Robert Craigie

Retroviral DNA integration is mediated by nucleoprotein complexes (intasomes) comprising a pair of viral DNA ends synapsed by a tetramer of integrase. Current integrase inhibitors act on intasomes rather than free integrase protein. Structural and functional studies of intasomes are essential to understand their mechanism of action and how the virus can escape by mutation. To date, prototype foamy virus (PFV) is the only retrovirus for which high-resolution structures of intasomes have been determined. In the PFV intasome structure, only the core domains of the outer subunits are ordered; the N-terminal domain, C-terminal domain, and N-terminal extension domain are disordered. Are these “missing domains” required for function or are they dispensable? We have devised a strategy to assemble “hetero-intasomes” in which the outer domains are not present as a tool to assess the functional role of the missing domains for catalysis of integration. We find that the disordered domains of outer subunits are not required for intasome assembly or catalytic activity as catalytic core domains can substitute for the outer subunits in the case of both PFV and HIV-1 intasomes.

AIDS has been transformed from a death sentence to a manageable disease for many patients with access to combination antiviral therapy. It is informative to look back on some of the key advances that have led to this transformation. The arsenal of tools currently available to clinicians now includes inhibitors of the viral reverse transcriptase, protease and integrase enzymes. The author discusses some of the key advances that have led to this transformation with an emphasis on the role of basic science in developing integrase inhibitors. Many of the stepping-stones could not easily have been foreseen to lead to medical advances. Treatments for diseases that are yet to emerge will likely depend on the progress made in basic science today.

The DNA cutting and joining reactions of HIV-1 integration are catalyzed by integrase (IN), a viral protein that functions as a tetramer bridging the two viral DNA ends (intasome). Two major obstacles for biochemical and structural studies of HIV-1 intasomes are 1) the low efficiency of assembly with oligonucleotide DNA substrates, and 2) the non-specific aggregation of both intasomes and free IN in the reaction mixture. By fusing IN with a small non-specific DNA binding protein, Sulfolobus solfataricus chromosomal protein Sso7d (PDB: 1BNZ), we have engineered a highly soluble and hyperactive IN. Unlike wild-type IN, it efficiently catalyzes intasome assembly and concerted integration with oligonucleotide DNA substrates. The fusion IN protein also functions to integrate viral reverse transcripts during HIV-infection. The hyperactive HIV-1 IN may assist in facilitating future biochemical and structural studies of HIV-1 intasomes. Understanding the mechanistic basis of the Sso7d-IN fusion protein could provide insight into the factors that have hindered biophysical studies of wild-type HIV-1 IN and intasomes.

Integrase is the key enzyme that mediates integration of retroviral DNA into cellular DNA which is essential for viral replication. Inhibitors of HIV‐1 that target integrase recognize the nucleoprotein complexes formed by integrase and viral DNA substrate (intasomes) rather than the free enzyme. Atomic resolution structures of HIV‐1 intasomes are therefore required to understand the mechanisms of inhibition and drug resistance. To date, prototype foamy virus (PFV) is the only retrovirus for which such structures have been determined. We show that PFV strand transfer complexes (STC) can be assembled on product DNA without going through the normal forward reaction pathway. The finding that a retroviral STC can be assembled in this way may provide a powerful tool to alleviate the obstacles that impede structural studies of nucleoprotein intermediates in HIV‐1 DNA integration.

Integration of viral DNA into cellular DNA is an essential step in the replication cycle of HIV and other retroviruses. The first antiviral drugs that target integrase, the viral enzyme that catalyzes DNA integration, have recently been approved and more are in the pipeline. These drugs bind to an intermediate in DNA integration called the intasome, in which a pair of viral DNA ends are synapsed by a tetramer of integrase, rather than free integrase enzyme. We discuss the biochemical mechanism of integration, which is now quite well understood, and recent progress towards obtaining atomic-resolution structures of HIV intasomes in complex with inhibitors. Such structures are ultimately required to understand the detailed mechanism of inhibition and the mechanisms by which mutations in integrase confer resistance. The path from early biochemical studies to therapeutic inhibitors of integrase highlights the value of basic science in fighting human diseases.

Retroviruses are distinguished from other viruses by two characteristic steps in the viral replication cycle. The first is reverse transcription, which results in the production of a double-stranded DNA copy of the viral RNA genome, and the second is integration, which results in covalent attachment of the DNA copy to host cell DNA. The initial catalytic steps of the integration reaction are performed by the virus-encoded integrase (IN) protein. The chemistry of the IN-mediated DNA breaking and joining steps is well worked out, and structures of IN-DNA complexes have now clarified how the overall complex assembles. Methods developed during these studies were adapted for identification of IN inhibitors, which received FDA approval for use in patients in 2007. At the chromosomal level, HIV integration is strongly favored in active transcription units, which may promote efficient viral gene expression after integration. HIV IN binds to the cellular factor LEDGF/p75, which promotes efficient infection and tethers IN to favored target sites. The HIV integration machinery must also interact with many additional host factors during infection, including nuclear trafficking and pore proteins during nuclear entry, histones during initial target capture, and DNA repair proteins during completion of the DNA joining steps. Models for some of the molecular mechanisms involved have been proposed, but important details remain to be clarified.

Integration of viral DNA into the host genome is an essential step in retroviral replication that is mediated by a stable nucleoprotein complex comprising a tetramer of integrase bridging the two ends of the viral DNA in a stable synaptic complex (SSC) or intasome. Assembly of HIV‐1 intasomes requires several hundred base pairs of nonspecific internal DNA in addition to the terminal viral DNA sequence that is protected in footprinting experiments. We find that only one of the viral DNA ends in the intasome requires long‐nonspecific internal DNA for intasome assembly. Although intasomes are unstable in solution when the nonspecific internal DNA is cut off after assembly, they are stable in agarose gels. These complexes are indistinguishable from SSCs with nonspecific internal DNA in Förster resonance energy transfer (FRET) experiments suggesting the interactions with the viral DNA and integrase tetramer are the same regardless of the presence of nonspecific internal DNA. We discuss models of how the internal DNA contributes to intasome assembly and stability. FRET is exquisitely sensitive to the distance between the fluorophores and given certain assumptions can be translated to distance measurements. We anticipated that a set of such distance constraints would provide a map of the DNA path within the intasome. In reality, the constraints we could impose from the FRET data were quite weak allowing a wide envelope for the possible path. We discuss the difficulties of converting the FRET signal to absolute distance within nucleoprotein complexes.

The HIV-1 integrase enzyme is essential for integrating the viral DNA into the host chromosome. Infection is aborted in the absence of integration, making integrase an attractive antiviral target. Recently approved inhibitors of integrase bind tightly to integrase assembled in a nucleoprotein complex with the viral DNA ends (intasome), but have only low affinity for free integrase. High-resolution structures of HIV-1 intasomes are therefore required to understand the detailed mechanisms of inhibition and resistance. Although the structure of the HIV-1 intasome has not yet been determined, the structure of the related prototype foamy virus (PFV) intasome was recently solved. A new study [1] exploits the PFV structure to model the HIV-1 intasome. The model provides the most reliable picture to date of the active site region of the HIV-1 intasome and is an important advance in studies of inhibition of this essential HIV-1 enzyme.