Duane P. Grandgenett

A 32,000-dalton nucleic acid-binding protein from avian retravirus cores possesses DNA endonuclease activity.

Abstract A 32,000-dalton nucleic acid-binding protein (p32), possessing DNA endonuclease activity, has been identified in avian myeloblastosis virus (AMV) and Rous sarcoma virus (Prague B strain) cores. The p32 nucleic acid-binding protein was purified from AMV virions by phosphocellulose, poly (U)-Sepharose 4B, and poly(C)-agarose chromatography and glycerol gradient centrifugation. A DNA endonuclease activity was found associated with p32 throughout purification, but the protein possessed no detectable exonuclease activity with natural or synthetic DNA or RNA substrates. In the presence of Mg 2+ , the p32-associated DNA endonuclease activity was able to convert a variety of supercoiled DNA molecules to the relaxed form by introducing single-stranded nicks into the DNA. There was only one nick per supercoiled Escherichia coli ColE 1 DNA strand whether the molecular ratio of p32 to DNA was 12, 60, 130, or 260 to 1. Under the same reaction conditions, the p32-associated endonuclease activity was much less efficient in nicking single- or double-stranded linear DNA molecules. Alkaline sucrose gradient centrifugation analysis of ColE 1 DNA which was nicked by p32 and subsequently digested by EcoRI endonuclease suggested that a limited number of preferred regions for p32 endonuclease activity existed on this DNA. This site-specificity was lost in the presence of Mn 2+ , but the efficiency of the DNA endonuclease activity was increased approximately 10-fold.

Recombinant Human Immunodeficiency Virus Type 1 Integrase Exhibits a Capacity for Full-Site Integration In Vitro That Is Comparable to That of Purified Preintegration Complexes from Virus-Infected Cells

ABSTRACT Retrovirus preintegration complexes (PIC) in virus-infected cells contain the linear viral DNA genome (∼10 kbp), viral proteins including integrase (IN), and cellular proteins. After transport of the PIC into the nucleus, IN catalyzes the concerted insertion of the two viral DNA ends into the host chromosome. This successful insertion process is termed “full-site integration.” Reconstitution of nucleoprotein complexes using recombinant human immunodeficiency virus type 1 (HIV-1) IN and model viral DNA donor substrates (∼0.30 to 0.48 kbp in length) that are capable of catalyzing efficient full-site integration has proven difficult. Many of the products are half-site integration reactions where either IN inserts only one end of the viral donor substrate into a circular DNA target or into other donors. In this report, we have purified recombinant HIV-1 IN at pH 6.8 in the presence of MgSO4 that performed full-site integration nearly as efficiently as HIV-1 PIC. The size of the viral DNA substrate was significantly increased to 4.1 kbp, thus allowing for the number of viral DNA ends and the concentrations of IN in the reaction mixtures to be decreased by a factor of ∼10. In a typical reaction at 37°C, recombinant HIV-1 IN at 5 to 10 nM incorporated 30 to 40% of the input DNA donor into full-site integration products. The synthesis of full-site products continued up to ∼2 h, comparable to incubation times used with HIV-1 PIC. Approximately 5% of the input donor was incorporated into the circular target producing half-site products with no significant quantities of other integration products produced. DNA sequence analysis of the viral DNA-target junctions derived from wild-type U3 and U5 coupled reactions showed an ∼70% fidelity for the HIV-1 5-bp host site duplications. Recombinant HIV-1 IN successfully utilized a mutant U5 end containing additional nucleotide extensions for full-site integration demonstrating that IN worked properly under nonideal active substrate conditions. The fidelity of the 5-bp host site duplications was also high with these coupled mutant U5 and wild-type U3 donor ends. These studies suggest that recombinant HIV-1 IN is at least as capable as native IN in virus particles and approaching that observed with HIV-1 PIC for catalyzing full-site integration.

Unraveling retrovirus integration

Compared with prokaryotic mobile DNA elements, the study of recombination mechanisms and proteins involved in the integration of vertebrate retroviruses is in its infancy. Genetic and biochemical studies have provided critical insights into defining the role in integration of both the retrovirus-encoded DNA endonuclease and the cisacting long terminal repeat (LTR) sequences of linear viral DNA. The hallmarks of retrovirus integration are its direct dependence on viral proteins, the exclusive utilization of linear viral DNA termini for integration, and the short duplication of cellular sequences at the site of insertion into the host genome. These properties are also characteristic of the integration of retrotransposons, such as Tyl, 2, and 3 elements in S. cerevisiae and Drosophila copia DNA elements. Following entry of the virus into the cell, the retroviral RNA genome is reverse transcribed into DNA. Missense mutations have demonstrated that a DNA binding endonuclease, designated integration protein (IN), is essential for integration of the newly synthesized viral DNA. IN is encoded in the 3’ portion of the viral polymerase gene and is proteolytically processed from a larger precursor to yield subunits of 32 kd (avian viruses and human immunodeficiency viruses) or 46 kd (murine leukemia virus; MuLV). Similarly, Ty elements encode a protein, pSO-TYB, that has a region homologous to IN. Mutations in this region effectively blocks integration of Tyl linear DNA in vivo and in vitro but does not prevent synthesis of reverse transcripts. (IN is not encoded in the 3’portion of the Tyl polymerase gene.) The size of the enzymatically active, proteolytically processed endonuclease of p90-TYB is yet to be defined. Interaction of retrovirus IN with the LTR sequences of linear viral DNA appears essential for normal integration. In vitro recombination assays and in vivo biochemical and genetic analyses have led to the following conclusions. Linear viral DNA is the immediate precursor to the integrated form of viral DNA (Brown et al., 1987). Genetic analysis of viruses with altered LTRs established the importance of these sequences for integration (Roth et al., 1989). After linear blunt-ended DNA is synthesized by reverse transcriptase, IN removes 2 bases from the 3’-hydroxyl termini of both strands (Fujiwara and Mizuuchi, 1988; Brown et al., 1989; Roth et al., 1989). This 3’-recessed DNA appears to be the immediate precursor to the integrated form of the DNA (see part A of figure), in which the 3’ ends are covalently attached to the S’phosphoryl ends of the host DNA (see part B of figure). The yeast Tyl DNA element appears to employ a similar nonhomologous integration mechanism (Eichinger and Boeke, 1988, Minireview

Efficient Concerted Integration by Recombinant Human Immunodeficiency Virus Type 1 Integrase without Cellular or Viral Cofactors

ABSTRACT 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 ∼30 nM IN (20 min at 37°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 ∼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 (∼125 μg/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.

Transcriptional Coactivator LEDGF/p75 Modulates Human Immunodeficiency Virus Type 1 Integrase-Mediated Concerted Integration

ABSTRACT Human transcriptional coactivator p75/lens epithelium-derived growth factor (LEDGF) binds human immunodeficiency virus type 1 (HIV-1) integrase (IN). We studied the effects of LEDGF on the assembly and activity of HIV-1 synaptic complexes, which, upon association with a target, mediate concerted integration of viral DNA substrates in vitro. We found that while augmenting single-ended viral DNA integration into target DNA, the host factor was able to either stimulate or abrogate concerted integration in a concentration-dependent manner. LEDGF modestly stimulated (two- to threefold) concerted integration at low molar ratios to IN (<1). The modest stimulation was independent of solution conditions and several different viral DNA substrates. In solution, concerted integration was inhibited if the molar ratios of LEDGF to IN were >1, apparently due to the disruption of IN-IN interactions essential for the formation of active synaptic complexes prior to their association with a circular target. The isolated IN binding domain of LEDGF was sufficient to stimulate and inhibit concerted integration, as observed with full-length protein, albeit at lower efficiencies. Our data show that LEDGF differentially affects IN-DNA complexes mediating single-ended viral DNA integration and synaptic complexes mediating concerted integration. Synaptic complexes associated with target, termed strand transfer complexes, are resistant to disruption by high concentrations of LEDGF. The results suggest that LEDGF may influence HIV-1 integration in vivo.

Inhibition of Human Immunodeficiency Virus Type 1 Concerted Integration by Strand Transfer Inhibitors Which Recognize a Transient Structural Intermediate

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) integrase (IN) inserts the viral DNA genome into host chromosomes. Here, by native agarose gel electrophoresis, using recombinant IN with a blunt-ended viral DNA substrate, we identified the synaptic complex (SC), a transient early intermediate in the integration pathway. The SC consists of two donor ends juxtaposed by IN noncovalently. The DNA ends within the SC were minimally processed (∼15%). In a time-dependent manner, the SC associated with target DNA and progressed to the strand transfer complex (STC), the nucleoprotein product of concerted integration. In the STC, the two viral DNA ends are covalently attached to target and remain associated with IN. The diketo acid inhibitors and their analogs effectively inhibit HIV-1 replication by preventing integration in vivo. Strand transfer inhibitors L-870,810, L-870,812, and L-841,411, at low nM concentrations, effectively inhibited the concerted integration of viral DNA donor in vitro. The inhibitors, in a concentration-dependent manner, bound to IN within the SC and thereby blocked the docking onto target DNA, which thus prevented the formation of the STC. Although 3′-OH recessed donor efficiently formed the STC, reactions proceeding with this substrate exhibited marked resistance to the presence of inhibitor, requiring significantly higher concentrations for effective inhibition of all strand transfer products. These results suggest that binding of inhibitor to the SC occurs prior to, during, or immediately after 3′-OH processing. It follows that the IN-viral DNA complex is “trapped” by the strand transfer inhibitors via a transient intermediate within the cytoplasmic preintegration complex.

DNase Protection Analysis of Retrovirus Integrase at the Viral DNA Ends for Full-Site Integration In Vitro

ABSTRACT Retrovirus intasomes purified from virus-infected cells contain the linear viral DNA genome and integrase (IN). Intasomes are capable of integrating the DNA termini in a concerted fashion into exogenous target DNA (full site), mimicking integration in vivo. Molecular insights into the organization of avian myeloblastosis virus IN at the viral DNA ends were gained by reconstituting nucleoprotein complexes possessing intasome characteristics. Assembly of IN–4.5-kbp donor complexes capable of efficient full-site integration appears cooperative and is dependent on time, temperature, and protein concentration. DNase I footprint analysis of assembled IN-donor complexes capable of full-site integration shows that wild-type U3 and other donors containing gain-of-function attachment site sequences are specifically protected by IN at low concentrations (<20 nM) with a defined outer boundary mapping ∼20 nucleotides from the ends. A donor containing mutations in the attachment site simultaneously eliminated full-site integration and DNase I protection by IN. Coupling of wild-type U5 ends with wild-type U3 ends for full-site integration shows binding by IN at low concentrations probably occurs only at the very terminal nucleotides (<10 bp) on U5. The results suggest that assembly requires a defined number of avian IN subunits at each viral DNA end. Among several possibilities, IN may bind asymmetrically to the U3 and U5 ends for full-site integration in vitro.

Crystal structure of the Rous sarcoma virus intasome

Integration of the reverse-transcribed viral DNA into the host genome is an essential step in the life cycle of retroviruses. Retrovirus integrase catalyses insertions of both ends of the linear viral DNA into a host chromosome. Integrase from HIV-1 and closely related retroviruses share the three-domain organization, consisting of a catalytic core domain flanked by amino- and carboxy-terminal domains essential for the concerted integration reaction. Although structures of the tetrameric integrase–DNA complexes have been reported for integrase from prototype foamy virus featuring an additional DNA-binding domain and longer interdomain linkers, the architecture of a canonical three-domain integrase bound to DNA remained elusive. Here we report a crystal structure of the three-domain integrase from Rous sarcoma virus in complex with viral and target DNAs. The structure shows an octameric assembly of integrase, in which a pair of integrase dimers engage viral DNA ends for catalysis while another pair of non-catalytic integrase dimers bridge between the two viral DNA molecules and help capture target DNA. The individual domains of the eight integrase molecules play varying roles to hold the complex together, making an extensive network of protein–DNA and protein–protein contacts that show both conserved and distinct features compared with those observed for prototype foamy virus integrase. Our work highlights the diversity of retrovirus intasome assembly and provides insights into the mechanisms of integration by HIV-1 and related retroviruses.

Requirement of the avian retrovirus pp32 DNA binding protein domain for replication

The NH2-terminal amino acid sequence of the pp32 DNA binding protein has been determined, thus establishing its precise coding region in the polymerase gene of Rous sarcoma virus. Specific mutations were constructed in molecularly cloned Prague A DNA near the NH2- and COOH-termini of pp32 and the effects were assayed by transfection on chick embryo fibroblasts. Out-of-frame deletions at both sites and an in-frame deletion near the NH2 terminus rendered the DNA noninfectious and transformation negative. Single point mutations near the NH2 terminus reduced the transfection efficiency and the rate of virus replication. Biochemical studies indicated that the RNA-directed DNA polymerase and RNase H activities of the mutant viruses were not affected but the processing of the viral beta polypeptide was altered.

Molecular and Genetic Determinants of Rous Sarcoma Virus Integrase for Concerted DNA Integration

ABSTRACT 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 ∼30% of the input donor into full-site integration products after 10 min of incubation at 37°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 ∼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°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.