LEDGF/p75-Independent HIV-1 Replication Demonstrates a Role for HRP-2 and Remains Sensitive to Inhibition by LEDGINs
[['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of a permanent viral reservoir that evades the immune system and enables HIV to rebound once antiretroviral drugs are withdrawn is one of the major remaining hurdles to surmount the HIV epidemic .', 'Lentiviral integration is catalyzed by the viral enzyme IN in close association with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 [1]–[7] .', 'LEDGF is encoded by the PSIP1 gene , which generates the splice variants LEDGF/p52 and LEDGF/p75 [8] .', '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 C-terminus [9] and fails to interact with HIV-1 IN [10] , [11] , whereas LEDGF/p75 contains an IBD ( aa 347–429 ) capable of interacting with lentiviral IN [3] , [12] , [13] .', 'The cofactor tethers IN to the host cell chromatin , protects it from proteolytic degradation , stimulates its enzymatic activity in vitro and in living cells [1] , [10] , [13]–[16] and determines HIV-1 integration site distribution [2] , [11] , [17] , [18] .', '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 ) [2] , [5] , [6] , [11] , [17] , [19] , [20] .', '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 [5] , [6] , [20] , which was attributed to imperfect RNAi-mediated KD of LEDGF/p75 , with minute amounts of LEDGF/p75 being sufficient to support HIV-1 replication [5] , [6] .', '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 [21] .', 'Data obtained from MEFs isolated from these embryos indicated a strong yet incomplete block in integration of HIV-based lentiviral vectors ( LV ) [17] .', '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 [11] .', '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 [22] , Hepatoma-derived growth factor related protein 2 ( HRP-2 ) , was shown to interact with HIV-1 IN [12] .', 'Although HRP-2 overexpression relocated IN from the cytoplasm to the nucleus in LEDGF/p75-depleted cells [23] , the IN–HRP-2 interaction was weaker than the IN-LEDGF/p75 interaction [12] .', 'Neither transient [20] , [24] nor stable HRP-2 KD [6] 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 that in the absence of LEDGF/p75 HRP-2 can function as an alternative molecular tether of HIV integration .', 'Allosteric HIV-1 IN inhibitors that target the LEDGF/p75-IN interaction interface ( LEDGINs ) and potently block HIV-1 replication [25] are in preclinical development .', 'The existence of alternative cellular cofactors , such as HRP-2 , or alternative escape routes might hamper the clinical development of this class of compounds .', 'To answer these questions , we have generated a human somatic LEDGF/p75 KO cell line .', 'We demonstrate that laboratory-adapted HIV strains are capable of replicating in the absence of LEDGF/p75 but show a drastic replication defect .', 'We show that this residual replication in the absence of LEDGF/p75 is predominantly mediated by HRP-2 .', 'Finally , we demonstrate that LEDGINs remained fully active even in the absence of LEDGF/p75 corroborating their allosteric mechanism of action .'], ['To clarify the role of LEDGF/p75 during spreading HIV-1 infection , we generated a human somatic KO in Nalm-6 cells , a human pre-B acute lymphoblastic leukemia cell line [26] , [27] .', 'We eliminated the LEDGF/p75 isoform while preserving the LEDGF/p52 splice variant .', 'Deletion of exon 11 to 14 in the PSIP1 gene fuses exon 10 to exon 15 resulting in a frame shift that yields a truncated LEDGF/p75 in which the C-terminus , including the IBD ( aa 326–530 ) is replaced by a 9 aa tail ( Figure S1A , referred to as LEDGFKO ) .', 'Targeting plasmids were designed carrying the genomic flanking regions of LEDGF/p75 exon 11 and 14 , interspersed with a floxed selection cassette ( Figure 1A ) .', 'Following transfection of wild-type Nalm-6 cells ( Nalm+/+ ) with the first targeting plasmid and subsequent selection , three heterozygous clones ( cl ) ( denoted as Nalm+/c; cl 31 , cl 97 and cl 147 , respectively ) were obtained ( Figure 1B ) .', 'We continued with Nalm+/c cl 31 .', 'Transfection of Nalm+/c cl 31 with the second targeting plasmid resulted in the selection of a homozygous KO clone carrying both resistance cassettes ( Nalmc/c 31 cl 73 ) .', 'Selection cassettes were removed by Cre-mediated excision , resulting in seven LEDGF/p75 KO clones , referred to as Nalm−/− cl 1-7 .', 'Correct homologous recombination of the genomic region was verified via genomic PCR ( Figure 1C ) , Southern blot analysis ( Figure 1D ) and sequencing of the genomic and mRNA region ( Figure S1A ) .', 'The absence of wild-type LEDGF/p75 in the KO cells was corroborated by RT-PCR ( Figure S1B and S1C ) , qRT-PCR ( Figure S1D ) and Western blot analysis ( Figure 1E , arrow ) .', 'A band of 52 kDa appears in the Nalm+/c and Nalm−/− cell lines; it corresponds to the truncated form , LEDGFKO ( Figure 1E , arrowhead ) , and is absent in wild-type cells .', 'Throughout the manuscript Nalm−/− cl 1 and cl 2 monoclonal cell lines are used .', 'Wild-type Nalm-6 cells , referred to as Nalm+/+ , were used as controls , next to Nalm+/c cl 31 , referred to as Nalm+/c , the closest clonal ancestor of the Nalm−/− cells .', 'We first evaluated whether the LEDGF/p75 KO cells ( Nalm−/− ) support transduction by a single round HIV-based viral vector .', 'We challenged the abovementioned engineered cell lines with a VSV-G pseudotyped HIV reporter virus encoding firefly luciferase under control of the viral long terminal repeat promoter ( HIV-fLuc ) .', 'Transduction efficiency ( RLU/µg protein ) was 6 . 7-fold lower in Nalm−/− cells ( cl 1 and cl 2 ) compared to control Nalm+/+ and Nalm+/c cells ( Figure 1F ) ( 15±3 . 7% residual reporter activity; n\u200a=\u200a10 ) .', 'Quantitative PCR revealed 2 . 4-fold lower integrated copies comparing Nalm−/− with Nalm+/c ( Figure 1G ) , whereas late RT products ( Figure S1E ) and 2-LTR circles remained unaffected ( Figure S1F ) .', 'Together these data indicate a block between reverse transcription and integration .', 'Since LEDGF/p75 determines lentiviral integration site selection , we analyzed the distribution of HIV-1 integration sites in the absence of LEDGF/p75 .', 'A total of 2535 HIV-1 integration sites were obtained in Nalm-6 cells of which 799 in Nalm−/− ( Table 1 ) .', 'Random control sites were generated computationally and matched to experimental sites with respect to the distance to the nearest MseI cleavage site ( matched random control , MRC ) [2] .', 'LEDGF/p75 KO significantly reduced the preference of HIV-1 to integrate in RefSeq genes ( P<0 . 0001 for comparison of Nalm−/− cl 1 or 2 with Nalm+/+ or Nalm+/c ) and instead , a preference for CpG islands ( P<0 . 05 for comparison of Nalm−/− cl 1 or 2 with Nalm+/+ or Nalm+/c and P<0 . 0001 for pooled comparison ) emerged ( Figure 1H and Table 1 ) .', 'Similar results were obtained using the Ensembl and UniGene annotation ( Figure S1G and S1H ) .', 'HIV-1 integration events in RefSeq genes remained nevertheless significantly favored over MRC in the KO cells ( P<0 . 0001 ) .', 'The target DNA consensus proved to be LEDGF/p75 independent ( compare Figure S1I with S1J ) .', 'The consensus sequence for the different cell lines was similar to that determined previously [28]–[30] .', 'In human LEDGF/p75 KD cells HIV-1 replication is hampered , but not completely blocked which can be attributed to the remaining minute amounts of LEDGF/p75 [5] , [6] , [20] .', 'Although single round viral vector transduction was severely reduced in LEDGF KO MEFs [11] , [17] , [21] , spreading HIV-1 infection in the absence of LEDGF/p75 could not be tested .', 'To test HIV-1 replication , we introduced the CD4 receptor into the Nalm-6 cells that express CXCR4 [31] , a co-receptor for HIV-1 replication .', 'All selected transgenic cell lines ( Nalm+/+ , Nalm+/c and Nalm−/− cl 1 and cl 2 ) showed similar growth rates ( Figure S6A and S6C ) and CD4 and CXCR4 expression levels ( Figure S6D and S6E ) .', 'We then challenged the respective cell lines with the laboratory strain HIVNL4 . 3 ( Figure 2A ) .', 'Both Nalm+/+ ( Figure S2A ) and Nalm+/c cells supported viral replication to the same extent ( Figure 2A ) .', 'Peak viral replication was consistently observed between day 7 and 9 post infection depending on the multiplicity of infection ( MOI; compare MOI 0 . 5 and 0 . 1 in Figure 2A ) .', 'In Nalm−/− cells infected with HIVNL4 . 3 , low-level p24 production was observed , eventually leading to a breakthrough albeit after a lag-period of 14 to 18 days compared to control cells ( Figure 2A , n\u200a=\u200a6 , a representative experiment is shown ) .', 'Comparable data showing this delay were obtained with another laboratory strain , HIVIIIb ( data not shown ) .', 'Next , we challenged the different cell lines with two clinical isolates of HIV-1 ( 93TH053 , denoted as #1 and 96USSN20 [32] , denoted as #2 ) .', 'Viral breakthrough was observed 17 to 20 days post infection in the control cell line ( Figure 2B and 2C ) .', 'In the first two weeks after infection of the KO cell lines only a discrete increase in p24 was observed; at 35 days after infection p24 levels were below detection limit ( Figure 2B and 2C ) .', 'We next evaluated whether the rise in p24 titers observed in Nalm−/− cells after challenge with laboratory HIV-1 strains could be explained by virus release from cells infected in the first round , rather than ongoing replication cycles .', 'Therefore we challenged Nalm+/c and Nalm−/− cells with HIVNL4 . 3 and resuspended the cells at 8 hrs post infection ( Figure S2B ) in fresh medium containing either zidovudine ( AZT ) , ritonavir ( RIT ) or no inhibitor .', 'AZT , a reverse transcriptase inhibitor , blocks infection of new cells but allows monitoring of virus release from already infected cells whereas RIT , a protease inhibitor , blocks processing of GAG-precursor processing in the virus released from infected cells .', 'In Nalm−/− cells as well as in control Nalm+/c cells the p24 production clearly decreased in the presence RIT or AZT .', 'The decrease in p24 in Nalm+/c without inhibitor at day 6 was due to the cytopathic effect of the virus .', 'This indicates that the p24 increase observed in Nalm−/− cells results from spreading infection and not solely from virus release from cells infected in the first round .', 'The observed delay in multiple round HIV-1 replication in the absence of LEDGF/p75 was further analyzed by quantification of the different HIV-1 DNA species at different time points after infection .', 'Late RT products at 10 hrs post infection and 2-LTR circles at 24 hrs post infection were comparable in Nalm+/c and Nalm−/− cells ( Figure S3A and S3B ) .', 'Addition of the IN strand transfer inhibitor ( INSTI ) raltegravir ( RAL ) in Nalm+/c and Nalm−/− cell lines resulted in a comparable increase in 2-LTR circles at 24 hrs post infection .', 'The number of integrated proviral copies ( Alu-qPCR , Figure S3C ) was severely reduced in the presence of RAL .', 'In Nalm−/− a reduction in the number of integrants was detected after 24 and 48 hrs compared to Nalm+/c cell lines .', 'We next characterized the virus harvested from Nalm−/− at day 18 after infection with the laboratory strain HIVNL4 . 3 ( referred to as HIV−/− ) .', 'Challenging Nalm+/c cells with this virus demonstrated that HIV−/− is replication competent ( Figure S2C , right panel , HIV−/− on Nalm+/c ) .', 'In addition , we evaluated whether HIV−/− virus was phenotypically adapted to the absence of LEDGF/p75 .', 'HIV−/− replication remained impaired in Nalm−/− compared to Nalm+/c cells ( Figure S2C , right panel ) .', 'The proviral IN sequence of HIV−/− was unaltered compared with the consensus sequence of HIVNL4 . 3 ( data not shown ) .', 'Control HIV harvested from Nalm+/c cells ( denoted as HIV+/c ) demonstrated a phenotype that was comparable to that of HIVNL4 . 3 ( Figure S2C , left panel ) .', 'Serial passaging ( N\u200a=\u200a10 ) of HIV-1 on LEDGF/p75 KO cells did not result in phenotypic adaptation or changes in the proviral IN sequence ( data not shown ) .', 'Although residual HIV-1 replication in KO cells was only detectable after infection with laboratory strains , we performed additional experiments to understand this phenotype .', 'Residual viral replication in the absence of LEDGF/p75 can either be explained by cofactor independent replication , or by the presence of a second cofactor that substitutes for LEDGF/p75 .', 'Like LEDGF/p75 , HRP-2 also harbors a PWWP-domain and an IBD-like domain shown to interact with HIV-1 IN in vitro [12] .', 'In order to determine whether HRP-2 can act as an alternative co-factor for HIV integration , we targeted the HRP-2 mRNA using miRNA-based short hairpins ( miR HRP-2 ) .', 'As controls we employed a vector lacking the miRNA expression cassette ( denoted as control ) ( Figure S7B ) .', 'We generated stable HRP-2 KD cells , termed Nalm+/+miR HRP-2 , Nalm+/cmiR HRP-2 and Nalm−/−miR HRP-2 and matched controls Nalm+/+control , Nalm+/ccontrol and Nalm−/−control .', 'HRP-2 KD cells showed 65 , 75 and 80% depletion of HRP-2 , respectively , as determined by qPCR ( Figure 3A ) .', 'No effect on cellular growth kinetics was observed ( data not shown ) .', 'Upon single round transduction with HIV-fLuc no difference was observed in Nalm+/c cells with or without HRP-2 KD ( Figure 3B , left panel ) , whereas luciferase activity was reduced 5-fold in the Nalm−/−control cell line ( 20 . 0±1 . 5% , n\u200a=\u200a3 ) due to LEDGF/p75 KO .', 'An additional 2 . 4-fold reduction was observed in Nalm−/−miR HRP-2 when compared to Nalm−/−control ( 8 . 4±0 . 6% , n\u200a=\u200a3 ) ( Figure 3B , left panel ) that correlated with a 2-fold reduction in integrated copies ( Figure 3B , right panel ) .', 'We next challenged these cells with the laboratory strain HIVNL4 . 3 at different MOI ( Figure 3C–E ) .', 'In the control Nalm+/+ and Nalm+/c cell lines , we observed a minor reduction in viral replication upon HRP-2 KD but only at lower MOI ( compare Figure 3C and 3D , E ) .', 'However , HRP-2 KD in LEDGF/p75 KO cells additionally inhibited HIV-1 replication 2- to 3-fold compared to control cells ( Figure 3C–E , compare Nalm−/−control and Nalm−/−miR HRP-2 , detail panel ) .', 'We generated a second LEDGF/p75 KO HRP-2 KD cell line to corroborate our results .', 'Single round transduction with HIV-fLuc resulted in an additional 4 . 7-fold reduction of luciferase reporter activity when compared with LEDGF KO cells ( Figure S5F ) , whereas HIVNL4 . 3 replication was affected 10-fold at day 8 post infection when comparing LEDGF/p75 KO and LEDGF/p75 KO HRP-2 KD cells ( compare Figure S5D with S5E , condition without compounds ) .', 'To corroborate that additional KD of HRP-2 results in an increased block of integration in LEDGF/p75 KO cells , we analyzed the number of integrated viral copies at 24 hrs and at 5 days post infection , the latter in the presence of RIT ( Figure 3F and 3G , respectively ) .', 'A 2-fold drop in proviral copies upon HRP-2 KD was observed .', 'To extend our findings in LEDGF/p75 KO cells , we tested whether HRP-2 KD resulted in additional reduction of viral replication in LEDGF/p75 KD HeLaP4 ( Figure S4 ) , PM1 ( Figure 4A–C ) and SupT1 ( Figure 4D–F ) cell lines .', 'First , wild-type HeLaP4 ( wild-type ) and LEDGF/p75 KD ( miR LEDGF ) cells [18] were transduced with miR HRP-2 or miR control vectors , the latter containing a miRNA-hairpin directed against monomeric red fluorescent protein ( DsRed ) mRNA [33] ( Figure S7C ) .', 'Following zeocin selection , single HRP-2 KD ( wild-type/miR HRP-2 ) and double KD ( miR LEDGF/miR HRP-2 ) cells showed 20–25% of residual HRP-2 mRNA levels compared to the control cell lines ( wild-type , wild-type/miR control and miR LEDGF/miR control cells ) as determined by qPCR ( Figure S4A and S4C ) .', 'Loss of HRP-2 protein was corroborated by Western blot analysis and immunocytochemistry ( data not shown ) .', 'Of note , LEDGF/p75 levels remained unaffected upon additional HRP-2 KD ( data not shown ) and growth rates of the respective cell lines were comparable ( Figure S6B and S6C ) .', 'KD of HRP-2 in wild-type HeLaP4 cells did not affect multiple round HIV-1 replication ( Figure S4B ) , confirming previous findings by Llano et al . [6] .', 'LEDGF/p75 KD on the other hand reduced HIV-fLuc transduction 5-fold ( luciferase reporter activity\u200a=\u200a19 . 2±3 . 5% of wild-type ) ( Figure S4D ) .', 'Additional KD of HRP-2 in LEDGF/p75-depleted cells diminished HIV-fLuc reporter activity an additional 3-fold , to 6 . 3±2% of control cells ( miR LEDGF/miR control ) ( Figure S4D ) .', 'This reduction was accompanied with a 2-fold decrease in the number of integrated copies ( Figure S4E ) .', 'Transfection of the cell lines with the plasmid encoding HIV-fLuc ( pHIV-fLuc ) did not demonstrate any difference ( Figure S4F ) , ruling out transcriptional effects upon HRP-2 KD .', 'Next , we infected double KD ( miR LEDGF/miR HRP-2 ) cells and control ( miR LEDGF/miR control ) cells together with wild-type and LEDGF/p75 back-complemented ( LEDGF BC ) cells with the laboratory strain HIVNL4 . 3 ( Figure S4G ) .', 'Viral replication was inhibited in miR LEDGF cells and rescued upon LEDGF/p75 back-complementation ( Figure S4G , compare wild-type and LEDGF BC ) .', 'Additional KD of HRP-2 in LEDGF/p75 depleted cells ( miR LEDGF/miR HRP-2 ) inhibited viral replication more than LEDGF/p75 KD alone ( miR LEDGF/miR control ) .', 'The latter demonstrated a breakthrough around day 30 post infection ( Figure S4G , open diamonds ) , whereas cells with double KD did not demonstrate viral breakthrough ( Figure S4G , open squares ) .', 'Analysis was ended at 48 days post infection .', 'Comparable data were obtained in HeLaP4 cell lines generated with other LV constructs ( Figure S7B and S7D ) using hygromycin B selection or eGFP sorting ( data not shown ) .', 'The additional block of HIV-1 replication upon HRP-2 KD in LEDGF/p75 depleted cell lines was also measured by quantifying the number of integrated proviral copies .', 'At day 39 , 45 and 48 post infection the number of integrated copies was low in double KD ( miR LEDGF/miR HRP-2 ) cells compared to the control LEDGF/p75 KD ( miR LEDGF/miR control ) cells ( Figure S4H ) with proviruses numbering 0 . 032 ( ±0 . 012 ) and 0 . 038 ( ±0 . 012 ) per RNaseP genomic copy on day 39 and 48 respectively , compared to 1 . 39 ( ±0 . 18 ) and 0 . 79 ( ±0 . 23 ) in the control LEDGF/p75 KD cell lines .', 'In addition , we quantified different HIV-1 DNA species at different time points post infection in wild-type , LEDGF/p75 KD ( miR LEDGF/miR control ) and double KD cells ( miR LEDGF/miR HRP-2 ) .', 'We observed no difference in late RT products at 10 hrs post infection ( Figure S4I ) .', 'The number of 2-LTR circles in LEDGF/p75 KD ( miR LEDGF/miR control ) and both LEDGF/p75 and HRP-2 KD ( miR LEDGF/miR HRP-2 ) cells was elevated compared to wild-type cells ( Figure S4J ) .', 'Together with the data in the LEDGF/p75 KO cells , these data indicate that HRP-2 KD blocks HIV-1 at a step between reverse transcription and integration but only after potent depletion of LEDGF/p75 .', 'Next , we expanded our findings to relevant T-cell lines , PM1 and SupT1 .', 'We generated cell lines with stable KD of LEDGF/p75 , HRP-2 or both , together with their respective controls ( constructs shown in Figure S7B ) .', 'For PM1 cells KD efficiency was 85–92% for LEDGF/p75 ( Figure 4A ) and 79–81% for HRP-2 ( Figure 4B ) , for SupT1 cells it amounted to 81–88% for LEDGF/p75 ( Figure 4D ) and 75–80% for HRP-2 ( Figure 4E ) .', 'In both cell lines HRP-2 KD alone did not affect HIV-1 replication , whereas a clear reduction in HIV-1 replication was observed upon LEDGF/p75 KD ( Figure 4C and 4F , left panel , for PM1 and SupT1 respectively ) .', 'Consistent with our findings in LEDGF/p75 KO cells and LEDGF/p75 depleted HeLaP4 cells , also in PM1 and SupT1 cells , HRP-2 KD in LEDGF/p75 depleted cells further hampered HIV-1 replication ( Figure 4C and 4F , detail panels , for PM1 and SupT1 respectively ) .', 'Recently , we reported a new class of antiretrovirals termed LEDGINs that bind to the LEDGF/p75 binding pocket of HIV-1 IN and block HIV-1 integration and replication in cell culture [25] .', 'We assayed their activity in the LEDGF/p75 KO cells .', 'We challenged Nalm+/+ and Nalm+/c cells together with Nalm−/− cells with the laboratory strain HIVIIIb in the presence of different concentrations of LEDGIN 7 [25] .', 'LEDGIN 7 blocked HIV-1 replication in all cell lines in a concentration dependent manner ( Figure 5A and 5C ) .', 'Similar data were obtained with the laboratory strain HIVNL4 . 3 ( Figure S5A ) .', 'The toxicity profile in Nalm-6 cells corresponded to that elaborated previously in MT4 cells [25] .', 'No significant toxicity was observed in the concentrations used ( data not shown ) .', 'Of note , LEDGINs were also active against HIV harvested from LEDGF/p75 KO cells ( HIV−/− , data not shown ) .', 'RAL served as a positive control , demonstrating equal inhibition of HIV-1 replication in the different cell lines ( Figure 5B and 5D ) .', 'Dose response curves ( Figure 5E and 5F ) enabled determination of IC50 values , listed in Table S1 .', 'We have shown that residual replication of HIV-1 laboratory strains in LEDGF/p75 KO cells is predominantly mediated by HRP-2 and that LEDGINs block residual HIV-1 replication in KO cells .', 'This can be explained by allosteric inhibition of LEDGINs or by the fact that binding of LEDGINs to the IN-surface also impedes the interaction with HRP-2 or a combination of both .', 'We evaluated whether LEDGINs inhibit the HRP-2-IN interaction in an AlphaScreen assay .', 'Since IN binds HRP-2 via its IBD ( aa 470–593 ) [12] in vitro , we measured the interaction between recombinant HIV-1 IN and the C-terminal part of HRP-2 ( aa 448–670 ) .', 'We generated maltose binding protein ( MBP ) tagged fusions containing either the C-terminal end of LEDGF/p75 ( aa 325–530 ) or HRP-2 ( aa 448–670 ) .', "These recombinant proteins , MBP-LEDGF/p75325–530 and MBP-HRP-2448–670 , bound to His6-IN with apparent KD's of 6 . 6 nM ( ±4 . 6 nM ) or 89 . 8 nM ( ±18 . 1 nM ) , respectively ( Figure 6A ) .", 'In line with previous observations [25] , LEDGINs inhibited the IN-LEDGF325–530 interaction ( Figure 6B; IC50\u200a=\u200a2 . 60±0 . 99 µM ) .', 'LEDGINs also inhibited the IN-HRP-2448–670 interaction , albeit with a 10-fold lower IC50 ( Figure 6B; IC50\u200a=\u200a0 . 23±0 . 14 µM ) .', 'This 10-fold increased potency for LEDGIN 7 to block interaction of IN with MBP-HRP-2448–670 compared to MBP-LEDGF325–530 correlates well with the 13-fold lower affinity of MBP-HRP-2448–670 for IN , as shown in Figure 6A .', 'Next , we evaluated whether LEDGINs remain active in LEDGF/p75 KO HRP-2 KD cells .', 'The residual HIV-1 replication was sensitive to inhibition by LEDGINs ( Figure S5E ) .'], ['Since the identification of LEDGF/p75 as a binding partner of HIV-1 IN in 2003 [1] , we and other groups have demonstrated its importance for HIV-1 replication [3]–[7] , [10] , [11] , [34] , [35] .', 'Our current understanding of the mechanism of action proposes LEDGF/p75 to act as a molecular tether between the lentiviral preintegration complex and the host cell chromatin; the chromatin reading capacity of LEDGF/p75 thereby determines integration site distribution [2] , [11] , [17] , [18] .', 'Given the methodological restrictions associated with the RNAi and mouse KO studies of the past , we decided to investigate the role of LEDGF/p75 in HIV-1 replication by generating a human somatic LEDGF/p75 KO cell line .', 'A second rationale for this study follows the recent development of LEDGINs , small molecules that efficiently target the interaction between HIV-1 IN and LEDGF/p75 by interaction with the LEDGF/p75 binding pocket in HIV-1 IN [25] .', 'Since LEDGINs block HIV-1 replication , the interest in the question whether or not LEDGF/p75 is essential for viral replication was revived .', 'Our studies demonstrate that residual HIV-1 replication in LEDGF/p75 KO cells can be observed using laboratory-adapted HIV-1 strains ( Figure 2A ) .', 'These observations are reminiscent to data obtained in LEDGF/p75 KD cell lines [5] , [6] , [20] , although important differences can be noticed .', 'First , when clinical HIV-1 isolates were used , we observed sterilizing infections in LEDGF/p75 KO cells ( Figure 2B and 2C ) .', 'Sterilizing infection has never been reported with RNAi mediated LEDGF/p75 KD .', 'Although the effect might be in part explained by a lower infectivity of these clinical isolates , it emphasizes the importance of LEDGF/p75 for HIV-1 replication .', 'In addition , LEDGF/p75 KO results in a more pronounced shift of HIV-1 integration out of RefSeq genes when compared to control cells ( 25 . 7% difference when comparing LEDGF/p75 KO to control cells; Table S3 , see column 8 ) , whereas integration in LEDGF/p75 KD cells was only moderately affected ( 1 . 6–8 . 4% compared to control cells , Table S3 , see column 8 ) [2] .', 'A next application of our KO cell line was the investigation of the role of HRP-2 in HIV-1 replication .', 'The cellular function of HRP-2 is currently unknown .', 'Like LEDGF/p75 , HRP-2 contains a PWWP domain at its N-terminus [12] , [22] , [36] , [37] and a basic C-terminus , that harbors an IBD-like domain .', 'GST pull-downs showed that the homologous IBD region in HRP-2 ( amino acids 470–593 ) interacts with IN [12] .', 'Vanegas and colleagues reported earlier that HRP-2 overexpression relocated HIV-1 IN from the cytoplasm to the nucleus in LEDGF/p75 depleted cells [23] .', 'Although HRP-2 was investigated previously as a potential alternative for LEDGF/p75 , no effect in multiple round HIV-1 replication was observed after HRP-2 KD alone or in combination with LEDGF/p75 KD [6] , [20] , [24] .', 'However , these observations may have been obscured by the remaining LEDGF/p75 after incomplete RNAi mediated KD .', 'Therefore we revisited the mechanism of residual replication of HIV-1 laboratory strains in LEDGF/p75 KO cell lines .', 'We demonstrate that both single round transduction and multiple round replication is additionally hampered upon HRP-2 KD in LEDGF/p75 KO cells .', 'HIV-1 engages HRP-2 as an alternative for LEDGF/p75 , but this low affinity IN binding partner ( Figure 6A ) can only substitute for LEDGF/p75 after depletion of the latter ( Figure 3 , 4 and S4 ) , suggesting a dominant role for LEDGF/p75 over HRP-2 .', 'Several reasons can be proposed .', 'Cherepanov et al . [12] demonstrated that considerably less IN could be co-immunoprecipitated by HRP-2 than LEDGF/p75 , implying that the IN–HRP-2 interaction is weaker than the IN-LEDGF/p75 interaction .', 'In line with these observations , Vanegas et al . reported that Flag-LEDGF/p75 but not Flag-HRP-2 co-immunoprecipitated IN from cell lysates [23] .', 'Here we demonstrate using AlphaScreen technology that the IBD containing C-terminal end of HRP-2 has an approximately 13-fold lower affinity for HIV-1 IN than the corresponding part in LEDGF/p75 ( Figure 6A ) .', 'Next , LEDGF/p75 demonstrates a speckled nuclear localization pattern and binds to mitotic chromatin .', 'Vanegas et al . demonstrated that contrary to LEDGF/p75 , HRP-2 does not bind to mitotic chromatin [23] questioning its role as a chromatin-tethering molecule .', 'However , since LEDGF/p75 KD also affects viral replication in non-dividing macrophages [20] , the binding capacity of LEDGF/p75 to condensed mitotic chromatin might not be relevant for HIV-1 replication .', 'The preference of HIV-1 to integration in genes [38] is reduced upon LEDGF/p75 KO corroborating previous observations in LEDGF/p75 KD cells [2] , [11] , [17] , [18] and underscoring LEDGF/p75 as the major targeting factor for HIV-1 integration .', 'In line with this tethering role for LEDGF/p75 , chimeras carrying alternative chromatin binding motifs fused to IBD could retarget HIV-1 integration [18] , [39] , [40] .', 'In addition , De Rijck et al . [41] demonstrated that the LEDGF/p75 chromatin binding mirrors HIV-1 integration site distribution .', 'HIV-1 integration in RefSeq genes remained significantly different from MRC throughout ( P<0 . 0001 ) and more directed towards CpG islands in LEDGF/p75 KO cells .', 'Both observations support the idea of an alternative targeting mechanism for HIV-1 acting in the absence of LEDGF/p75 .', 'Since additional HRP-2 KD resulted in an additional 2-fold reduction in integrated copies compared to LEDGF/p75 depletion , HRP-2 is a candidate .', 'The integration site distribution pattern of HIV-1 derived vectors remained unaltered after additional HRP-2 KD in LEDGF/p75 KD HEK293T cells [2] , but LEDGF/p75 depletion may have been insufficient in those experiments .', 'Apart from LEDGF/p75 and HRP-2 , no other human protein contains a PWWP-domain in conjunction with an IBD .', 'However , other proteins or protein complexes could take over the tethering activity in the absence of LEDGF/p75 and HRP-2 by combining an IBD-like domain with a chromatin-binding function .', 'The IBD belongs to a family exemplified by the Transcription Factor IIS ( TFIIS ) N-terminal domain ( InterPro IPR017923 TFIIS_N ) ( [12] and based on an updated search using the HHpred algorithm [42] , [43] ) .', 'Sequence comparison of the respective predicted IN-binding loops of these domains , suggests it is however unlikely that IN binds to these IBD-like proteins as it does to the IBD of LEDGF/p75 or HRP-2 ( data not shown ) .', 'Therefore the residual HIV-1 replication observed in the LEDGF/p75 KO HRP-2 KD cells may', '1 ) still be HRP-2 mediated since the KD of HRP-2 is not complete ,', '2 ) be mediated by an unknown third cellular cofactor or complex , or', '3 ) occur independently from cellular cofactors .', 'The question remains whether HRP-2 is of any importance for HIV infection in patients ?', 'The HRP-2 phenotype only becomes evident in vitro using laboratory strains and upon strong depletion or KO of LEDGF/p75 .', 'Taking into account the lower affinity of HRP-2 for HIV-1 IN , interaction likely only takes place in the complete absence of LEDGF/p75 .', 'The LEDGF/p75IBD is highly conserved within humans and across species [12] .', 'Only a few SNPs have been identified [44] .', 'Although relative LEDGF/p75 and HRP-2 expression levels still need to be verified in relevant human cells , to date there is no evidence for LEDGF/p75 depletion in humans and a substituting role of HRP-2 in HIV-1 infection .', 'Previous reports demonstrated a moderate increase in 2-LTR circles upon LEDGF/p75 KD [5] , [6] , whereas 2-LTR circles were not significantly different in LEDGF KO MEFs [11] .', 'In this study , we observed no clear difference in the number of 2-LTR circles upon LEDGF/p75 KO .', 'Possibly , the complete absence of LEDGF/p75 affects other steps besides integration that might result in reduced nuclear import and circle formation .', 'Altern
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['Lens epithelium–derived 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 .', 'Here we report the generation of a human somatic LEDGF/p75 knockout cell line that allows the study of spreading HIV-1 infection in the absence of LEDGF/p75 .', 'By homologous recombination the exons encoding the LEDGF/p75 IBD ( exons 11 to 14 ) were knocked out .', 'In the absence of LEDGF/p75 replication of laboratory HIV-1 strains was severely delayed while clinical HIV-1 isolates were replication-defective .', 'The residual replication was predominantly mediated by the Hepatoma-derived growth factor related protein 2 ( HRP-2 ) , the only cellular protein besides LEDGF/p75 that contains an IBD .', 'Importantly , the recently described IN-LEDGF/p75 inhibitors ( LEDGINs ) remained active even in the absence of LEDGF/p75 by blocking the interaction with the IBD of HRP-2 .', 'These results further support the potential of LEDGINs as allosteric integrase inhibitors .']
['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 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 .', 'We show that the residual replication of laboratory strains is predominantly mediated by a LEDGF/p75-related protein , HRP-2 .', 'Interestingly , the recently developed 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 .']
['medicine', 'infectious diseases', 'drug research and development', 'drugs and devices', 'hiv', 'pharmacology', 'viral diseases']