LEDGF/p75 directly interacts with lentiviral integrase proteins and can modulate their enzymatic activities and chromosomal association. A novel genetic knockout model was established that allowed us for the first time to analyze HIV-1 integration in the absence of LEDGF/p75 protein. Supporting a crucial role for the cofactor in viral replication, HIV-1 vector integration and reporter gene expression were significantly reduced in LEDGF-null cells. Yet, integrase processed the viral cDNA termini normally and maintained its local target DNA sequence preference during integration. Preintegration complexes extracted from knockout cells moreover supported normal levels of DNA strand transfer activity in vitro. In contrast, HIV-1 lost its strong bias toward integrating into transcription units, displaying instead increased affinity for promoter regions and CpG islands. Our results reveal LEDGF/p75 as a critical targeting factor, commandeering lentiviruses from promoter- and/or CpG island-proximal pathways that are favored by other members of Retroviridae. Akin to yeast retrotransposons, disrupting the lentiviral targeting mechanism significantly perturbs overall integration.

HIV-1 replication relies on the proper functioning of specific viral proteins, three of which — protease, integrase, and reverse transcriptase with associated RNase H activity — are enzymes. Antiviral drugs that inhibit protease, integrase and reverse transcriptase (specifically, DNA polymerase) activities are approved for treating patients with AIDS. The highly active antiretroviral therapy (HAART) regimens use cocktails of three inhibitors to suppress HIV-1 replication and the outgrowth of drug-resistant virus strains. Replication of HIV-1 depends on a plethora of functional interactions between its proteins and those of the host. Certain other cellular proteins, referred to as restriction factors, counteract viral growth. TRIM5α, APOBEC3G, tetherin and SAMHD1 are examples of such restriction factors. In addition to enzyme active sites, critical viral protein–host protein interactions define targets for therapeutic intervention. Drugs can block interactions between the viral and host proteins that are needed for replication, as is the case for the approved entry inhibitor maraviroc, or they can enhance the effects of cellular restriction factors. Neutralization of the viral envelope glycoprotein gp120 by the adaptive immune system underscores the development strategies for AIDS vaccines. Structural biology studies yield three-dimensional glimpses of protein function at near atomic resolution. Such approaches form the cornerstone of modern efforts for the development of antiviral drugs and vaccines.

Integrase (IN) is an essential retroviral enzyme, and human transcriptional coactivator p75, which is also referred to as lens epithelium-derived growth factor (LEDGF), is the dominant cellular binding partner of HIV-1 IN. Here, we report the crystal structure of the dimeric catalytic core domain of HIV-1 IN complexed to the IN-binding domain of LEDGF. Previously identified LEDGF hotspot residues anchor the protein to both monomers at the IN dimer interface. The principal structural features of IN that are recognized by the host factor are the backbone conformation of residues 168–171 from one monomer and a hydrophobic patch that is primarily comprised of α-helices 1 and 3 of the second IN monomer. Inspection of diverse retroviral primary and secondary sequence elements helps to explain the apparent lentiviral tropism of the LEDGF-IN interaction. Because the lethal phenotypes of HIV-1 mutant viruses unable to interact with LEDGF indicate that IN function is highly sensitive to perturbations of the structure around the LEDGF-binding site, we propose that small molecule inhibitors of the protein–protein interaction might similarly disrupt HIV-1 replication.

Lentiviruses can infect non-dividing cells, and various cellular transport proteins provide crucial functions for lentiviral nuclear entry and integration. We previously showed that the viral capsid (CA) protein mediated the dependency on cellular nucleoporin (NUP) 153 during HIV-1 infection, and now demonstrate a direct interaction between the CA N-terminal domain and the phenylalanine-glycine (FG)-repeat enriched NUP153 C-terminal domain (NUP153_C). NUP153_C fused to the effector domains of the rhesus Trim5α restriction factor (Trim-NUP153_C) potently restricted HIV-1, providing an intracellular readout for the NUP153_C-CA interaction during retroviral infection. Primate lentiviruses and equine infectious anemia virus (EIAV) bound NUP153_C under these conditions, results that correlated with direct binding between purified proteins in vitro. These binding phenotypes moreover correlated with the requirement for endogenous NUP153 protein during virus infection. Mutagenesis experiments concordantly identified NUP153_C and CA residues important for binding and lentiviral infectivity. Different FG motifs within NUP153_C mediated binding to HIV-1 versus EIAV capsids. HIV-1 CA binding mapped to residues that line the common alpha helix 3/4 hydrophobic pocket that also mediates binding to the small molecule PF-3450074 (PF74) inhibitor and cleavage and polyadenylation specific factor 6 (CPSF6) protein, with Asn57 (Asp58 in EIAV) playing a particularly important role. PF74 and CPSF6 accordingly each competed with NUP153_C for binding to the HIV-1 CA pocket, and significantly higher concentrations of PF74 were needed to inhibit HIV-1 infection in the face of Trim-NUP153_C expression or NUP153 knockdown. Correlation between CA mutant viral cell cycle and NUP153 dependencies moreover indicates that the NUP153_C-CA interaction underlies the ability of HIV-1 to infect non-dividing cells. Our results highlight similar mechanisms of binding for disparate host factors to the same region of HIV-1 CA during viral ingress. We conclude that a subset of lentiviral CA proteins directly engage FG-motifs present on NUP153 to affect viral nuclear import. Lentiviruses such as HIV-1 possess mechanisms to bypass the nuclear envelope and reach the nuclear interior for viral DNA integration. Numerous nuclear transport proteins are important for HIV-1 infection, suggesting the viral nucleoprotein complex enters the nucleus by passing through nuclear pore complexes. HIV-1 was previously found to utilize cellular nucleoporin (NUP) 153 protein in a manner determined by the viral capsid protein. Here, we show HIV-1 capsid directly binds NUP153 in a phenylalanine-glycine motif-dependent manner; such motifs form the general selectivity barrier that restricts transport through the nuclear pore. We find that NUP153 binds a hydrophobic pocket found on capsid proteins from both primate and equine lentiviruses, suggesting an evolutionary predilection for this interaction. The pocket on HIV-1 capsid also binds phenylalanine moieties present in a small molecule inhibitor of HIV-1 infection, as well as a separate host factor implicated in the nuclear import pathway. We found that these molecules compete for NUP153 binding, providing insight into their mechanisms of action during HIV-1 infection. These results demonstrate a previously unknown interaction important for HIV-1 nuclear trafficking, and posit direct binding of viral capsids with phenylalanine-glycine motifs as a novel example of viral hijacking of a fundamental cellular process.

Integration is vital to retroviral replication and influences the establishment of the latent HIV reservoir. HIV-1 integration favors active genes, which is in part determined by the interaction between integrase and lens epithelium-derived growth factor (LEDGF)/p75. Because gene targeting remains significantly enriched, relative to random in LEDGF/p75 deficient cells, other host factors likely contribute to gene-tropic integration. Nucleoporins 153 and 358, which bind HIV-1 capsid, play comparatively minor roles in integration targeting, but the influence of another capsid binding protein, cleavage and polyadenylation specificity factor 6 (CPSF6), has not been reported. In this study we knocked down or knocked out CPSF6 in parallel or in tandem with LEDGF/p75. CPSF6 knockout changed viral infectivity kinetics, decreased proviral formation, and preferentially decreased integration into transcriptionally active genes, spliced genes, and regions of chromatin enriched in genes and activating histone modifications. LEDGF/p75 depletion by contrast preferentially altered positional integration targeting within gene bodies. Dual factor knockout reduced integration into genes to below the levels observed with either single knockout and revealed that CPSF6 played a more dominant role than LEDGF/p75 in directing integration to euchromatin. CPSF6 complementation rescued HIV-1 integration site distribution in CPSF6 knockout cells, but complementation with a capsid binding mutant of CPSF6 did not. We conclude that integration targeting proceeds via two distinct mechanisms: capsid-CPSF6 binding directs HIV-1 to actively transcribed euchromatin, where the integrase-LEDGF/p75 interaction drives integration into gene bodies.

Attacking HIV by stabilizing its capsid: Current HIV treatments require drugs that must be taken daily, and care would be improved with an effective drug that is long-acting. GS-6207 (Lenacapavir) is a drug developed by Gilead Sciences that shows potential for a 6-month dosing interval and is in phase 2/3 clinical trials. Bester et al. describe structural and biophysical studies that provide a basis for the potent antiviral activity of GS-6207. The HIV capsid is cone shaped, and GS-6207 binds two neighboring capsid subunits and stabilizes the curved capsid. GS-6207 also interferes with capsid binding of cofactors that play a role in viral infection. This insight into GS-6207 activity provides a platform for the rational development of improved long-acting therapies. Science , this issue p. 360