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The integrase (IN) enzyme of retrovirus prototype foamy virus (PFV) consists of four domains: amino terminal extension (NED), amino terminus (NTD), catalytic core (CCD), and carboxyl terminus domains (CTD). A tetramer of PFV IN with two viral DNA ends forms the functional intasome. Two inner monomers are catalytically active while the CCDs of the two outer monomers appear to play only structural roles. The NED, NTD, and CTD of the outer monomers are disordered in intasome structures. Truncation mutants reveal that integration to a supercoiled plasmid increases without the outer monomer CTDs present. Deletion of the outer CTDs enhances the lifetime of the intasome compared to full length (FL) IN or deletion of the outer monomer NTDs. High ionic strength buffer or several additives, particularly protocatechuic acid (PCA), enhance the integration of FL intasomes by preventing aggregation. These data confirm previous studies suggesting the disordered outer domains of PFV intasomes are not required for intasome assembly or integration. Instead, the outer CTDs contribute to aggregation of PFV intasomes which may be inhibited by high ionic strength buffer or the small molecule PCA.

Integration of the reverse-transcribed viral DNA into the host genome is an essential step in the life cycle of retroviruses. Retrovirus integrase catalyses insertions of both ends of the linear viral DNA into a host chromosome. Integrase from HIV-1 and closely related retroviruses share the three-domain organization, consisting of a catalytic core domain flanked by amino- and carboxy-terminal domains essential for the concerted integration reaction. Although structures of the tetrameric integrase-DNA complexes have been reported for integrase from prototype foamy virus featuring an additional DNA-binding domain and longer interdomain linkers, the architecture of a canonical three-domain integrase bound to DNA remained elusive. Here we report a crystal structure of the three-domain integrase from Rous sarcoma virus in complex with viral and target DNAs. The structure shows an octameric assembly of integrase, in which a pair of integrase dimers engage viral DNA ends for catalysis while another pair of non-catalytic integrase dimers bridge between the two viral DNA molecules and help capture target DNA. The individual domains of the eight integrase molecules play varying roles to hold the complex together, making an extensive network of protein-DNA and protein-protein contacts that show both conserved and distinct features compared with those observed for prototype foamy virus integrase. Our work highlights the diversity of retrovirus intasome assembly and provides insights into the mechanisms of integration by HIV-1 and related retroviruses.

Retroviral integrase catalyses the integration of viral DNA into host target DNA, which is an essential step in the life cycle of all retroviruses. Previous structural characterization of integrase-viral DNA complexes, or intasomes, from the spumavirus prototype foamy virus revealed a functional integrase tetramer, and it is generally believed that intasomes derived from other retroviral genera use tetrameric integrase. However, the intasomes of orthoretroviruses, which include all known pathogenic species, have not been characterized structurally. Here, using single-particle cryo-electron microscopy and X-ray crystallography, we determine an unexpected octameric integrase architecture for the intasome of the betaretrovirus mouse mammary tumour virus. The structure is composed of two core integrase dimers, which interact with the viral DNA ends and structurally mimic the integrase tetramer of prototype foamy virus, and two flanking integrase dimers that engage the core structure via their integrase carboxy-terminal domains. Contrary to the belief that tetrameric integrase components are sufficient to catalyse integration, the flanking integrase dimers were necessary for mouse mammary tumour virus integrase activity. The integrase octamer solves a conundrum for betaretroviruses as well as alpharetroviruses by providing critical carboxy-terminal domains to the intasome core that cannot be provided in cis because of evolutionarily restrictive catalytic core domain-carboxy-terminal domain linker regions. The octameric architecture of the intasome of mouse mammary tumour virus provides new insight into the structural basis of retroviral DNA integration.

Retroviral integrase (IN) is responsible for two consecutive reactions, which lead to insertion of a viral DNA copy into a host cell chromosome. Initially, the enzyme removes di- or trinucleotides from viral DNA ends to expose 3'-hydroxyls attached to the invariant CA dinucleotides (3'-processing reaction). Second, it inserts the processed 3'-viral DNA ends into host chromosomal DNA (strand transfer). Herein, we report a crystal structure of prototype foamy virus IN bound to viral DNA prior to 3'-processing. Furthermore, taking advantage of its dependence on divalent metal ion cofactors, we were able to freeze trap the viral enzyme in its ground states containing all the components necessary for 3'-processing or strand transfer. Our results shed light on the mechanics of retroviral DNA integration and explain why HIV IN strand transfer inhibitors are ineffective against the 3'-processing step of integration. The ground state structures moreover highlight a striking substrate mimicry utilized by the inhibitors in their binding to the IN active site and suggest ways to improve upon this clinically relevant class of small molecules.

To establish productive infection, a retrovirus must insert a DNA replica of its genome into host cell chromosomal DNA. This process is operated by the intasome, a nucleoprotein complex composed of an integrase tetramer (IN) assembled on the viral DNA ends. The intasome engages chromosomal DNA within a target capture complex to carry out strand transfer, irreversibly joining the viral and cellular DNA molecules. Although several intasome/transpososome structures from the DDE(D) recombinase superfamily have been reported, the mechanics of target DNA capture and strand transfer by these enzymes remained unclear. Here we report crystal structures of the intasome from prototype foamy virus in complex with target DNA, elucidating the pre-integration target DNA capture and post-catalytic strand transfer intermediates of the retroviral integration process. The cleft between IN dimers within the intasome accommodates chromosomal DNA in a severely bent conformation, allowing widely spaced IN active sites to access the scissile phosphodiester bonds. Our results resolve the structural basis for retroviral DNA integration and provide a framework for the design of INs with altered target sequences.

Although parasite-host co-speciation is a long-held hypothesis, convincing evidence for long-term co-speciation remains elusive, largely because of small numbers of hosts and parasites studied and uncertainty over rates of evolutionary change. Co-speciation is especially rare in RNA viruses, in which cross-species transfer is the dominant mode of evolution. Simian foamy viruses (SFVs) are ubiquitous, non-pathogenic retroviruses that infect all primates. Here we test the co-speciation hypothesis in SFVs and their primate hosts by comparing the phylogenies of SFV polymerase and mitochondrial cytochrome oxidase subunit II from African and Asian monkeys and apes. The phylogenetic trees were remarkably congruent in both branching order and divergence times, strongly supporting co-speciation. Molecular clock calibrations revealed an extremely low rate of SFV evolution, 1.7 x 10(-8) substitutions per site per year, making it the slowest-evolving RNA virus documented so far. These results indicate that SFVs might have co-speciated with Old World primates for at least 30 million years, making them the oldest known vertebrate RNA viruses.

The presence of closed circular forms of the linear DNA genome of human foamy virus (HFV) has not been established. The ability of the HFV integrase (IN) to catalyse the integration of these circular forms (termed 2 long terminal repeat (LTR) circles) was investigated, with a view to producing a novel hybrid vector. To this end, a construct was made containing, in addition to the enhanced green fluorescent protein (eGFP) marker gene, the last 27 bp of the 3' U5 LTR region of HFV fused to the first 28 bp of the 5' U3 LTR, the latter representing a 2LTR circle. Marker gene expression following transfection of both 293 and 293T cells indicated that the level of integration was not significantly increased by the HFV IN. Moreover, correctly integrated provirus-like forms of the input plasmid could not be detected by PCR. Taken together, these results show that the HFV IN is not able to integrate a circular molecule containing an LTR junction and, hence, the technique is not exploitable as a tool to produce hybrid vectors for gene therapy.