Structure-based mutagenesis of the integrase-LEDGF/p75 interface uncouples a strict correlation between in vitro protein binding and HIV-1 fitness

AbstractLEDGF/p75 binding-defective IN mutant viruses were previously characterized as replication-defective, yet RNAi did not reveal an essential role for the host factor in HIV-1 replication. Correlative analyses of protein binding and viral fitness were expanded here by targeting 12 residues at the IN-LEDGF/p75 binding interface. Whereas many of the resultant viruses were defective, the majority of the INs displayed wild-type in vitro integration activities. Though an overall trend of parallel loss of LEDGF/p75 binding and HIV-1 infectivity was observed, a strict correlation was not. His-tagged INA128Q, derived from a phenotypically wild-type virus, failed to pull-down LEDGF/p75, but INA128Q was effectively recovered in a reciprocal GST pull-down assay. Under these conditions, INH171A, also derived from a phenotypically wild-type virus, interacted less efficiently than a previously described interaction-defective mutant, INQ168A. Thus, the relative affinity of the in vitro IN-LEDGF/p75 interaction is not a universal predictor of IN mutant viral fitness

Biochemical and genetic analyses of integrase-interacting proteins lens epithelium-derived growth factor (LEDGF)/p75 and hepatoma-derived growth factor related protein 2 (HRP2) in preintegration complex function and HIV-1 replication

AbstractHuman immunodeficiency virus type 1 (HIV-1) integrase (IN) functions in cells within the context of high molecular weight preintegration complexes (PICs). Lens epithelium-derived growth factor (LEDGF) transcriptional coactivator/p75 and hepatoma-derived growth factor related protein 2 (HRP2) tightly bind to HIV-1 IN and stimulate its integration activity in vitro. Here, we show that each recombinant host cell factor efficiently reconstitutes the in vitro activity of HIV-1 PICs disrupted for functional integration by pre-treatment with high concentrations of salt. Mutational analysis reveals that both the IN-binding and DNA-binding activities of LEDGF/p75 contribute to functional PIC reconstitution. We also investigate a role(s) for these proteins in HIV-1 infection by using short-interfering RNA. HIV-1 infection was essentially unaffected in HeLa-P4 cells depleted for LEDGF/p75, HRP2, or both proteins. We conclude that cells knocked-out for LEDGF/p75 and/or HRP2 will be useful genetic tools to address the roles of these host cell factors in HIV-1 replication

Lys-34, Dispensable for Integrase Catalysis, Is Required for Preintegration Complex Function and Human Immunodeficiency Virus Type 1 Replication

Retroviral integrases (INs) function in the context of preintegration complexes (PICs). Two conserved Lys residues in the N-terminal domain of human immunodeficiency virus type 1 (HIV-1) IN were analyzed here for their roles in integration and virus replication. Whereas HIV-1(K46A) grew like the wild type, HIV-1(K34A) was dead. Yet recombinant IN(K34A) protein functioned in in vitro integration assays, and Vpr-IN(K34A) efficiently transcomplemented the infectivity defect of an IN active site mutant virus in cells. HIV-1(K34A) was therefore similar to a number of previously characterized mutant viruses that failed to replicate despite encoding catalytically competent IN. To directly analyze mutant PIC function, a sensitive PCR-based integration assay was developed. HIV-1(K34A) and related mutants failed to support detectable levels (<1% of wild type) of integration. We therefore concluded that mutations like K34A disrupted higher-order interactions important for PIC function/maturation compared to the innate catalytic activity of IN enzyme

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 2α (LAP2α), 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 LAP2α 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

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

Human Immunodeficiency Virus Type 1 cDNAs Produced in the Presence of APOBEC3G Exhibit Defects in Plus-Strand DNA Transfer and Integration▿ †

Encapsidation of host restriction factor APOBEC3G (A3G) into vif-deficient human immunodeficiency virus type 1 (HIV-1) blocks virus replication at least partly by C-to-U deamination of viral minus-strand DNA, resulting in G-to-A hypermutation. A3G may also inhibit HIV-1 replication by reducing viral DNA synthesis and inducing viral DNA degradation. To gain further insight into the mechanisms of viral inhibition, we examined the metabolism of A3G-exposed viral DNA. We observed that an overall 35-fold decrease in viral infectivity was accompanied by a five- to sevenfold reduction in viral DNA synthesis. Wild-type A3G induced an additional fivefold decrease in the amount of viral DNA that was integrated into the host cell genome and similarly reduced the efficiency with which HIV-1 preintegration complexes (PICs) integrated into a target DNA in vitro. The A3G C-terminal catalytic domain was required for both of these antiviral activities. Southern blotting analysis of PICs showed that A3G reduced the efficiency and specificity of primer tRNA processing and removal, resulting in viral DNA ends that are inefficient substrates for integration and plus-strand DNA transfer. However, the decrease in plus-strand DNA transfer did not account for all of the observed decrease in viral DNA synthesis associated with A3G. These novel observations suggest that HIV-1 cDNA produced in the presence of A3G exhibits defects in primer tRNA processing, plus-strand DNA transfer, and integration