Overexpression of the Lens Epithelium-Derived Growth Factor/p75 Integrase Binding Domain Inhibits Human Immunodeficiency Virus Replication

Abstract

We initially identified lens epithelium-derived growth factor/p75 (LEDGF/p75) as a binding partner of human immunodeficiency virus type 1 (HIV-1) integrase. To investigate the role of LEDGF/p75 in HIV replication and its potential as a new antiviral target, we stably overexpressed two different fragments containing the integrase binding domain (IBD) of LEDGF/p75 fused to enhanced green fluorescent protein (eGFP). HIV-1 replication was severely inhibited by overexpression of the eGFP-IBD fusion proteins, while no inhibition was observed in cell lines overexpressing the interaction-deficient D366A mutant. Quantitative PCR pinpointed the block to the integration step, whereas nuclear import was not affected. Competition of the IBD fusion proteins with endogenous LEDGF/p75 for binding to integrase led to a potent defect in HIV-1 replication in both HeLaP4-and MT-4-derived cell lines. A previously described diketo acid-resistant HIV-1 strain remained fully susceptible to inhibition, suggesting that this strategy will also work in patients who harbor strains resistant to the current experimental integrase inhibitors. These data support LEDGF/p75 as an important cofactor for HIV replication and provide proof of concept for the LEDGF/p75-integrase interaction as a novel target for treating HIV-1 infection.

Integrase, LEDGF/p75 and HIV replication

“…Many chromatin-associated proteins are highly mobile, such that they rapidly and continuously exchange among chromatin binding sites in a stop-and-go manner [20,21]. The inferred competition between endogenous p75 and IBD fusion proteins would be consistent with a situation whereby p75 interacts in such a manner with both the PIC and chromatin [96,97].…”

“…The interaction between p75 and IN is direct [73] and it is confined to only one of the seven retroviral genera, lentivirinae [75,87,100]. A number of laboratories established the role of p75 in chromatin tethering, mapped relevant functional domains, and worked to answer the question of virological relevance [74][75][76][77][78][79][80][81][82][85][86][87][88][89][90][91]93,[95][96][97][100][101][102][103][104][105][108][109][110]. The first indication of the protein's significance was the clarification it provided about the vexing issue of the intracellular trafficking of retroviral integrase proteins.…”

“…The loss of a single negative charge through an Asp → Asn change at residue 366 (D366N) disrupts binding to IN. Mutants such as D366N and D366A have been useful for demonstrating specificity of effects in HIV replication experiments [90,[95][96][97]101,105,109].…”

“…Faced with these ambiguities, investigators developed three basic experimental approaches: intensified RNAi [96], p75 knockout mice [101]), and over-expression of the IBD [96,97,109]. Prior RNAi experiments had used either siRNA transfection or plasmid-based shRNAs.…”

“…Over-expression of chimeric GFP-IBD fusion proteins has also resulted in substantial inhibition of viral replication, which is specifically abrogated by mutating Asp366 at the heart of the CCD-IBD interface [96,97,109]. (Over-expressing the IBD alone has not proved feasible, because of instability that N-terminal fusion partners appear to mitigate).…”