Zoom lens epitheliumCderived growth factor (LEDGF/p75) is a cellular cofactor of
Zoom lens epitheliumCderived growth factor (LEDGF/p75) is a cellular cofactor of HIV-1 integrase (IN) that interacts with IN through its IN binding domain (IBD) and tethers the viral pre-integration complex to the host cell chromatin. results further support the potential of LEDGINs as allosteric integrase inhibitors. Author Summary Like other viruses, HIV has a limited genome and needs to exploit the machinery of the host cell to complete its replication cycle. The elucidation of virus-host interactions not only sheds light on pathogenesis but also provides opportunities in a limited number of cases to develop novel antiviral drugs. A prototypical example is the interaction between the cellular protein LEDGF/p75 and HIV-1 AV-412 integrase (IN). Here we generated a human somatic LEDGF/p75 knockout cell line to demonstrate that HIV-1 replication is highly dependent on its cofactor. Rabbit polyclonal to beta defensin131 We show that the residual replication of laboratory strains is predominantly mediated by a LEDGF/p75-related protein, HRP-2. Interestingly, the recently developed AV-412 HIV-1 IN inhibitors that target the LEDGF/p75-IN interaction interface, LEDGINs, remain active even in the absence of LEDGF/p75. We demonstrate that LEDGINs efficiently block the interaction between IN and HRP-2. In case HIV-1 would be able to bypass LEDGF/p75-dependent replication using HRP-2 as an alternative tether, LEDGINs would remain fully active. Introduction Integration of viral DNA into the host cell genome is a critical step during HIV replication. A stably inserted provirus is essential for productive infection and archives the genetic information of HIV in the host cell. The presence of a permanent viral reservoir that evades the immune system and enables HIV to rebound once antiretroviral drugs are withdrawn is one of the major remaining hurdles to surmount the HIV epidemic. Lentiviral integration is catalyzed by the viral enzyme IN in close association with the cellular cofactor LEDGF/p75 C. LEDGF is encoded by the gene, which generates the splice variants LEDGF/p52 and LEDGF/p75 . Both share an N-terminal region of 325 residues containing an ensemble of chromatin binding elements, such as the PWWP and AT hook domain, yet differ at the C-terminus. LEDGF/p52 contains 8 amino acids at its AV-412 C-terminus  and fails to interact with HIV-1 IN , , whereas LEDGF/p75 contains an IBD (aa 347C429) capable of interacting with lentiviral IN , , . The cofactor tethers IN to the host cell chromatin, protects it from proteolytic degradation, stimulates its enzymatic activity and in living cells , , C and determines HIV-1 integration site distribution , , , . The role of LEDGF/p75 in HIV-1 replication was studied using RNA interference (RNAi) targeting LEDGF/p75 or using LEDGF KO murine embryonic fibroblasts (MEF) , , , , , , . Although both strategies point to a key role for LEDGF/p75 in lentiviral replication, they resulted in somewhat conflicting conclusions. Potent RNAi-mediated knockdown (KD) of LEDGF/p75 reduced HIV-1 replication, yet residual replication was observed , , , which was attributed to imperfect RNAi-mediated KD of LEDGF/p75, with minute amounts of LEDGF/p75 being sufficient to support HIV-1 replication , . Whether LEDGF/p75 is essential for HIV-1 replication or not could not be addressed by this approach. Later, two LEDGF KO mice were generated. Since mouse cells are not permissive to spreading HIV-1 infection, HIV-based viral vectors were used. The first effort resulted in mouse LEDGF KO clones following insertion of a gene trap . Data obtained from MEFs isolated from these embryos indicated a strong yet incomplete block in integration of HIV-based lentiviral vectors (LV) . Next, a Cre-conditional LEDGF KO mouse was generated. Challenge of the KO MEFs with LV resulted in reduced but not annihilated reporter gene expression . Although analysis was restricted to single round assays, both studies suggest LEDGF/p75 not to be essential for HIV-1 replication, with the cofactor being involved in integration site selection rather than in promoting integration. Here we present the generation of the first human somatic LEDGF/p75 KO cell line to finally answer the question whether LEDGF/p75 is required for spreading infection of various HIV strains. Besides LEDGF/p75, a second member of the hepatoma-derived growth factor related protein family , Hepatoma-derived growth factor related protein 2 (HRP-2), was shown to interact with HIV-1 IN . Although HRP-2 overexpression relocated IN from the cytoplasm to the nucleus in LEDGF/p75-depleted cells , the INCHRP-2 interaction was weaker than the IN-LEDGF/p75 interaction . Neither transient ,  nor stable HRP-2 KD  reduced HIV-1 replication even after reduction of LEDGF/p75, suggesting that HRP-2 is not involved in HIV replication. However, it has not been excluded.