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PubMed2003

New classes of HIV drugs on the horizon. A review of the presentation at the satellite symposium "New hope: advancing care in HIV infection" at the 15th annual Association of Nurses in AIDS Care conference, November 2002.

Despite the success of combination HAART in improving the clinical prognosis of HIV-infected patients, therapeutic options are limited for many patients, particularly those with extensive treatment experience. Resistance, cross-resistance, and toxicity combine to threaten the durability of antiviral response, necessitating the development of novel agents. The HIV life cycle has many potential targets for inhibition in addition to the current reverse transcriptase and protease targets. These include the HIV integrase, nucleocapsid, and Tat proteins and the viral entry process. Entry inhibitors can be classified as attachment, coreceptor, and fusion inhibitors, according to the stage in the entry process at which they act. Most advanced in development of these is the fusion inhibitor enfuvirtide. Enfuvirtide is a potent inhibitor of HIV fusion whose novel mechanism of action and extracellular nature mean that it will induce limited cross-resistance to currently approved antiretrovirals and fewer toxicities.

Animals↗
PubMed1990

Characterization of an internally initiated integrase protein of HIV-1 produced in E. coli.

In E. coli cells transformed by an expression vector for the production of the protease (PR) integrase (IN) of HIV-1, three vitally encoded proteins were produced: an 11-kDa protein and a 32-kDa protein identified by immunoassays as the mature PR and IN protein, respectively, and an additional protein 15-kDa in size that reacted strongly with an antiserum recognizing a region in the carboxyl half of the IN protein. The kinetics of its synthesis indicated that it was not a degradation product of p32-IN, rather it probably arose from internal initiation at an AUG codon in the middle of the IN gene. Amino terminal sequence analysis of the first 70 residues demonstrated a perfect match with those predicted from the nucleotide sequence, beginning with the methionine codon at position 154 of the integrase gene.

Amino Acid Sequence↗
PubMed2006

Modulating target site selection during human immunodeficiency virus DNA integration in vitro with an engineered tethering factor.

The mechanisms controlling retroviral integration have been the topic of intense interest, in part because of adverse clinical events that occurred during retrovirus-mediated human gene therapy. Here we investigate the use of artificial tethering interactions to constrain retroviral integration site selection in an in vitro model. During normal infection, HIV DNA integration is favored in active cellular transcription units. One component of the targeting mechanism is the cellular LEDGF/p75 protein. LEDGF/p75 binds tightly to HIV integrase (IN) protein, and depletion of LEDGF/p75 from target cells results in reduced integration in transcription units, suggesting integration targeting by a tethering mechanism. We constructed and analyzed fusions of LEDGF/p75 or its IN-binding domain (IBD) to the DNA-binding domain of phage lambda repressor protein (lambdaR). In the presence of the lambdaR-LEDGF/p75 fusions, increased strand transfer by IN was seen in target DNA near lambdaR-binding sites in vitro . These data support the idea that a direct interaction between LEDGF/p75 and IN can mediate targeting via a tethering mechanism, and provide proof of concept for the idea that protein-protein interactions might be engineered to constrain integration site selection during human gene therapy.

Base Sequence↗
PubMedDate not supplied

Mechanistic aspects of HIV-1 reverse transcription initiation.

During reverse transcription, the positive-strand HIV-1 RNA genome is converted into a double-stranded DNA copy which can be permanently integrated into the host cell genome. Recent analyses show that HIV-1 reverse transcription is a highly regulated process. The initiation reaction can be distinguished from a subsequent elongation reaction carried out by a reverse transcription complex composed of (at least) heterodimeric reverse transcriptase, cellular tRNA(lys3) and HIV-1 genomic RNA sequences. In addition, viral factors including Tat, Nef, Vif, Vpr, IN and NCp7, cellular proteins, and TAR RNA and other RNA stem-loop structures appear to influence this complex and contribute to the efficiency of the initiation reaction. As viral resistance to many antiretroviral compounds is a continuing problem, understanding the ways in which these factors influence the reverse transcription complex will likely lead to novel antiretroviral strategies.

Animals↗
PubMed2000

Identification of efficiently cleaved substrates for HIV-1 protease using a phage display library and use in inhibitor development.

The recognition sequences for substrate cleavage by aspartic protease of HIV-1 are diverse and cleavage specificities are controlled by complex interactions between at least six amino acids around the cleavage site. We have identified 45 efficiently cleaved peptide substrates of HIV-1 protease (PR) using substrate phage display, an approach that can elucidate both context-dependent and context-independent preferences at individual subsites of a protease substrate. Many of the selected peptides were cleaved more efficiently and had lower K(m) values than physiologically relevant substrates of HIV-1 PR. Therefore, mutations occurring in the cleavage sites of the Gag and Gag-pol polyproteins of HIV-1 could significantly lower the K(m) values to better compete against drugs for protease binding while maintaining cleavage rates necessary for viral replication. The most efficiently cleaved peptide substrate derived from these phage, Ac-GSGIF*LETSL-NH(2), was cleaved 60 times more efficiently and had a K(m) approximately 260 times lower than a nine-amino-acid peptide based on the natural reverse transcriptase/integrase cleavage site when assayed at pH 5.6, 0.2 M NaCl. The peptide substrates selected served as frameworks for synthesis of tight binding reduced amide inhibitors of HIV-1 PR. The results show that the most efficiently cleaved substrates serve as the best templates for synthesis of the tightest binding inhibitors. Thus, defining changes in substrate preferences for drug-resistant proteases may aid in the development of more efficacious inhibitors.

Amides↗
PubMed2005

A very large diversity space of synthetically accessible compounds for use with drug design programs.

We have constructed a very large virtual diversity space containing more than 10(13) chemical compounds. The diversity space is built from about 400 combinatorial libraries, which have been expanded by choosing sizeable collections of suitable R-groups that can be attached to each link point of their scaffolds. These R-group collections have been created by selecting reagents that have drug-like properties from catalogs of available chemicals. As members of known combinatorial libraries, the compounds in the diversity space are in general synthetically accessible and useful as potential drug leads. Hence, the diversity space can be used as a vast source of compounds by a de novo drug design program. For example, we have used such a program to generate inhibitors of HIV integrase enzyme that exhibited activity in the micromolar range.

Algorithms↗
PubMed1996

Cell protein cross-linking by erbstatin and related compounds.

The protein-tyrosine kinase inhibitor and stable erbstatin analogue methyl 2,5-dihydroxycinnamate (4) cross-links cell proteins by a non-physiological chemical mechanism (Stanwell et al., Cancer Res 55: 4950-4956, 1995). To determine the structural requirements for this effect, erbstatin (1) and fifteen related compounds, including caffeic acid phenylethyl ester (9) were synthesized and examined for their ability to induce cross-linking of cellular protein at concentrations ranging from low micromolar up to 1000 microM. Tests were conducted in NIH3T3 fibroblasts as well as mouse keratinocytes. Potent cross-linking of cellular protein was observed for a number of analogues, including erbstatin, at concentrations as low as 10-50 microM. The inactivity of methoxy and fluoro as compared with their corresponding dihydroxylated counterparts indicated that free aromatic hydroxyls were essential for cross-linking. Additionally, compounds containing phenyl rings with 1,4-dihydroxy substituents were more potent than those having 1,2-dihydroxylated patterns. As with the prototype compound 4, cross-linking was induced at both 37 degrees and 4 degrees, suggesting a chemical rather than physiological mechanism. Consistent with the data, a mechanism of action is proposed which involves initial oxidation to reactive quinone intermediates that subsequently cross-link protein nucleophiles via multiple 1,4-Michael-type additions. Similar alkylation of protein by protein-tyrosine kinase inhibitors, such as herbimycin A, has been invoked. While the latter benzoquinoid ansamycin antibiotics contain performed quinone moieties, results of the present study suggest that other hydroxylated kinase inhibitors can potentially participate in similar phenomena. A large number of potential therapeutics, including HIV integrase inhibitors, possess polyhydroxylated nuclei. The non-specific nature of the protein cross-linking reaction demonstrated for these erbstatin analogues, and the fact that cross-linking can occur at micromolar concentrations, may limit the therapeutic usefulness of such compounds to specific applications.

Animals↗
PubMed1988

Biosynthesis and analysis of a genetically engineered HIV-1 reverse transcriptase/endonuclease polyprotein in Escherichia coli.

Elimination of the protease domain from the polymerase open reading frame (pol) of the human immunodeficiency virus type 1 (HIV-1) leads, in Escherichia coli, to synthesis and accumulation of a non-processed 98-kDa reverse transcriptase/endonuclease (RT/ENDO) polyprotein. A partially purified preparation of this reverse RT/ENDO polyprotein displays little or no RT activity. Introduction of the pol protease domain as a separate transcriptional unit on the same plasmid restores the processing program, generating correctly sized RT and ENDO polypeptides. Concomitant with restoration of processing is the reappearance of RT activity. These results suggest that for HIV-1 RT to be active, it must be matured from the pol polyprotein.

Endodeoxyribonucleases↗
PubMed2003

Construction and characterisation of a full-length infectious molecular clone from a fast replicating, X4-tropic HIV-1 CRF02.AG primary isolate.

Based on our previous analysis of HIV-1 isolates from Cameroon, we constructed a full-length infectious molecular clone from a primary isolate belonging to the CRF02.AG group of recombinant viruses which dominate the HIV-epidemic in West and Central Africa. The virus derived by transfection of the proviral clone pBD6-15 replicated with similar efficiency compared to its parental isolate and used CXCR4 as coreceptor as well. Furthermore, HIV-1 BD6-15 exhibited similar replication properties and virus yield as the reference B-type HIV-1 strain NL4-3. Sequence analysis revealed open reading frames for all structural and accessory genes apart from vpr. Phylogenetic and bootscanning analyses confirmed that BD6-15 clusters with CRF02.AG recombinant strains from West and Central Africa with similar cross-over points as described for the CRF02.AG prototype strain lbNG. Thus, pBD6-15 represents the first non-subtype B infectious molecular clone of a fast replicating, high producer, X4-tropic primary HIV-1 isolate, which had only been briefly passaged in primary cells.

Adult↗
PubMed1997

Specificity in V(D)J recombination: new lessons from biochemistry and genetics.

Recent in vitro work on V(D)J recombination has helped to clarify its mechanism. The first stage of the reaction, which can be reproduced with the purified RAG1 and RAG2 proteins, is a site-specific cleavage that generates the same broken DNA species found in vivo. The cleavage reaction is closely related to known types of transpositional recombination, such as that of HIV integrase. All the site specificity of V(D)J recombination, including the 12/23 rule, is determined by the RAG proteins. The later steps largely overlap with the repair of radiation-induced DNA double-strand breaks, as indicated by the identity of several newly characterized factors involved in repair. These developments open the way for a thorough biochemical study of V(D)J recombination.

Animals↗
PubMed2003

MS2Assign, automated assignment and nomenclature of tandem mass spectra of chemically crosslinked peptides.

In a previous report (Young et al., Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5802-5806), we provided a proof-of-principle for fold recognition of proteins using a homobifunctional amine-specific chemical crosslinking reagent in combination with mass spectrometry analysis and homology modeling. In this current work, we propose a systematic nomenclature to describe the types of peptides that are generated after proteolysis of crosslinked proteins, their fragmentation by tandem mass spectrometry, and an automated algorithm for MS/MS spectral assignment called "MS2Assign." Several examples are provided from crosslinked peptides and proteins including HIV-integrase, cytochrome c, ribonuclease A, myoglobin, cytidine 5-monophosphate N-acetylneuraminic acid synthetase, and the peptide thymopentin. Tandem mass spectra were obtained from various crosslinked peptides using post source decay MALDI-TOF and collision induced dissociation on a quadrupole-TOF instrument, along with their automated interpretation using MS2Assign. A variety of possible outcomes are described and categorized according to the number of modified lysines and/or peptide chains involved, as well as the presence of singly modified (dead-end) lysine residues. In addition, the proteolysis and chromatographic conditions necessary for optimized crosslinked peptide recovery are presented.

Algorithms↗
PubMed1998

Hairpin coding end opening is mediated by RAG1 and RAG2 proteins.

Despite the importance of hairpin opening in antigen receptor gene assembly, the molecular machinery that mediates this reaction has not been defined. Here, we show that RAG1 plus RAG2 can open DNA hairpins. Hairpin opening by RAGs is not sequence specific, but in Mg2+, hairpin opening occurs only in the context of a regulated cleavage complex. The chemical mechanism of hairpin opening by RAGs resembles RSS cleavage and 3' end processing by HIV integrase and Mu transposase in that these reactions can proceed through alcoholysis. Mutations in either RAG1 or RAG2 that interfere with RSS cleavage also interfere with hairpin opening, suggesting that RAGs have a single active site that catalyzes several distinct DNA cleavage reactions.

Alcohols↗
PubMed2006

The inner-nuclear-envelope protein emerin regulates HIV-1 infectivity.

Primate lentiviruses such as human immunodeficiency type 1 (HIV-1) have the capacity to infect non-dividing cells such as tissue macrophages. In the process, viral complementary DNA traverses the nuclear envelope to integrate within chromatin. Given the intimate association between chromatin and the nuclear envelope, we examined whether HIV-1 appropriates nuclear envelope components during infection. Here we show that emerin, an integral inner-nuclear-envelope protein, is necessary for HIV-1 infection. Infection of primary macrophages lacking emerin was abortive in that viral cDNA localized to the nucleus but integration into chromatin was inefficient, and conversion of viral cDNA to non-functional episomal cDNA increased. HIV-1 cDNA associated with emerin in vivo, and the interaction of viral cDNA with chromatin was dependent on emerin. Barrier-to-autointegration factor (BAF), the LEM (LAP, emerin, MAN) binding partner of emerin, was required for the association of viral cDNA with emerin and for the ability of emerin to support virus infection. Therefore emerin, which bridges the interface between the inner nuclear envelope and chromatin, may be necessary for chromatin engagement by viral cDNA before integration.

Cells, Cultured↗
PubMed2004

True reversal of Mu integration.

We describe a high-temperature (75 degrees C) transition in the Mu integration complex that causes efficient and true reversal of the integration reaction. A second reversal pathway, first described as 'foldback' reversal for the HIV integrase, was also observed upon disassembly/reassembly of the Mu complex at normal temperatures. Both true and foldback reversal severed only one or the other of the two integrated Mu ends, and each exhibited distinct metal ion specificities. Our results directly implicate an altered transposase configuration in the Mu strand transfer complex that inhibits reversal, thereby regulating the directionality of transposition.

Bacteriophage mu↗
PubMed1997

Indeterminate human immunodeficiency virus type 1 western blot may indicate an abortive infection in some low-risk blood donors.

BACKGROUND: The infectious status of persons with an indeterminate human immunodeficiency virus type 1 (HIV-1) Western blot must be established. STUDY DESIGN AND METHODS: Evaluation of the CD4 and CD8 T-cell subsets and the expression of HIV-1-integrated sequences by Southern blot and polymerase chain reaction were studied in a group of low-risk subjects with an indeterminate Western blot. RESULTS: From a total of 45,000 blood donors and 50 patients with chronic renal failure on hemodialysis who were tested during the period of 1985 through 1990, 50 sera (0.1%) had an indeterminate Western blot. A low CD4:CD8 ratio (0.7-1.2) was detected in 14 of 24 tested subjects, whereas the unfractionated and adherence-enriched cells of 7 (32%) and 5 (23%) of 22 patients, respectively, could be stained with a p24 monoclonal antibody. A transient positive culture was detected in 3 of 20 subjects, but these viral isolates could not be transmitted to CEM-A310 cells. Ultracentrifuged culture supernatants hybridized under high-stringency conditions with genomic gag-pol (4 cases), env (3 cases), and tat (1 case) cDNA fragments of the HXB2 HIV-1 clone. In one case, DNA obtained from adherent but not unfractionated mononuclear cells contained 3.3- and 3.9-kb env- and gag-pol-related HIV-1 sequences, respectively; these sequences were heavier than expected. Polymerase chain reaction analysis for gag and pol but not env sequences was positive in 1 and 2 of 7 cases, respectively. A female patient with a positive viral culture and who was positive for pol in polymerase chain reaction demonstrated a full seroconversion 19 months later. CONCLUSION: The results strongly suggest that, rarely, some low-risk subjects with indeterminate Western blot results might be infected with low-level replicative strains or HIV-related viruses; thus, an exhaustive immunologic and virologic workup is needed for the investigation of these subjects.

Adolescent↗
PubMed1999

Asymmetric DDE (D35E)-like sequences in the RAG proteins: implications for V(D)J recombination and retroviral pathogenesis.

Experimental evidence suggests that the mechanism of vertebrate V(D)J recombination catalyzed by the vertebrate RAG proteins is similar to both retroviral integration and the transposition of IS630/Tc1-family transposons. The mechanism of both retroviral integration and IS630/Tc1 element transposition is well characterized and utilizes a functional metal ion binding site termed the DDE (or D35E) motif. We have previously identified a DDE-like region in the RAG-2 protein and a similar region within the RAG-1 protein. In this work, we propose that interference between DDE-like regions in the RAG proteins and the DDE-site of the HIV integrase may be a mechanism of retroviral pathogenesis in cells in which both the RAG proteins and retroviral integrase are co-expressed.

Amino Acid Sequence↗