The SET complex acts as a barrier to autointegration of HIV-1.

Retroviruses and retrotransposons are vulnerable to a suicidal pathway known as autointegration, which occurs when the 3'-ends of the reverse transcript are activated by integrase and then attack sites within the viral DNA. Retroelements have diverse strategies for suppressing autointegration, but how HIV-1 protects itself from autointegration is not well-understood. Here we show that knocking down any of the components of the SET complex, an endoplasmic reticulum-associated complex that contains 3 DNases (the base excision repair endonuclease APE1, 5'-3' exonuclease TREX1, and endonuclease NM23-H1), inhibits HIV-1 and HIV-2/SIV, but not MLV or ASV, infection. Inhibition occurs at a step in the viral life cycle after reverse transcription but before chromosomal integration. Antibodies to SET complex proteins capture HIV-1 DNA in the cytoplasm, suggesting a direct interaction between the SET complex and the HIV preintegration complex. Cloning of HIV integration sites in cells with knocked down SET complex components revealed an increase in autointegration, which was verified using a novel semi-quantitative nested PCR assay to detect autointegrants. When SET complex proteins are knocked down, autointegration increases 2-3-fold and chromosomal integration correspondingly decreases approximately 3-fold. Therefore, the SET complex facilitates HIV-1 infection by preventing suicidal autointegration.

Identification and characterization of PWWP domain residues critical for LEDGF/p75 chromatin binding and human immunodeficiency virus type 1 infectivity.

Lens epithelium-derived growth factor (LEDGF)/p75 functions as a bimodal tether during lentiviral DNA integration: its C-terminal integrase-binding domain interacts with the viral preintegration complex, whereas the N-terminal PWWP domain can bind to cellular chromatin. The molecular basis for the integrase-LEDGF/p75 interaction is understood, while the mechanism of chromatin binding is unknown. The PWWP domain is homologous to other protein interaction modules that together comprise the Tudor clan. Based on primary amino acid sequence and three-dimensional structural similarities, 24 residues of the LEDGF/p75 PWWP domain were mutagenized to garner essential details of its function during human immunodeficiency virus type 1 (HIV-1) infection. Mutating either Trp-21 or Ala-51, which line the inner wall of a hydrophobic cavity that is common to Tudor clan members, disrupts chromatin binding and virus infectivity. Consistent with a role for chromatin-associated LEDGF/p75 in stimulating integrase activity during infection, recombinant W21A protein is preferentially defective for enhancing integration into chromatinized target DNA in vitro. The A51P mutation corresponds to the S270P change in DNA methyltransferase 3B that causes human immunodeficiency, centromeric instability, and facial anomaly syndrome, revealing a critical role for this amino acid position in the chromatin binding functions of varied PWWP domains. Our results furthermore highlight the requirement for a conserved Glu in the hydrophobic core that mediates interactions between other Tudor clan members and their substrates. This initial systematic mutagenesis of a PWWP domain identifies amino acid residues critical for chromatin binding function and the consequences of their changes on HIV-1 integration and infection.

Subunit-specific protein footprinting reveals significant structural rearrangements and a role for N-terminal Lys-14 of HIV-1 Integrase during viral DNA binding.

To identify functional contacts between HIV-1 integrase (IN) and its viral DNA substrate, we devised a new experimental strategy combining the following two methodologies. First, disulfide-mediated cross-linking was used to site-specifically link select core and C-terminal domain amino acids to respective positions in viral DNA. Next, surface topologies of free IN and IN-DNA complexes were compared using Lys- and Arg-selective small chemical modifiers and mass spectrometric analysis. This approach enabled us to dissect specific contacts made by different monomers within the multimeric complex. The foot-printing studies for the first time revealed the importance of a specific N-terminal domain residue, Lys-14, in viral DNA binding. In addition, a DNA-induced conformational change involving the connection between the core and C-terminal domains was observed. Site-directed mutagenesis experiments confirmed the importance of the identified contacts for recombinant IN activities and virus infection. These new findings provided major constraints, enabling us to identify the viral DNA binding channel in the active full-length IN multimer. The experimental approach described here has general application to mapping interactions within functional nucleoprotein complexes.

LEDGF/p75 functions downstream from preintegration complex formation to effect gene-specific HIV-1 integration.

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.

Wild-type levels of human immunodeficiency virus type 1 infectivity in the absence of cellular emerin protein.

Preintegration complexes (PICs) mediate retroviral integration, and recent results indicate an important role for the inner nuclear membrane protein emerin in orienting human immunodeficiency virus type 1 (HIV-1) PICs to chromatin for integration. Two other host cell proteins, the barrier-to-autointegration factor (BAF) and lamina-associated polypeptide 2alpha (LAP2alpha), seemed to play a similar preintegrative role for Moloney murine leukemia virus (MMLV) in addition to HIV-1. In contrast, we determined efficient HIV-1 and MMLV infection of HeLa-P4 cells following potent down-regulation of emerin, BAF, or LAP2alpha protein by using short interfering RNA. Mouse embryo fibroblasts ablated for emerin protein through gene knockout support the same level of HIV-1 infection as cells derived from wild-type littermate control animals. As the expression of human emerin in mouse knockout cells fails to affect the level of infectivity achieved in its absence, we conclude that HIV-1 efficiently infects cells in the absence of emerin protein and, by extension, that emerin is not a universally important regulator of HIV-1 infectivity.