Irreversible inhibition of human immunodeficiency virus type 1 integrase by dicaffeoylquinic acids

Human immunodeficiency virus type 1 (HIV-1) and other retroviruses require integration of a double-stranded DNA copy of the RNA genome into the host cell chromosome for productive infection. The viral enzyme, integrase, catalyzes the integration of retroviral DNA and represents an attractive target for developing antiretroviral agents. We identified several derivatives of dicaffeoylquinic acids (DCQAs) that inhibit HIV-1 replication in tissue culture and catalytic activities of HIV-1 integrase in vitro. The specific step at which DCQAs inhibit the integration in vitro and the mechanism of inhibition were examined in the present study. Titration experiments with different concentrations of HIV-1 integrase or DNA substrate found that the effect of DCQAs was exerted on the enzyme and not the DNA. In addition to HIV-1, DCQAs also inhibited the in vitro activities of MLV integrase and truncated variants of feline immunodeficiency virus integrase, suggesting that these compounds interacted with the central core domain of integrase. The inhibition on retroviral integrases was relatively specific, and DCQAs had no effect on several other DNA-modifying enzymes and phosphoryltransferases. Kinetic analysis and dialysis experiments showed that the inhibition of integrase by DCQAs was irreversible. The inhibition did not require the presence of a divalent cation and was unaffected by preassembling integrase onto viral DNA. The results suggest that the irreversible inhibition by DCQAs on integrase is directed toward conserved amino acid residues in the central core domain during catalysis.     

A hard way to the nucleus

As a member of the Retrovirus family, human immunodeficiency virus (HIV), a causative agent of AIDS, replicates by integrating its genome into the host cell's nuclear DNA. However, in contrast to most retroviruses that depend on mitotic dissolution of the nuclear envelope to gain access to the host cell's genome, the HIV pre-integration complex can enter the nucleus of the target cell during the interphase. Such capacity greatly enhances HIV replication and allows the virus to productively infect terminally differentiated nonproliferating cells, such as macrophages. Infection of macrophages is a critical factor in the pathogenesis of diseases caused by HIV-1 and other lentiviruses. The mechanisms responsible for this unusual feature of HIV have enticed researchers since the early 90s, when the first characterization of the HIV-1 pre-integration complex was reported. Several viral factors, including matrix protein, integrase, viral protein R, and central DNA flap, have been proposed as regulators of HIV-1 nuclear import, only to be later shown as nonessential for this process. As a result, after more than a decade of intense research, there is still no consensus on which HIV-1 and cellular proteins control this critical step in HIV-1 replication. In this review, we will discuss recent advances and suggest possible solutions to the controversial issue of HIV-1 nuclear import.     

Integration is not necessary for expression of human immunodeficiency virus type 1 protein products.

A common feature in the life cycle of cytocidal retroviruses, including human immunodeficiency virus type 1 (HIV-1), is the accumulation of large amounts of unintegrated viral DNA. As yet, the role of unintegrated viral DNA in the cytopathogenesis of cytocidal retrovirus infections remains unresolved. HIV-1 mutants which were deleted in the integrase/endonuclease gene and which were unable to establish an integrated form of the virus were constructed. Despite an inability to integrate, these mutants were fully competent templates for HIV-1 core and envelope antigen production. HIV-1 antigen could be detected in the supernatants of lymphocyte cultures infected with HIV-1 integrase mutants. However, an inability to rescue infectious virus from these cultures indicated that HIV-1 integration was required for the production of infectious HIV-1. On the basis of the ability of unintegrated HIV-1 DNA to serve as a template for HIV-1 antigen production, it is plausible that unintegrated viral DNA can contribute to the HIV-1 antigen pool during HIV-1 replication.     

Integrase-lexA fusion proteins incorporated into human immunodeficiency virus type 1 that contains a catalytically inactive integrase gene are functional to mediate integration

Purified fusion proteins made up of a retroviral integrase and a sequence-specific DNA-binding protein have been tested in in vitro assays for their ability to direct integration into specific target sites. To determine whether these fusion proteins can be incorporated into human immunodeficiency virus type 1 (HIV-1) and are functional to mediate integration, we used an in trans approach to deliver various integrase-LexA proteins to an integrase-defective virus containing an integrase mutation at aspartate residue 64. Integrase-LexA, integrase-LexA DNA-binding domain, or N- or C-terminally truncated integrase-LexA proteins were fused to the HIV-1 accessory protein, Vpr. Coexpression of the Vpr fusion proteins and an integrase-defective HIV-1 molecular clone by a producer cell line resulted in efficient incorporation of the fusion protein into the integrase-mutated virus. In addition, each of these viruses was infectious and capable of performing integration, as determined by two independent cellular assays that measure reporter gene expression. With the exception of the N-terminally truncated integrase fused to LexA, which was at about 1%, all of the fusion proteins restored integration to a similar level, at 17 to 24% of that of the wild-type virus. The low level observed with the N-terminally truncated integrase fused to LexA is consistent with previous results implying that the N terminus of integrase is involved in multiple steps of the retroviral life cycle. These data indicate that the integrase-fusion proteins retain catalytic function in the integrase-mutated viruses and demonstrate the feasibility of incorporating integrase fusion proteins into HIV-1 for the development of site-directed retroviral vectors.     

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.     

Weak palindromic consensus sequences are a common feature found at the integration target sites of many retroviruses

Integration into the host genome is one of the hallmarks of the retroviral life cycle and is catalyzed by virus-encoded integrases. While integrase has strict sequence requirements for the viral DNA ends, target site sequences have been shown to be very diverse. We carefully examined a large number of integration target site sequences from several retroviruses, including human immunodeficiency virus type 1, simian immunodeficiency virus, murine leukemia virus, and avian sarcoma-leukosis virus, and found that a statistical palindromic consensus, centered on the virus-specific duplicated target site sequence, was a common feature at integration target sites for these retroviruses.     

DNA damage sensors ATM, ATR, DNA-PKcs, and PARP-1 are dispensable for human immunodeficiency virus type 1 integration

Integration of a DNA copy of the viral RNA genome is a crucial step in the life cycle of human immunodeficiency virus type 1 (HIV-1) and other retroviruses. While the virally encoded integrase is key to this process, cellular factors yet to be characterized are suspected to participate in its completion. DNA damage sensors such as ATM (ataxia-telangiectasia mutated), ATR (ATM- and Rad3-related), DNA-PK (DNA-dependent protein kinase), and PARP-1 [poly(ADP-ribose) polymerase 1] play central roles in responses to various forms of DNA injury and as such could facilitate HIV integration. To test this hypothesis, we examined the susceptibility to infection with wild-type HIV-1 and to transduction with a vesicular stomatitis virus G protein (VSV-G)-pseudotyped HIV-1-derived lentiviral vector of human cells stably expressing small interfering RNAs against ATM, ATR, and PARP-1. We found that integration normally occurred in these knockdown cells. Similarly, the VSV-G-pseudotyped HIV-1-based vector could effectively transduce ATM and PARP-1 knockout mouse cells as well as human cells deficient for DNA-PK. Finally, treatment of target cells with the ATM and ATR inhibitors caffeine and wortmannin was without effect in these infectivity assays. We conclude that the DNA repair enzymes ATM, ATR, DNA-PKcs, and PARP-1 are not essential for HIV-1 integration.     

Fusion proteins consisting of human immunodeficiency virus type 1 integrase and the designed polydactyl zinc finger protein E2C direct integration of viral DNA into specific sites

In order to establish a productive infection, a retrovirus must integrate the cDNA of its RNA genome into the host cell chromosome. While this critical process makes retroviruses an attractive vector for gene delivery, the nonspecific nature of integration presents inherent hazards and variations in gene expression. One approach to alleviating the problem involves fusing retroviral integrase to a sequence-specific DNA-binding protein that targets a defined chromosomal site. We prepared proteins consisting of wild-type or truncated human immunodeficiency virus type 1 (HIV-1) integrase fused to the synthetic polydactyl zinc finger protein E2C. The purified fusion proteins bound specifically to the 18-bp E2C recognition sequence as analyzed by DNase I footprinting. The fusion proteins were catalytically active and biased integration of retroviral DNA near the E2C-binding site in vitro. The distribution was asymmetric, and the major integration hot spots were localized within a 20-bp region upstream of the C-rich strand of the E2C recognition sequence. Integration bias was not observed with target plasmids bearing a mutated E2C-binding site or when HIV-1 integrase and E2C were added to the reaction as separate proteins. The results demonstrate that the integrase-E2C fusion proteins offer an efficient approach and a versatile framework for directing the integration of retroviral DNA into a predetermined DNA site.     

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.     

Requirement of active human immunodeficiency virus type 1 integrase enzyme for productive infection of human T-lymphoid cells.

The human immunodeficiency virus type 1 (HIV-1) integrase enzyme exhibits significant amino acid sequence conservation with integrase proteins of other retroviruses. We introduced specific amino acid substitutions at a number of the conserved residue positions of recombinant HIV-1 integrase. Some of these substitutions resulted in proteins which were not able to be purified in the same manner as the wild-type enzyme, and these were not studied further. The remaining mutant enzymes were assessed for their abilities to perform functions characteristic of the integrase protein. These included specific removal of the terminal dinucleotides from oligonucleotide substrates representative of the viral U5-long terminal repeat, nonspecific cleavage of oligonucleotide substrates, and mediation of the strand transfer (integration) reaction. Substitution at position 43, within the protein's zinc finger motif region, resulted in an enzyme with reduced specificity for cleavage of the terminal dinucleotide. In addition, a double substitution of aspartic acid and glutamine for valine and glutamic acid, respectively, at positions 151 and 152 within the D,D(35)E motif region rendered the integrase protein inactive for all of its functions. The introduction of this double substitution into an infectious HIV-1 provirus yielded a mutant virus that was incapable of productively infecting human T-lymphoid cells in culture.     

LEDGF/p75 Proteins with Alternative Chromatin Tethers Are Functional HIV-1 Cofactors

LEDGF/p75 can tether over-expressed lentiviral integrase proteins to chromatin but how this underlies its integration cofactor role for these retroviruses is unclear. While a single integrase binding domain (IBD) binds integrase, a complex N-terminal domain ensemble (NDE) interacts with unknown chromatin ligands. Whether integration requires chromatin tethering per se, specific NDE-chromatin ligand interactions or other emergent properties of LEDGF/p75 has been elusive. Here we replaced the NDE with strongly divergent chromatin-binding modules. The chimeras rescued integrase tethering and HIV-1 integration in LEDGF/p75-deficient cells. Furthermore, chromatin ligands could reside inside or outside the nucleosome core, and could be protein or DNA. Remarkably, a short Kaposi''s sarcoma virus peptide that binds the histone 2A/B dimer converted GFP-IBD from an integration blocker to an integration cofactor that rescues over two logs of infectivity. NDE mutants were corroborative. Chromatin tethering per se is a basic HIV-1 requirement and this rather than engagement of particular chromatin ligands is important for the LEDGF/p75 cofactor mechanism.     

Retroviral Integrase Proteins and HIV-1 DNA Integration

Retroviral integrases catalyze two reactions, 3′-processing of viral DNA ends, followed by integration of the processed ends into chromosomal DNA. X-ray crystal structures of integrase-DNA complexes from prototype foamy virus, a member of the Spumavirus genus of Retroviridae, have revealed the structural basis of integration and how clinically relevant integrase strand transfer inhibitors work. Underscoring the translational potential of targeting virus-host interactions, small molecules that bind at the host factor lens epithelium-derived growth factor/p75-binding site on HIV-1 integrase promote dimerization and inhibit integrase-viral DNA assembly and catalysis. Here, we review recent advances in our knowledge of HIV-1 DNA integration, as well as future research directions.     

Substrate features important for recognition and catalysis by human immunodeficiency virus type 1 integrase identified by using novel DNA substrates.

The integrase encoded by human immunodeficiency virus type 1 (HIV-1) is required for integration of viral DNA into the host cell chromosome. In vitro, integrase mediates a concerted cleavage-ligation reaction (strand transfer) that results in covalent attachment of viral DNA to target DNA. With a substrate that mimics the strand transfer product, integrase carries out disintegration, the reverse of the strand transfer reaction, resolving this integration intermediate into its viral and target DNA parts. We used a set of disintegration substrates to study the catalytic mechanism of HIV-1 integrase and the interaction between the protein and the viral and target DNA sequence. One substrate termed dumbbell consists of a single oligonucleotide that can fold to form a structure that mimics the integration intermediate. Kinetic analysis using the dumbbell substrate showed that integrase turned over, establishing that HIV-1 integrase is an enzyme. Analysis of the disintegration activity on the dumbbell substrate and its derivatives showed that both the viral and target DNA parts of the molecule were required for integrase recognition. Integrase recognized target DNA asymmetrically: the target DNA upstream of the viral DNA joining site played a much more important role than the downstream target DNA in protein-DNA interaction. The site of transesterification was determined by both the DNA sequence of the viral DNA end and the structure of the branched substrate. Using a series of disintegration substrates with various base modifications, we found that integrase had relaxed structural specificity for the hydroxyl group used in transesterification and could tolerate distortion of the double-helical structure of these DNA substrates.     

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.