Viral DNA Termini Research Articles

Retroviral Integrases Promote Fraying of Viral DNA Ends

In the initial step of integration, retroviral integrase (IN) introduces precise nicks in the degenerate, short inverted repeats at the ends of linear viral DNA. The scissile phosphodiester bond is located immediately 3' of a highly conserved CA/GT dinucleotide, usually 2 bp from the ends. These nicks create new recessed 3'-OH viral DNA ends that are required for joining to host cell DNA. Previous studies have indicated that unpairing, "fraying," of the viral DNA ends by IN contributes to end recognition or catalysis. Here, we report that end fraying can be detected independently of catalysis with both avian sarcoma virus (ASV) and human immunodeficiency virus type 1 (HIV-1) IN proteins by use of fluorescence resonance energy transfer (FRET). The results were indicative of an IN-induced intramolecular conformational change in the viral DNA ends (cis FRET). Fraying activity is tightly coupled to the DNA binding capabilities of these enzymes, as follows: an inhibitor effective against both IN proteins was shown to block ASV IN DNA binding and end fraying, with similar dose responses; ASV IN substitutions that reduced DNA binding also reduced end fraying activity; and HIV-1 IN DNA binding and end fraying were both undetectable in the absence of a metal cofactor. Consistent with our previous results, end fraying is sequence-independent, suggesting that the DNA terminus per se is a major structural determinant for recognition. We conclude that frayed ends represent a functional intermediate in which DNA termini can be sampled for suitability for endonucleolytic processing.

Retroviral intasome assembly and inhibition of DNA strand transfer

Integrase is an essential retroviral enzyme that binds both termini of linear viral DNA and inserts them into a host cell chromosome. The structure of full-length retroviral integrase, either separately or in complex with DNA, has been lacking. Furthermore, although clinically useful inhibitors of HIV integrase have been developed, their mechanism of action remains speculative. Herein we report a crystal structure of full-length integrase from the prototype foamy virus in complex with its cognate DNA. The structure reveals the organization of the retroviral intasome comprising an integrase tetramer tightly associated with a pair of viral DNA ends. All three canonical integrase structural domains are involved in extensive protein-DNA and protein-protein interactions. Binding of strand transfer inhibitors displaces the reactive viral DNA end from the active site, disarming the viral nucleoprotein complex. Our findings define the structural basis of retroviral DNA integration and will allow modeling of the HIV-1 intasome to aid in the development of antiretroviral drugs.

Molecular Interactions between HIV-1 Integrase and the Two Viral DNA Ends within the Synaptic Complex that Mediates Concerted Integration

A macromolecular nucleoprotein complex in retrovirus-infected cells, termed the preintegration complex, is responsible for the concerted integration of linear viral DNA genome into host chromosomes. Isolation of sufficient quantities of the cytoplasmic preintegration complexes for biochemical and biophysical analysis is difficult. We investigated the architecture of HIV-1 nucleoprotein complexes involved in the concerted integration pathway in vitro. HIV-1 integrase (IN) non-covalently juxtaposes two viral DNA termini forming the synaptic complex, a transient intermediate in the integration pathway, and shares properties associated with the preintegration complex. IN slowly processes two nucleotides from the 3′ OH ends and performs the concerted insertion of two viral DNA ends into target DNA. IN remains associated with the concerted integration product, termed the strand transfer complex. The synaptic complex and strand transfer complex can be isolated by native agarose gel electrophoresis. In-gel fluorescence resonance energy transfer measurements demonstrated that the energy transfer efficiencies between the juxtaposed Cy3 and Cy5 5′-end labeled viral DNA ends in the synaptic complex (0.68±0.09) was significantly different from that observed in the strand transfer complex (0.07±0.02). The calculated distances were 46±3 Å and 83±5 Å, respectively. DNaseI footprint analysis of the complexes revealed that IN protects U5 and U3 DNA sequences up to ∼32 bp from the end, suggesting two IN dimers were bound per terminus. Enhanced DNaseI cleavages were observed at nucleotide positions 6 and 9 from the terminus on U3 but not on U5, suggesting independent assembly events. Protein–protein cross-linking of IN within these complexes revealed the presence of dimers, tetramers, and a larger multimer (>120 kDa). Our results suggest a new model where two IN dimers individually assemble on U3 and U5 ends before the non-covalent juxtaposition of two viral DNA ends, producing the synaptic complex.

Cheminformatics methods for novel nanopore analysis of HIV DNA termini.

BackgroundChannel current feature extraction methods, using Hidden Markov Models (HMMs) have been designed for tracking individual-molecule conformational changes. This information is derived from observation of changes in ionic channel current blockade "signal" upon that molecule's interaction with (and occlusion of) a single nanometer-scale channel in a "nanopore detector". In effect, a nanopore detector transduces single molecule events into channel current blockades. HMM analysis tools described are used to help systematically explore DNA dinucleotide flexibility, with particular focus on HIV's highly conserved (and highly flexible/reactive) viral DNA termini. One of the most critical stages in HIV's attack is the binding between viral DNA and the retroviral integrase, which is influenced by the dynamic-coupling induced high flexibility of a CA/TG dinucleotide positioned precisely two base-pairs from the blunt terminus of the duplex viral DNA. This suggests the study of a family of such CA/TG dinucleotide molecules via nanopore measurement and cheminformatics analysis.ResultsHMMs are used for level identification on the current blockades, HMM/EM with boosted variance emissions are used for level projection pre-processing, and time-domain FSAs are used to parse the level-projected waveform for kinetic information. The observed state kinetics of the DNA hairpins containing the CA/TG dinucleotide provides clear evidence for HIV's selection of a peculiarly flexible/interactive DNA terminus.

Strategy to Discriminate between High and Low Affinity Bindings of Human Immunodeficiency Virus, Type 1 Integrase to Viral DNA

The last decade has contributed to our understanding of the three-dimensional structure of the human immunodeficiency virus, type 1 (HIV-1) integrase (IN) and to the description of how the enzyme catalyzes the viral DNA integration into the host DNA. Recognition of the viral DNA termini by IN is sequence-specific, and that of the host DNA does not require particular sequence, although in physicochemical studies IN fails to discriminate between the two interactions. Here, such discrimination was allowed thanks to a model system using designed oligonucleotides and peptides as binding structures. Spectroscopic (circular dichroism, NMR, and fluorescence anisotropy) techniques and biochemical (enzymatic and filter binding) assays clearly indicated that the amphipathic helix alpha4, located at the catalytic domain surface, is responsible for the specific high affinity binding of the enzyme to viral DNA. Analogues of the alpha4 peptide having increased helicity and still bearing the biologically relevant lysines 156 and 159 on the DNA binding face, and oligonucleotides conserving an intact attachment site, are required to achieve high affinity complexes (Kd of 1.5 nm). Data corroborate previous in vivo results obtained with mutated viruses.

Efficient concerted integration by recombinant human immunodeficiency virus type 1 integrase without cellular or viral cofactors.

Replication of retroviruses requires integration of the linear viral DNA genome into the host chromosomes. Integration requires the viral integrase (IN), located in high-molecular-weight nucleoprotein complexes termed preintegration complexes (PIC). The PIC inserts the two viral DNA termini in a concerted manner into chromosomes in vivo as well as exogenous target DNA in vitro. We reconstituted nucleoprotein complexes capable of efficient concerted (full-site) integration using recombinant wild-type human immunodeficiency virus type I (HIV-1) IN with linear retrovirus-like donor DNA (480 bp). In addition, no cellular or viral protein cofactors are necessary for purified bacterial recombinant HIV-1 IN to mediate efficient full-site integration of two donor termini into supercoiled target DNA. At about 30 nM IN (20 min at 37 degrees C), approximately 15 and 8% of the input donor is incorporated into target DNA, producing half-site (insertion of one viral DNA end per target) and full-site integration products, respectively. Sequencing the donor-target junctions of full-site recombinants confirms that 5-bp host site duplications have occurred with a fidelity of about 70%, similar to the fidelity when using IN derived from nonionic detergent lysates of HIV-1 virions. A key factor allowing recombinant wild-type HIV-1 IN to mediate full-site integration appears to be the avoidance of high IN concentrations in its purification (about 125 microg/ml) and in the integration assay (<50 nM). The results show that recombinant HIV-1 IN may not be significantly defective for full-site integration. The findings further suggest that a high concentration or possibly aggregation of IN is detrimental to the assembly of correct nucleoprotein complexes for full-site integration.

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.

Evaluation of retroviral ribonuclease H activity.

Reverse transcription is the process whereby the single-stranded RNA genome of a retrovirus is converted into double-stranded DNA by the DNA polymerase and ribonuclease H (RNase H) activities of virus-coded reverse transcriptase (RT) (,). The former activity of reverse transcriptase has been studied extensively, both as a potential target for antiviral therapy and a tool for molecular biology. However, increasing attention has been directed recently toward understanding the roles of RNase H activity, both nonspecific and highly specialized, in reverse transcription (reviewed in ref. ). A complete summary of the process is depicted in Fig. 1. Quantitatively, the most important RNase H function is to degrade the RNA strand of ∼10,000 bp RNA-DNA replication intermediate, eliminating the need for strand-displacement during synthesis of the second or (+) strand. This degradation is largely nonspecific, yet several cleavage events demand a high degree of precision, such as scission of the RNA strand at specific positions near the 3− termini of small, purine-rich stretches of RNA called polypurine tracts (PPTs). These RNAs serve as primers for (+) strand synthesis, but must also be removed by the enzyme once initiation has occurred. The (−;) strand primer, a host-derived tRNA, is removed after its 3′-terminus is copied during (+) strand synthesis. If the (+) and (−) strand primers are not precisely removed, the resulting errors would alter the 5′- and 3′-termini of viral DNA (Fig. 1 E). This would inhibit integration of viral DNA into the host-cell genome and perhaps adversely affect the viability of the virus at later stages of its life cycle.

Substrate Recognition by Retroviral Integrases

Substrate recognition by the retroviral IN enzyme is critical for retroviral integration. To catalyze this recombination event, IN must recognize and act on two types of substrates, viral DNA and host DNA, yet the necessary interactions exhibit markedly different degrees of specificity. Although particular sequences at the viral DNA termini are recognized by IN, many host DNA sequences can serve as the target for integration. Over the last decade, both in vitro and in vivo data have contributed to our understanding of how IN recognizes its substrates. This review provides an overview of the sequence and structure requirements for recognition of viral and host DNA by different retroviral INs and discusses recent progress in mapping protein domains involved in these interactions.

Inhibitors of Human Immunodeficiency Virus Integrase

Integration of the viral DNA into a host cell chromosome is an essential step for HIV replication and maintenance of persistent infection. Two viral factors are essential for integration: the viral DNA termini (the att sites) and IN. Accruing knowledge of the IN structure, catalytic mechanisms, and interactions with other proteins can be used to design strategies to block integration. A large number of inhibitors have been identified that can be used as leads for the development of potent and selective anti-IN drugs with antiviral activity.

Footprints on the viral DNA ends in moloney murine leukemia virus preintegration complexes reflect a specific association with integrase.

Retroviral DNA integration is mediated by the preintegration complex, a large nucleoprotein complex derived from the core of the infecting virion. We previously have used Mu-mediated PCR to probe the nucleoprotein organization of Moloney murine leukemia virus preintegration complexes. A region of protection spans several hundred base pairs at each end of the viral DNA, and strong enhancements are present near the termini. Here, we show that these footprints reflect a specific association between integrase and the viral DNA ends in functional preintegration complexes. Barrier-to-autointegration factor, a cellular protein that blocks autointegration of Moloney murine leukemia virus DNA, also plays an indirect role in generating the footprints at the ends of the viral DNA. We have exploited Mu-mediated PCR to examine the effect of mutations at the viral DNA termini on complex formation. We find that a replication competent mutant with a deletion at one end of the viral DNA still exhibits a strong enhancement about 20 bp from the terminus of the mutant DNA end. The site of the enhancement therefore appears to be at a fixed distance from the ends of the viral DNA. We also find that a mutation at one end of the viral DNA, which renders the virus incompetent for replication, abolishes the enhancements and protection at both the U3 and U5 ends. A pair of functional viral DNA ends therefore are required to interact before the chemical step of 3' end processing.

Highly potent synthetic polyamides, bisdistamycins, and lexitropsins as inhibitors of human immunodeficiency virus type 1 integrase.

Alignment of the available human immunodeficiency virus type 1 (HIV-1) viral DNA termini [U5 and U3 long terminal repeats (LTRs)] shows a high degree of conservation and the presence of a stretch of five or six consecutive adenine and thymine (AT) sequences approximately 10 nucleotides away from each LTR end. A series of AT-selective minor-groove binders, including distamycin and bisdistamycins, bisnetropsins, novel lexitropsins, and the classic monomeric DNA binders Hoechst 33258, 4'-diamino-2-phenylindole, pentamidine, berenil, spermine, and spermidine, were tested for their inhibitory activities against HIV-1 integrase (IN). Although netropsin, distamycin, and all other monomeric DNA binders showed weak activities in the range of 50-200 microM, some of the polyamides, bisdistamycins, and lexitropsins were remarkably active at nanomolar concentrations. Bisdistamycins were 200 times less potent when the conserved AAAAT stretch present in the U5 LTR was replaced with GGGGG, consistent with the preferred binding of these drugs to AT sequences. DNase I footprinting of the U5 LTR further demonstrated the selectivity of these bisdistamycins for the conserved AT sequence. The tested compounds were more potent in Mg+2 than in Mn+2 and inhibited IN50-212 deletion mutant in disintegration assays and the formation of IN/DNA complexes. The lexitropsins also were active against HIV-2 IN. Some of the synthetic polyamides exhibited significant antiviral activity. Taken together, these data suggest that selective targeting of the U5 and U3 ends of the HIV-1 LTRs can inhibit IN function. Polyamides might represent new leads for the development of antiviral agents against acquired immune deficiency syndrome.

Displacement of viral DNA termini from stable HIV-1 integrase nucleoprotein complexes induced by secondary DNA-binding interactions.

The human immunodeficiency virus type-1 (HIV-1) integrase is known to form a highly stable interaction with the termini of the linear, pre-integrated retroviral genome, where it catalyzes the 3'-OH processing and strand transfer processes required for their coordinated integration into host DNA. Here, we determine that the association of HIV-1 integrase with the viral DNA termini leads to the formation of two classes of nucleoprotein complexes with distinct properties in vitro. Both bound states are intrinsically stable and highly resistant to exonuclease digestion, but nonetheless they exhibit different stabilities in the presence of single-stranded polynucleotides. While a population of preassembled complexes tolerates elevated polynucleotide concentrations, the remainder forms an unstable ternary (integrase-substrate-polynucleotide) intermediate, leading to the rapid expulsion of the otherwise tightly bound substrate. The distribution of complexes between the two states is influenced by the preincubation time and temperature, increases in either of which favor the formation of the challenge-resistant species. Challenge-resistant complexes are formed more efficiently with Mn2+ than with Mg2+ and are sensitive to the length rather than the sequence of the DNA substrate. Due to the delayed appearance of the challenge-resistant form after the initial stable binding of the DNA substrate, our results may be indicative of a structural change in the preassembled complex which thereby modulates its response to exogenous DNA targets.

Disruption of the terminal base pairs of retroviral DNA during integration.

Integrase catalyzes two essential steps in the integration of the retroviral genome--end processing and strand transfer--both of which require the interaction of integrase with viral att sites located at the ends of viral genomic DNA. These two different polynucleotidyl transfer reactions are apparently carried out by a single active site. The end product of these reactions, the integrated provirus, does not undergo transposition and remains a stable part of the host cell genome. A central question in understanding the mechanism of integration is how a single active site accomplishes two distinct polynucleotidyl transfer reactions. We propose that integrase distorts DNA substrates to accommodate both reactions within the active site. Evidence is provided for disruption of base-pairing at the terminus of viral DNA during end processing. Furthermore, we show that this end fraying is a required step in end processing and that it appears to occur after initial binding of the viral DNA end. This requirement for base-pair disruption may account for the inability of integrase to use internal sites on DNA molecules as viral att sites. The specificity of integrase for DNA ends solves a problem posed by the long terminal repeat structure of the viral genome, and may help to prevent transposition of integrated proviruses.

Influence of subterminal viral DNA nucleotides on differential susceptibility to cleavage by human immunodeficiency virus type 1 and visna virus integrases.

A comparison of the extents of site-specific cleavage of U5 and U3 viral DNA termini by the integrases of human immunodeficiency virus type 1 and visna virus guided the quantitative testing of oligonucleotide substrates containing specific base substitutions. The simultaneous exchange of positions 5 and 6 between U3 substrates switched the patterns of differential susceptibility to the two integrases. The activity of visna virus integrase was more dependent on the identity of position 5 adjacent to the invariant CA bases than on position 6, whereas human immunodeficiency virus type 1 integrase appeared to interact even more critically with position 6. Although the paired natural substrates of most lentiviral integrases match at positions 7 and 8, these bases were not important for susceptibility of U5 substrates. In fact, the final six U5 positions contained all of the sequence information necessary for susceptibility. These results suggest that constraints other than integration influence the terminal inverted repeats of retroviral DNA.

Nonspecific alcoholysis, a novel endonuclease activity of human immunodeficiency virus type 1 and other retroviral integrases

Retroviral integrase (IN) exhibits a previously unrecognized endonuclease activity which we have termed nonspecific alcoholysis. This action occurred at every position in nonviral DNA sequences except those near 5' ends and is clearly distinguished from, and was not predicted by, the site-specific alcoholysis activity previously described for IN at the processing site near viral DNA termini. The integrases of human immunodeficiency virus type 1, visna virus, and Rous sarcoma virus exhibited different target site preferences in this new assay. The isolated central domain of human immunodeficiency virus type 1 IN preferred the same sites as the full-length protein. Nonspecific alcoholysis may provide insights into the structure and function of IN and other endonucleases and suggests that stimulators of some activities possessed by retroviral enzymes should be sought as antiviral agents.

Telomeres and telomerase in human hematologic neoplasia.

Telomeres are the physical ends of eukaryotic chromosomes. Telomeric DNA sequences are highly conserved in all well-characterized eukaryotic nuclear chromosomes and differ greatly from the termini of linear viral, extranuclear plasmid, or mitochondrial DNA. Human telomeric DNA consists of 2-15 kb of a tandemly repeated sequence (TTAGGG)n, oriented 5'-3' toward the end of the chromosome. The evolutionary conservation of this repetitive DNA sequence implies that the sequence is essential to cellular function. These repeated sequences are synthesized by an RNA-dependent DNA polymerase, telomerase, which is composed of an essential RNA and a few proteins. Human telomerase has been proposed to be repressed in somatic tissues, and human telomeres become shorter during somatic development and with increasing age. Telomeres in tumors are even shorter, and loss of telomeric DNA during tumorigenesis may contribute to the genome instability associated with transformed cells. This article reviews the structure and function of telomeres and the recent studies on human hematologic cells.

2′-Deoxyadenylyl-(3′→5′)-isodideoxyadenosine, a unique dinucleotide: Synthesis, enzymology, and conformational studies

The synthesis, enzymatic stability toward nucleases, and conformational properties of a unique dinucleotide, 2′-deoxyadenylyl-(3′→5′)-isodideoxyadenosine ( 2), a model system for the terminus of HIV viral DNA on incorporation of the L-related isomeric dideoxynucleoside, (S,S)-IsoddA, is reported. This is the first example of a dinucleotide bearing an isomeric nucleoside component.

Activities and substrate specificity of the evolutionarily conserved central domain of retroviral integrase

The retroviral integrase (IN) is a virus-encoded enzyme that is essential for insertion of viral DNA into the host chromosome.In order to map and define the properties of a minimal functional domain for this unique viral enzyme, a series of N- and C-terminal deletions of both Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV) INs were constructed. The RSV IN deletion mutants were first tested for their ability to remove two nucleotides from the end of a substrate representing the terminus of viral DNA in order to assess the contribution of N and C regions towards this reaction, referred to as processing. The results suggest that C-terminal amino acids of the intact RSV protein are required to maintain specificity of the processing reaction. Though deficient for processing, the RSV deletion mutants exhibited a secondary endonucleolytic activity that was indistinguishable from that of wild-type IN, demonstrating that all retained some enzymatic activity. RSV, and a larger set of HIV-1, IN deletion mutants were then tested for their ability to perform an intramolecular, concerted cleavage- ligation reaction using an oligodeoxynucleotide substrate that mimics the intermediate viral-host DNA junction found prior to the final step of covalent closure. The composite results from such analyses define a minimal functional central region of ∼140 amino acids for each enzyme that includes the highly conserved D,D(35)E domain. Results with HIV-1 and HIV-2 IN also indicate that the efficiency of concerted cleavage- ligation depends upon the presence of CA/GT base pairs within the viral component of the DNA substrate at the reaction site. Even the isolated central region of HIV-1 IN exhibited this sequence requirement for optimal activity. We conclude that this evolutionarily conserved central region of IN not only encodes residues that are required for the catalytic activity of the enzyme but also harbors some or all of the determinants responsible for recognition of the CA/GT dinucleotides that are present at the ends of all retroviral DNAs.

Retroviral integration: in vitro host site selection by avian integrase.

Viral integrase catalyzes the integration of the linear viral DNA genome into the chromatin of the infected host cell, an essential step in the life cycle of retroviruses. The reaction produces a characteristic small duplication of host sequences at the site of integration, implying that there is a close juxtaposition of the viral DNA ends during a concerted integration event. We have used an in vitro assay to measure the concerted integration of virus-like plasmid DNA into naked lambda DNA catalyzed by virion purified avian integrase. In contrast to in vivo avian integration, which has strong fidelity for a 6-bp duplication, purified avian integrase in the context of this assay produced a distribution of duplication sizes, with the 6-bp size dominating. The metal cofactor Mg2+ induced increased fidelity for the 6-bp duplication relative to that with Mn2+. The immediate sequence of the host site may also influence duplication size in that we found sites that sustained multiple independent integration events producing the same duplication size. Additionally, for each set of cloned integration sites (5, 6, and 7 bp), a unique but similar symmetrical pattern of G/C and A/T sequence biases was found. Using duplex oligonucleotides as target substrates, we tested the significance of the 6-bp G/C and A/T pattern for site selection. In the context of this assay, which is likely dominated by the integration of only one viral end, the 6-bp pattern was not preferred. Instead, integration was predominantly into the 3' ends of the oligonucleotides. The combined results of the lambda and oligonucleotide assays indicated that although host site selection has properties in common with recognition of the viral DNA termini, the nonrandom sequence preferences seen for host site selection were not identical to the sequence requirements for long terminal repeat recognition.