Biomedical subjects
Robert Craigie
Publications and source records attributed to Robert Craigie.
Retroviral DNA integration: reaction pathway and critical intermediates.
The key DNA cutting and joining steps of retroviral DNA integration are carried out by the viral integrase protein. Structures of the individual domains of integrase have been determined, but their organization in the active complex with viral DNA is unknown. We show that HIV-1 integrase forms stable synaptic complexes in which a tetramer of integrase is stably associated with a pair of viral DNA ends. The viral DNA is processed within these complexes, which go on to capture the target DNA and integrate the viral DNA ends. The joining of the two viral DNA ends to target DNA occurs sequentially, with a stable intermediate complex in which only one DNA end is joined. The integration product also remains stably associated with integrase and likely requires disassembly before completion of the integration process by cellular enzymes. The results define the series of stable nucleoprotein complexes that mediate retroviral DNA integration.
Seeing is believing: structure of the catalytic domain of HIV-1 integrase in complex with human LEDGF/p75.
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Structural basis for DNA bridging by barrier-to-autointegration factor.
The ability of barrier-to-autointegration factor (BAF) to bind and bridge DNA in a sequence-independent manner is crucial for its role in retroviral integration and a variety of cellular processes. To better understand this behavior, we solved the crystal structure of BAF bound to DNA. The structure reveals that BAF bridges DNA using two pairs of helix-hairpin-helix motifs located on opposite surfaces of the BAF dimer without changing its conformation.
Processing of viral DNA ends channels the HIV-1 integration reaction to concerted integration.
Retroviral DNA made by reverse transcription is blunt-ended, and the viral integrase protein must remove two nucleotides from each 3' end prior to integration into chromosomal DNA. Under most reaction conditions for integration in vitro, the majority of the reaction products are "half-site" products that result from integration of only one viral DNA end into one strand of the target DNA. Preprocessed DNA substrates are more efficient substrates for half-site reactions than are blunt-ended substrates, which require the removal of two nucleotides prior to integration. In contrast, we find that blunt-ended DNA is a better substrate for the biologically relevant reaction of concerted integration of pairs of viral DNA ends. The reaction pathway is channeled to concerted integration, and half-site integration products are reduced with blunt-ended DNA substrate that must first be processed by integrase. In addition, the terminal nucleotide requirements for concerted integration are more stringent than for the half-site reaction. Longer DNA is more efficient for the concerted reaction than is shorter DNA that is capable of efficient half-site integration. This suggests that nonspecific interactions of integrase with viral DNA distant from the termini contribute to the assembly of a complex that is competent for concerted integration. Finally, differential effects of mutation of a residue in the C-terminal domain of integrase on concerted versus half-site integration implicate protein-protein interactions involving this domain as important for concerted integration.
Mass spectrometric analysis of the HIV-1 integrase-pyridoxal 5'-phosphate complex reveals a new binding site for a nucleotide inhibitor.
HIV-1 integrase (IN) is an important target for designing new antiviral therapies. Screening of potential inhibitors using recombinant IN-based assays has revealed a number of promising leads including nucleotide analogs such as pyridoxal 5'-phosphate (PLP). Certain PLP derivatives were shown to also exhibit antiviral activities in cell-based assays. To identify an inhibitory binding site of PLP to IN, we used the intrinsic chemical property of this compound to form a Schiff base with a primary amine in the protein at the nucleotide binding site. The amino acid affected was then revealed by mass spectrometric analysis of the proteolytic peptide fragments of IN. We found that an IC(50) concentration (15 mum) of PLP modified a single IN residue, Lys(244), located in the C-terminal domain. In fact, we observed a correlation between interaction of PLP with Lys(244) and the compound's ability to impair formation of the IN.DNA complex. Site-directed mutagenesis studies confirmed an essential role of Lys(244) for catalytic activities of recombinant IN and viral replication. Molecular modeling revealed that Lys(244) together with several other DNA binding residues provides a plausible pocket for a nucleotide inhibitor-binding site. To our knowledge, this is the first report indicating that a small molecule inhibitor can impair IN activity through its binding to the protein C terminus. At the same time, our findings highlight the importance of structural analysis of the full-length protein.
LAP2alpha and BAF collaborate to organize the Moloney murine leukemia virus preintegration complex.
Integration of viral DNA into the host genome is an essential step in retroviral replication. The viral DNA made by reverse transcription is a component of the preintegration complex (PIC) that also contains the viral integrase protein, the enzyme that integrates the viral DNA. Several other viral and cellular proteins are present in the PIC, but their functional roles are less well established. Barrier-to-autointegration factor (BAF) is a cellular protein component of the PIC that blocks autointegration of the viral DNA and stimulates intermolecular integration. In uninfected cells, BAF interacts with members of the LEM family of inner nuclear membrane and nucleoplasmic proteins. Here, we demonstrate that one of the LEM proteins, lamina-associated polypeptide 2alpha (LAP2alpha), is a component of the PIC. LAP2alpha stabilizes the association of BAF with the PIC to stimulate intermolecular integration and suppress autointegration. To further understand the role of LAP2alpha, we established LAP2alpha-knockdown cell lines. Depletion of LAP2alpha significantly inhibited viral replication. Our results demonstrate a critical contribution of LAP2alpha to the nucleoprotein organization of the PIC and to viral replication.
Identification of an inhibitor-binding site to HIV-1 integrase with affinity acetylation and mass spectrometry.
We report a methodology that combines affinity acetylation with MS analysis for accurate mapping of an inhibitor-binding site to a target protein. For this purpose, we used a known HIV-1 integrase inhibitor containing aryl di-O-acetyl groups (Acetylated-Inhibitor). In addition, we designed a control compound (Acetylated-Control) that also contained an aryl di-O-acetyl group but did not inhibit HIV-1 integrase. Examination of the reactivity of these compounds with a model peptide library, which collectively contained all 20 natural amino acids, revealed that aryl di-O-acetyl compounds effectively acetylate Cys, Lys, and Tyr residues. Acetylated-Inhibitor and Acetylated-Control exhibited comparable chemical reactivity with respect to these small peptides. However, these two compounds differed markedly in their interactions with HIV-1 integrase. In particular, Acetylated-Inhibitor specifically acetylated K173 at its inhibitory concentration (3 microM) whereas this site remained unrecognized by Acetylated-Control. Our data enabled creation of a detailed model for the integrase:Acetylated-Inhibitor complex, which indicated that the inhibitor selectively binds at an architecturally critical region of the protein. The methodology reported herein has a generic application for systems involving a variety of ligand-protein interactions.
MoMLV reverse transcriptase regulates its own expression.
A precise ratio of Gag:Gag-Pol expression is required for assembly of infectious retroviral virions. In this issue of Cell, Orlova et al. show that MoMLV reverse transcriptase binds the translation release factor eRF1, and that this interaction promotes translation readthrough to make Gag-Pol.
Barrier-to-autointegration factor: major roles in chromatin decondensation and nuclear assembly.
Barrier-to-autointegration factor (BAF) is a DNA-bridging protein, highly conserved in metazoans. BAF binds directly to LEM (LAP2, emerin, MAN1) domain nuclear membrane proteins, including LAP2 and emerin. We used site-directed mutagenesis and biochemical analysis to map functionally important residues in human BAF, including those required for direct binding to DNA or emerin. We also tested wild-type BAF and 25 point mutants for their effects on nuclear assembly in Xenopus egg extracts, which contain approximately 12 microM endogenous BAF dimers. Exogenous BAF caused two distinct effects: at low added concentrations, wild-type BAF enhanced chromatin decondensation and nuclear growth; at higher added concentrations, wild-type BAF completely blocked chromatin decondensation and nuclear growth. Mutants fell into four classes, including one that defines a novel functional surface on the BAF dimer. Our results suggest that BAF, unregulated, potently compresses chromatin structure, and that BAF interactions with both DNA and LEM proteins are critical for membrane recruitment and chromatin decondensation during nuclear assembly.
Regulatory mechanisms by which barrier-to-autointegration factor blocks autointegration and stimulates intermolecular integration of Moloney murine leukemia virus preintegration complexes.
Retroviral integration is mediated by a preintegration complex (PIC) which contains the viral DNA made by reverse transcription together with associated protein factors. Prior to association with target DNA, the PIC must avoid suicidal intramolecular integration of its viral DNA (autointegration). We have demonstrated that barrier-to-autointegration factor (BAF) blocks the autointegration of Moloney murine leukemia virus (MoMLV) PICs in vitro. In this study, we show that BAF is an authentic component of MoMLV. Analysis of the sedimentation properties of initial, salt-stripped, and BAF-reconstituted PICs reveals that the viral DNA within the PIC is reversibly compacted by BAF, consistent with the functional role of BAF in protecting the viral DNA from autointegration. Furthermore, we find that BAF can promote the association of PICs with target DNA. Thus, our data suggest that BAF plays critical roles in promoting preferential intermolecular integration by both blocking autointegration and stimulating the capture of target DNA.