketo acid-related inhibitors in cells. Thus, amino acid residues involved in recognition of the LTRs are among these positions that change in development of drug resistance.     

Integration of avian sarcoma virus specific DNA in mammalian chromatin

Constitutive heterochromatin and euchromatin fractions from normal and avian sarcoma virus transformed cells of Mus musculur and Microtus agrestis were isolated in order to characterize the site of integration of the viral specific DNA sequences. The transformed mouse (BALB/c 3T3-B77) and M. agrestis (UMMA-RSV-21) cell lines, as well as a revertant clone of the M. agrestis (UMMA-RSV-R-4) were found to have integrated 1–2 viral copies per diploid genome. The number of viral copies was studied by the technique of DNA-DNA hybridization in solution, and in all cases the viral sequences were located in the euchromatin fraction.     

Molecular and genetic determinants of rous sarcoma virus integrase for concerted DNA integration

Site-directed mutagenesis of recombinant Rous sarcoma virus (RSV) integrase (IN) allowed us to gain insights into the protein-protein and protein-DNA interactions involved in reconstituted IN-viral DNA complexes capable of efficient concerted DNA integration (termed full-site). At 4 nM IN, wild-type (wt) RSV IN incorporates approximately 30% of the input donor into full-site integration products after 10 min of incubation at 37 degrees C, which is equivalent to isolated retrovirus preintegration complexes for full-site integration activity. DNase I protection analysis demonstrated that wt IN was able to protect the viral DNA ends, mapping approximately 20 bp from the end. We had previously mapped the replication capabilities of several RSV IN mutants (A48P and P115S) which appeared to affect viral DNA integration in vivo. Surprisingly, recombinant RSV A48P IN retained wt IN properties even though the virus carrying this mutation had significantly reduced integrated viral DNA in comparison to wt viral DNA in virus-infected cells. Recombinant RSV P115S IN also displayed all of the properties of wt RSV IN. Upon heating of dimeric P115S IN in solution at 57 degrees C, it became apparent that the mutation in the catalytic core of RSV IN exhibited the same thermolabile properties for 3' OH processing and strand transfer (half-site and full-site integration) activities consistent with the observed temperature-sensitive defect for integration in vivo. The average half-life for inactivation of the three activities were similar, ranging from 1.6 to 1.9 min independent of the IN concentrations in the assay mixtures. Wt IN was stable under the same heat treatment. DNase I protection analysis of several conservative and nonconservative substitutions at W233 (a highly conserved residue of the retrovirus C-terminal domain) suggests that this region is involved in protein-DNA interactions at the viral DNA attachment site. Our data suggest that the use of recombinant RSV IN to investigate efficient full-site integration in vitro with reference to integration in vivo is promising.     

Efficient concerted integration of retrovirus-like DNA in vitro by avian myeloblastosis virus integrase.

We report the efficient concerted integration of a linear virus-like DNA donor into a 2.8 kbp circular DNA target by integrase (IN) purified from avian myeloblastosis virus. The donor was 528 bp, contained recessed 3' OH ends, was 5' end labeled, and had a unique restriction site not found in the target. Analysis of concerted (full-site) and half-site integration events was accomplished by restriction enzyme analysis and agarose gel electrophoresis. The donor also contained the SupF gene that was used for genetic selection of individual full-site recombinants to determine the host duplication size. Two different pathways, involving either one donor or two donor molecules, were used to produce full-site recombinants. About 90% of the full-site recombinants were the result of using two donor molecules per target. These results imply that juxtapositioning an end from each of two donors by IN was more efficient than the juxtapositioning of two ends of a single donor for the full-site reaction. The formation of preintegration complexes containing integrase and donor on ice prior to the addition of target enhanced the full-site reaction. After a 30 min reaction at 37 degrees C, approximately 20-25% of all donor/target recombinants were the result of concerted integration events. The efficient production of full-site recombinants required Mg2+; Mn2+ was only efficient for the production of half-site recombinants. We suggest that these preintegration complexes can be used to investigate the relationships between the 3' OH trimming and strand transfer reactions.     

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.     

Rous sarcoma virus integrase protein: mapping functions for catalysis and substrate binding.

Rous sarcoma virus (RSV), like all retroviruses, encodes an integrase protein that is responsible for covalently joining the reverse-transcribed viral DNA to host DNA. We have probed the organization of functions within RSV integrase by constructing mutant derivatives and assaying their activities in vitro. We find that deletion derivatives lacking the amino-terminal 53 amino acids, which contain the conserved H-X(3-7)-H-X(23-32)-C-X(2)-C (HHCC) Zn(2+)-binding motif, are greatly impaired in their ability to carry out two reactions characteristic of integrase proteins: specific cleavage of the viral DNA termini and DNA strand transfer. Deletion mutants lacking the carboxyl-terminal 69 amino acids are also unable to carry out these reactions. However, all deletion mutants that retain the central domain are capable of carrying out disintegration, an in vitro reversal of the normal DNA strand transfer reaction, indicating that the catalytic center probably lies within this central region. Another conserved motif, D-X(39-58)-D-X(35)-E, is found in this central domain. These findings with RSV integrase closely parallel previous findings with human immunodeficiency virus integrase, indicating that a modular catalytic domain is a general feature of this family of proteins. Surprisingly, and unlike results obtained so far with human immunodeficiency virus integrase, efficient strand transfer activity can be restored to a mutant RSV integrase lacking the amino-terminal HHCC domain by fusion to various short peptides. Furthermore, these fusion proteins retain the substrate specificity of RSV integrase. These data support a model in which the integrase activities required for strand transfer in vitro, including substrate recognition, multimerization, and catalysis, all lie primarily outside the amino-terminal HHCC domain.     

Translation of avian sarcoma virus RNA in Xenopus laevis oocytes.

Evidence for the synthesis and processing of Pr76 (precursor to group-specific antigens p27, p19, and $\text{p12}\text{15}$, upon injection of avian sarcoma virus 70S or 35S RNA into $\text{Xenopus}$ oocytes has been presented. Further, we show that tRNA^trp primer, bound to 35S RNA, does not block translation of virion RNA under these conditions.     

Subcellular localization of avian sarcoma virus and human immunodeficiency virus type 1 integrases.

The composition and subcellular trafficking of subviral preintegration complexes are reported to vary among the different retroviruses. The process by which the avian sarcoma virus (ASV) preintegration complex gains access to target chromatin remains unknown. Here we report that ASV integrase (IN) expressed as a fusion to beta-galactosidase accumulates in the nuclei of transfected COS-1 cells. In contrast, human immunodeficiency type 1 (HIV-1) IN-beta-galactosidase fusions expressed similarly are predominantly cytoplasmic. To identify the region of ASV IN that specifies nuclear localization, various subdomains of the protein were expressed as beta-galactosidase fusions and their subcellular locations were assessed cytochemically and by indirect immunofluorescence. These analyses showed that the ASV IN protein possesses a functional nuclear localization signal that spans amino acids 206 to 235 and displays limited homology with known nuclear transport signals.     

A substitution in rous sarcoma virus integrase that separates its two biologically relevant enzymatic activities

Retroviral integrase prepares viral DNA for integration by removing 2 nucleotides from each end of unintegrated DNA in a reaction referred to as processing. However, it has been known since the processing assay was first described that avian integrases frequently nick 3 nucleotides, as well as 2 nucleotides, from viral DNA ends when reaction mixtures contain Mn2+. We now report that specificity for the biologically relevant "-2" site is enhanced when the serine at amino acid 124 of Rous sarcoma virus (RSV) integrase is replaced by alanine, valine, glycine, lysine, or aspartate. The protein with a serine-to-aspartate substitution exhibited especially high fidelity for the correct site, as evidenced by a ratio of -2 nicks to -3 nicks that was more than 40-fold greater than that for the wild-type enzyme in reactions with Mn2+. Even with Mg2+, the substituted proteins exhibited greater specificity than the wild type, especially the S124D protein. Moreover, this protein was more efficient than the wild type at processing viral DNA ends. Unexpectedly, however, the S124D protein was significantly impaired at catalyzing the insertion of viral DNA ends in reactions with Mn2+ and joining was undetectable in reactions with Mg2+. Thus, the S124D protein has separated the processing and joining activities of integrase. Similar results were found for human immunodeficiency virus integrase with the analogous substitution. No proteins with comparable properties have been described. Moreover, RSV virions containing integrase with the S124D mutation were unable to replicate in cell cultures. Together, these data suggest that integrase has evolved to have submaximal processing activity so that it can also catalyze DNA joining.