Robert Craigie

Structure of a two-domain fragment of HIV-1 integrase: implications for domain organization in the intact protein.

Retroviral integrase, an essential enzyme for replication of human immunodeficiency virus type‐1 (HIV‐1) and other retroviruses, contains three structurally distinct domains, an N‐terminal domain, the catalytic core and a C‐terminal domain. To elucidate their spatial arrangement, we have solved the structure of a fragment of HIV‐1 integrase comprising the N‐terminal and catalytic core domains. This structure reveals a dimer interface between the N‐terminal domains different from that observed for the isolated domain. It also complements the previously determined structure of the C‐terminal two domains of HIV‐1 integrase; superposition of the conserved catalytic core of the two structures results in a plausible full‐length integrase dimer. Furthermore, an integrase tetramer formed by crystal lattice contacts bears structural resemblance to a related bacterial transposase, Tn5, and exhibits positively charged channels suitable for DNA binding.

Identification of discrete functional domains of HIV-1 integrase and their organization within an active multimeric complex.

HIV‐1 integrase protein possesses the 3′ processing and DNA strand transfer activities that are required to integrate HIV DNA into a host chromosome. The N‐, C‐terminal and core domains of integrase are necessary for both activities in vitro. We find that certain pairs of mutant integrase proteins, which are inactive when each protein is assayed alone, can support near wild type levels of activity when both proteins are present together in the reaction mixture. This complementation implies that HIV‐1 integrase functions as a multimer and has enabled us to probe the organization of the functional domains within active mixed multimers. We have identified a minimal set of functional integrase domains that are sufficient for 3′ processing and DNA strand transfer and find that some domains are contributed in trans by separate monomers within the functional complex.

Solution structure of the N-terminal zinc binding domain of HIV-1 integrase

The solution structure of the N-terminal zinc binding domain (residues 1–55; IN1–55) of HIV-1 integrase has been solved by NMR spectroscopy. IN1–55 is dimeric, and each monomer comprises four helices with the zinc tetrahedrally coordinated to His 12, His 16, Cys 40 and Cys 43. IN1–55 exists in two interconverting conformational states that differ with regard to the coordination of the two histidine side chains to zinc. The different histidine arrangements are associated with large conformational differences in the polypeptide backbone (residues 9–18) around the coordinating histidines. The dimer interface is predominantly hydrophobic and is formed by the packing of the N-terminal end of helix 1, and helices 3 and 4. The monomer fold is remarkably similar to that of a number of helical DMA binding proteins containing a helix-turn-helix (HTH) motif with helices 2 and 3 of IN1–55 corresponding to the HTH motif. In contrast to the DNA binding proteins where the second helix of the HTH motif is employed for DNA recognition, IN1–55 uses this helix for dimerization.

Sequence specificity of viral end DNA binding by HIV‐1 integrase reveals critical regions for protein–DNA interaction

HIV‐1 integrase specifically recognizes and cleaves viral end DNA during the initial step of retroviral integration. The protein and DNA determinants of the specificity of viral end DNA binding have not been clearly identified. We have used mutational analysis of the viral end LTR sequence, in vitro selection of optimal viral end sequences, and specific photocrosslinking to identify regions of integrase that interact with specific bases in the LTR termini. The results highlight the involvement of the disordered loop of the integrase core domain, specifically residues Q148 and Y143, in binding to the terminal portion of the viral DNA ends. Additionally, we have identified positions upstream in the LTR termini which interact with the C‐terminal domain of integrase, providing evidence for the role of that domain in stabilization of viral DNA binding. Finally, we have located a region centered 12 bases from the viral DNA terminus which appears essential for viral end DNA binding in the presence of magnesium, but not in the presence of manganese, suggesting a differential effect of divalent cations on sequence‐specific binding. These results help to define important regions of contact between integrase and viral DNA, and assist in the formulation of a molecular model of this vital interaction.

HIV integrase, a brief overview from chemistry to therapeutics.

Retroviruses are a large and diverse family of RNA viruses that synthesize a DNA copy of their RNA genome after infection of the host cell. Integration of this viral DNA into host DNA is an essential step in the replication cycle of HIV and other retroviruses (reviewed in Refs. 1–3). The integrated viral DNA is transcribed to make the RNA genome of progeny virions and the template for translation of viral proteins. Following assembly, virions bud from the cell surface and subsequently infect previously uninfected cells, thus completing the replication cycle. An infecting retrovirus introduces a large nucleoprotein complex into the cytoplasm of the host cell. This complex, which is derived from the core of the infecting virion, contains two copies of the viral RNA together with a number of viral proteins, including reverse transcriptase and integrase. Reverse transcription of the viral RNA occurs within the complex to make a double-stranded DNA copy of the viral genome, the viral DNA substrate for integration. The viral DNA remains associated with both viral and cellular proteins in a nucleoprotein complex termed the preintegration complex. One constituent of the preintegration complex is the viral integrase protein, the key player in the integration of the viral DNA into the host genome. The other components of the preintegration complex that are transported to the nucleus along with the viral DNA and integrase, and their possible functions, have not been firmly established and are not discussed here. The critical DNA cutting and joining events that integrate the viral DNA are carried out by the integrase protein itself. Here we review our current knowledge of the molecular mechanism of this reaction and discuss some of the key issues that are yet to be understood.

The road to chromatin - nuclear entry of retroviruses.

Human immunodeficiency virus 1 (HIV-1) and other retroviruses synthesize a DNA copy of their genome after entry into the host cell. Integration of this DNA into the host cells genome is an essential step in the viral replication cycle. The viral DNA is synthesized in the cytoplasm and is associated with viral and cellular proteins in a large nucleoprotein complex. Before integration into the host genome can occur, this complex must be transported to the nucleus and must cross the nuclear envelope. This Review summarizes our current knowledge of how this journey is accomplished.

Critical contacts between HIV-1 integrase and viral DNA identified by structure-based analysis and photo-crosslinking.

Analysis of the crystal structure of HIV‐1 integrase reveals a cluster of lysine residues near the active site. Using site‐directed mutagenesis and photo‐crosslinking we find that Lys156 and Lys159 are critical for the functional interaction of integrase with viral DNA. Mutation of Lys156 or Lys159 to glutamate led to a loss of both 3′ processing and strand transfer activities in vitro while maintaining the ability to interact with nonspecific DNA and support disintegration. However, mutation of both residues to glutamate produced a synergistic effect eliminating nearly all nonspecific DNA interaction and disintegration activity. In addition, virus containing either of these changes was replication‐defective at the step of integration. Photo‐crosslinking, using 5‐iododeoxyuracil‐substituted oligonucleotides, suggests that Lys159 interacts at the N7 position of the conserved deoxyadenosine adjacent to the scissile phosphodiester bond of viral DNA. Sequence conservation throughout retroviral integrases and certain bacterial transposases (e.g. Tn10/IS10) supports the premise that within those families of polynucleotidyl transferases, these residues are strategic for DNA interaction.

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.

HIV integrase structure and function.

HIV integrase consists of three domains, the structures of which have been individually determined by X-ray crystallography or NMR spectroscopy. The core domain, spanning residues 50-212, is responsible for the catalytic activity of the enzyme. The crystal structure of a dimer of this domain shows similarity to other proteins that carry out polynucleotidyl transfer, including MuA transposase and RNase H. The small N-terminal domain folds into a dimeric helix-turn-helix structure, which is stabilized by the coordination of zinc with conserved His and Cys residues. The function of this domain is unclear; however, it is required for integration activity and enhances tetramerization in the context of the full-length integrase. The C-terminal domain, which has an SH3-like fold, is involved in DNA binding. The structure of this domain reveals a large saddle-shaped cleft that is formed by dimerization. This cleft contains a number of positively charged residues, and its dimensions are appropriate for accommodating a double-stranded DNA helix. Although the C-terminal domain was originally believed to be involved in target DNA binding, more recent evidence suggests that it may bind to both the ends of the viral DNA and to the target DNA. Although the individual domain structures provide some insights into the function of the protein, a more detailed understanding of the complete mechanism by which integrase binds, cleaves, and transfers DNA requires a greater knowledge of how these domains are arranged in the active multimer.

LAP2 binds to BAF⋅DNA complexes: requirement for the LEM domain and modulation by variable regions

LAP2 belongs to a family of nuclear membrane proteins sharing a 43 residue LEM domain. All LAP2 isoforms have the same N‐terminal ‘constant’ region (LAP2‐c), which includes the LEM domain, plus a C‐terminal ‘variable’ region. LAP2‐c polypeptide inhibits nuclear assembly in Xenopus extracts, and binds in vitro to barrier‐to‐autointegration factor (BAF), a DNA‐bridging protein. We tested 17 Xenopus LAP2‐c mutants for nuclear assembly inhibition, and binding to BAF and BAF·DNA complexes. LEM domain mutations disrupted all activities tested. Some mutations outside the LEM domain had no effect on binding to BAF, but disrupted activity in Xenopus extracts, suggesting that LAP2‐c has an additional unknown function required to inhibit nuclear assembly. Mutagenesis results suggest that BAF changes conformation when complexed with DNA. The binding affinity of LAP2 was higher for BAF·DNA complexes than for BAF, suggesting that these interactions are physiologically relevant. Nucleoplasmic domains of Xenopus LAP2 isoforms varied 9‐fold in their affinities for BAF, but all isoforms supershifted BAF·DNA complexes. We propose that the LEM domain is a core BAF‐binding domain that can be modulated by the variable regions of LAP2 isoforms.