Retroviral DNA integration

The synthesis and integration of DNA into the genome of its host cell is a normal step in the replication of the retroviruses. Previous studies have provided details concerning the structure of viral DNA and viral and host integration sites. More recent genetic and biochemical results have expanded our understanding considerably: it should soon be possible to describe the exact viral DNA sequence recognized during the integration reaction for several viruses. In addition, at least one of the viral proteins and enzymatic activities required in the reaction has been identified. Analysis of this apparently efficient and highly specific site-directed recombination event in eukaryotic cells promises to provide insights of both fundamental and practical interest.     

Retroviral DNA integration: structure of an integration intermediate

The structure of a presumptive DNA intermediate in the integration of retroviral DNA was studied in a cell-free reaction with exogenously added target DNA. The product made by viral core particles of Moloney murine leukemia virus (Mo-MLV) containing linear viral DNA has a structure consistent with an integration mechanism similar to that observed during bacteriophage Mu transposition. In this intermediate, the 3' ends of the LTR sequences are joined to the target DNA, while the 5' ends of the viral DNA remain unjoined. The 5' ends of the LTR sequences in the intermediate are exactly the same as those found in the unintegrated linear double-stranded viral DNA. This result demonstrates that the linear form of Mo-MLV DNA can integrate directly without prior circularization.     

Retroviral DNA integration and the DNA damage response

Retroviral DNA integration creates a discontinuity in the host cell chromatin and repair of this damage is required to complete the integration process. As integration and repair are essential for both viral replication and cell survival, it is possible that specific interactions with the host DNA repair systems might provide new cellular targets for human immunodeficiency virus therapy. Various genetic, pharmacological, and biochemical studies have provided strong evidence that postintegration DNA repair depends on components of the nonhomologous end-joining (NHEJ) pathway (DNA-PK (DNA-dependent protein kinase), Ku, Xrcc4, DNA ligase IV) and DNA damage-sensing pathways (Atr (Atm and Rad related), gamma-H2AX). Furthermore, deficiencies in NHEJ components result in susceptibility to apoptotic cell death following retroviral infection. Here, we review these findings and discuss other ways that retroviral DNA intermediates may interact with the host DNA damage signaling and repair pathways.     

Retroviral integration: Site matters

Here, we review genomic target site selection during retroviral integration as a multistep process in which specific biases are introduced at each level. The first asymmetries are introduced when the virus takes a specific route into the nucleus. Next, by co‐opting distinct host cofactors, the integration machinery is guided to particular chromatin contexts. As the viral integrase captures a local target nucleosome, specific contacts introduce fine‐grained biases in the integration site distribution. In vivo, the established population of proviruses is subject to both positive and negative selection, thereby continuously reshaping the integration site distribution. By affecting stochastic proviral expression as well as the mutagenic potential of the virus, integration site choice may be an inherent part of the evolutionary strategies used by different retroviruses to maximise reproductive success.     

Retroviral DNA integration: HIV and the role of LEDGF/p75

To replicate, a retrovirus must integrate a DNA copy of its RNA genome into a chromosome of the host cell. Integration is not random in the host genome but favors particular regions, and preferences differ among retroviruses. Several mechanisms might play a part in this favored integration targeting: (i) open chromatin might be preferentially accessible for viral DNA integration; (ii) DNA replication during cell division might facilitate access of integration complexes to favored sites; and (iii) cellular proteins bound to the host chromosome might tether integration complexes to favored regions. This review summarizes recent advances in understanding the mechanisms of retroviral integration, focusing on LEDGF/p75--the first cellular protein shown to have a role in directing HIV DNA integration. Studies on LEDGF/p75 indicate that it directs HIV integration site selection by a tethering interaction, whereas the chromatin accessibility or cell cycle models are less well supported. Understanding viral integration will help improve the safety of retrovirus-based vectors used in gene therapy.     

Crystal structures of I-SceI complexed to nicked DNA substrates: snapshots of intermediates along the DNA cleavage reaction pathway

I-SceI is a homing endonuclease that specifically cleaves an 18-bp double-stranded DNA. I-SceI exhibits a strong preference for cleaving the bottom strand DNA. The published structure of I-SceI bound to an uncleaved DNA substrate provided a mechanism for bottom strand cleavage but not for top strand cleavage. To more fully elucidate the I-SceI catalytic mechanism, we determined the X-ray structures of I-SceI in complex with DNA substrates that are nicked in either the top or bottom strands. The structures resemble intermediates along the DNA cleavage reaction. In a structure containing a nick in the top strand, the spatial arrangement of metal ions is similar to that observed in the structure that contains uncleaved DNA, suggesting that cleavage of the bottom strand occurs by a common mechanism regardless of whether this strand is cleaved first or second. In the structure containing a nick in the bottom strand, a new metal binding site is present in the active site that cleaves the top strand. This new metal and a candidate nucleophilic water molecule are correctly positioned to cleave the top strand following bottom strand cleavage, providing a plausible mechanism for top strand cleavage.     

PriA mediates DNA replication pathway choice at recombination intermediates

We report the reconstitution of the initial steps of the double-strand break-repair pathway where joint molecule formation between a duplex DNA fragment and a circular template by the combined action of RecA, RecBCD, and the single-stranded DNA binding protein provides the substrate for replication fork formation by the restart primosome and the DNA polymerase III holoenzyme. We show that PriA dictates the pathway of replication from the recombination intermediate by inhibiting a nonspecific, strand displacement DNA synthesis reaction and favoring the formation of a bona fide replication fork. Furthermore, we find that RecO and RecR significantly stimulate this recombination-directed DNA replication reaction, and that this stimulation is modulated by the presence of RecF, suggesting that the latter protein may also act as a regulator of the pathway of resolution of the recombination intermediate.     

Retroviral DNA Integration

DNA integration is a unique enzymatic process shared by all retroviruses and retrotransposons. During integration, double-stranded linear viral DNA is inserted into the host genome in a process catalyzed by the virus-encoded integrase (IN). The mechanism involves a series of nucleophillic attacks, the first of which removes the terminal 2 bases from the 3′ ends of the long terminal repeats and of the second which inserts the viral DNA into the host genome. IN specifically recognizes the DNA sequences at the termini of the viral DNA, juxtaposing both ends in an enzyme complex that inserts the viral DNA into a single site in a concerted manner. Small duplications of the host DNA, characteristic of the viral IN, are found at the sites of insertion. At least two host proteins, HMG-I(Y) and BAF, have been shown to increase the efficiency of the integration reaction.     

Increased recombination intermediates and homologous integration hot spots at DNA replication origins

We have studied the relationship between DNA replication and recombination in Schizosaccharomyces pombe using two-dimensional gel electrophoresis and functional analysis. Our results indicate that the activation of replication origins (ORIs) during the mitotic cell cycle is associated with the generation of joint DNA molecules between sister chromatids. The frequency of integration by homologous recombination was up to 50-fold higher than the genomic average within a narrow window overlapping the ars1 replication initiation site. The S. pombe rad22Delta, rhp51Delta, and rhp54Delta mutants, deficient in mitotic recombination, activate ORIs very inefficiently and accumulate abnormal replication intermediates. These results focus on the general link between replication and recombination previously found in several systems and suggest a role for recombination in the initiation of eukaryotic DNA replication.     

Retroviral integration profiles: their determinants and implications for gene therapy

Retroviruses have often been used for gene therapy because of their capacity for the long-term expression of transgenes via stable integration into the host genome. However, retroviral integration can also result in the transformation of normal cells into cancer cells, as demonstrated by the incidence of leukemia in a recent retroviral gene therapy trial in Europe. This unfortunate outcome has led to the rapid initiation of studies examining various biological and pathological aspects of retroviral integration. This review summarizes recent findings from these studies, including the global integration patterns of various types of retroviruses, viral and cellular determinants of integration, implications of integration for gene therapy and retrovirus-mediated infectious diseases, and strategies to shift integration to safe host genomic loci. A more comprehensive and mechanistic understanding of retroviral integration processes will eventually make it possible to generate safer retroviral vector platforms in the near future.     

Retroviral cDNA integration: stimulation by HMG I family proteins

To replicate, a retrovirus must synthesize a cDNA copy of the viral RNA genome and integrate that cDNA into a chromosome of the host. We have investigated the role of a host cell cofactor, HMG I(Y) protein, in integration of human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus (MoMLV) cDNA. Previously we reported that HMG I(Y) cofractionates with HIV-1 preintegration complexes (PICs) isolated from freshly infected cells. PICs depleted of required components by treatment with high concentrations of salt could be reconstituted by addition of purified HMG I(Y) in vitro. Here we report studies using immunoprecipitation that indicate that HMG I(Y) is associated with MoMLV preintegration complexes. In mechanistic studies, we show for both HIV-1 and MoMLV that each HMG I(Y) monomer must contain multiple DNA binding domains to stimulate integration by HMG I(Y)-depleted PICs. We also find that HMG I(Y) can condense model HIV-1 or MoMLV cDNA in vitro as measured by stimulation of intermolecular ligation. This reaction, like reconstitution of integration, depends on the presence of multiple DNA binding domains in each HMG I(Y) monomer. These data suggest that binding of multivalent HMG I(Y) monomers to multiple cDNA sites compacts retroviral cDNA, thereby promoting formation of active integrase-cDNA complexes.     

Retroviral integration into minichromosomes in vitro.

We describe here the use of chromatin as a target for retroviral integration in vitro. Extracts of cells newly infected with murine leukemia virus (MLV) provided the source of integration activity, and yeast TRP1ARS1 and SV40 minichromosomes served as simple models for chromatin. Both minichromosomes were used as targets for integration, with efficiencies comparable with that of naked DNA. In addition, under some reaction conditions the minichromosomes behaved as if they were used preferentially over naked DNAs in the same reaction. Mapping of integration sites by cloning and sequencing recombinants revealed that the integration machinery does not display a preference for nucleosome-free, nuclease-sensitive regions. The distributions of integration sites in TRP1ARS1 minichromosomes and a naked DNA counterpart were grossly similar, but in a detailed analysis the distribution in minichromosomes was found to be significantly more ordered: the sites displayed a periodic spacing of approximately 10 bp, many sites sustained multiple insertions and there was sequence bias at the target sites. These results are in accord with a model in which the integration machinery has preferential access to the exposed face of the nucleosomal DNA helix. The population of potential sites in chromatin therefore becomes more limited, in a manner dictated by the rotational orientation of the DNA sequence around the nucleosome core, and those sites are used more frequently than in naked DNA.     

Structure of the termini of DNA intermediates in the integration of retroviral DNA: dependence on IN function and terminal DNA sequence

Linear retroviral DNA, the major precursor to the integrated provirus of the murine leukemia viruses, contains a mixture of two structures at its ends: some termini are full-length and blunt, and some have recessed 3' strands. A temporal study of the end structures showed that the proportion of the DNA with recessed ends increases during the course of infection, and suggests that the blunt ends are precursors to the recessed ends. We have examined the DNA structures of the ends of retroviral mutants defective in the integration (IN) function. The results show that the formation of the recessed ends requires the presence of IN. Finally, we have analyzed the structures at the ends of mutant genomes with alterations in the terminal DNA sequence. The exact position of the recessed 3' end can be recessed one, two, or four nucleotides relative to the 5' end. In all cases the position of the recessed 3' end correlates perfectly with, and thus presumably determines, the site of joining to the target DNA.     

Reaction of nitric oxide with the turnover intermediates of cytochrome c oxidase: reaction pathway and functional effects

The reactions of nitric oxide (NO) with the turnover intermediates of cytochrome c oxidase were investigated by combining amperometric and spectroscopic techniques. We show that the complex of nitrite with the oxidized enzyme (O) is obtained by reaction of both the "peroxy" (P) and "ferryl" (F) intermediates with stoichiometric NO, following a common reaction pathway consistent with P being an oxo-ferryl adduct. Similarly to chloride-free O, NO reacted with P and F more slowly [k approximately (2-8) x 10(4) M(-1) s(-1)] than with the reduced enzyme (k approximately 1 x 10(8) M(-1) s(-1)). Recovery of activity of the nitrite-inhibited oxidase, either during turnover or after a reduction-oxygenation cycle, was much more rapid than nitrite dissociation from the fully oxidized enzyme (t(1/2) approximately 80 min). The anaerobic reduction of nitrite-inhibited oxidase produced the fully reduced but uncomplexed enzyme, suggesting that reversal of inhibition occurs in turnover via nitrite dissociation from the cytochrome a(3)-Cu(B) site: this finding supports the hypothesis that oxidase may have a physiological role in the degradation of NO into nitrite. Kinetic simulations suggest that the probability for NO to be transformed into nitrite is greater at low electron flux through oxidase, while at high flux the fully reduced (photosensitive) NO-bound oxidase is formed; this is fully consistent with our recent finding that light releases the inhibition of oxidase by NO only at higher reductant pressure [Sarti, P., et al. (2000) Biochem. Biophys. Res. Commun. 274, 183].     

Catalytic mechanisms and reaction intermediates along the hydrolytic pathway of a plant beta-D-glucan glucohydrolase.

BACKGROUND: Barley beta-D-glucan glucohydrolases represent family 3 glycoside hydrolases that catalyze the hydrolytic removal of nonreducing glucosyl residues from beta-D-glucans and beta-D-glucooligosaccharides. After hydrolysis is completed, glucose remains bound in the active site. RESULTS: When conduritol B epoxide and 2', 4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-glucopyranoside are diffused into enzyme crystals, they displace the bound glucose and form covalent glycosyl-enzyme complexes through the Odelta1 of D285, which is thereby identified as the catalytic nucleophile. A nonhydrolyzable S-glycosyl analog, 4(I), 4(III), 4(V)-S-trithiocellohexaose, also diffuses into the active site, and a S-cellobioside moiety positions itself at the -1 and +1 subsites. The glycosidic S atom of the S-cellobioside moiety forms a short contact (2.75 A) with the Oepsilon2 of E491, which is likely to be the catalytic acid/base. The glucopyranosyl residues of the S-cellobioside moiety are not distorted from the low-energy 4C(1) conformation, but the glucopyranosyl ring at the +1 subsite is rotated and translated about the linkage. CONCLUSIONS: X-ray crystallography is used to define the three key intermediates during catalysis by beta-D-glucan glucohydrolase. Before a new hydrolytic event begins, the bound product (glucose) from the previous catalytic reaction is displaced by the incoming substrate, and a new enzyme-substrate complex is formed. The second stage of the hydrolytic pathway involves glycosidic bond cleavage, which proceeds through a double-displacement reaction mechanism. The crystallographic analysis of the S-cellobioside-enzyme complex with quantum mechanical modeling suggests that the complex might mimic the oxonium intermediate rather than the enzyme-substrate complex.     

Possible genotoxicity of melanin synthesis intermediates: tyrosinase reaction products interact with DNA in vitro

Summary The actual cellular target of the cytotoxic intermediates of melanin synthesis is not yet known. In the present paper it is shown that eukaryotic DNA binds in vitro to soluble reaction products of tyrosinase (EC 1.14.18.1) and is physically modified, as ascertained by the following criteria: (a) buoyant density in cesium chloride density gradients; (b) polyacrylamide gel electrophoresis: (c) deoxyribonuclease (EC 3.1.4.5) test; (d) electron microscopy. The results reported here support the view that DNA itself may be a target for the cytotoxic intermediates of melanin synthesis.