Since the identification of LEDGF/p75 as a binding partner of HIV-1 IN in 2003 
, we and other groups have demonstrated its importance for HIV-1 replication 
. 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 
. 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 
. 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 (). These observations are reminiscent to data obtained in LEDGF/p75 KD cell lines 
, although important differences can be noticed. First, when clinical HIV-1 isolates were used, we observed sterilizing infections in LEDGF/p75 KO cells (). 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) 
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 
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 
. Vanegas and colleagues reported earlier that HRP-2 overexpression relocated HIV-1 IN from the cytoplasm to the nucleus in LEDGF/p75 depleted cells 
. 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 
. 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 () can only substitute for LEDGF/p75 after depletion of the latter (, and S4
), suggesting a dominant role for LEDGF/p75 over HRP-2. Several reasons can be proposed. Cherepanov et al. 
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 
. 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 (). 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 
questioning its role as a chromatin-tethering molecule. However, since LEDGF/p75 KD also affects viral replication in non-dividing macrophages 
, 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 
is reduced upon LEDGF/p75 KO corroborating previous observations in LEDGF/p75 KD cells 
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 
. In addition, De Rijck et al. 
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 
, 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) (
and based on an updated search using the HHpred algorithm 
). 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 
. Only a few SNPs have been identified 
. 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 
, whereas 2-LTR circles were not significantly different in LEDGF KO MEFs 
. 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. Alternatively, cellular pathways involved in 2-LTR formation may be affected. Opposing effects on circle formation by reduced import and reduced integration may finally result in an equal 2-LTR circle number. Alternatively, the sensitivity of 2-LTR circle quantification may be too low to detect a small increase.
In the last part of the manuscript we demonstrate that LEDGINs block the residual replication observed in LEDGF/p75 KO cell lines () and block the interaction in vitro
and IN (). illustrates how LEDGINs fit in the pocket at the IN core dimer interface. LEDGINs block the interaction with two interhelical loops of the IBDs of LEDGF/p75 () or HRP-2 (). The inhibition of the interaction with HRP-2 can explain why residual replication of HIV-1 in LEDGF/p75 KO cells is still sensitive to LEDGINs. Since LEDGF/p75 has been reported to act as an allosteric modulator of the IN activity in vitro
, it is plausible that inhibition of the LEDGF/p75-IN interaction not only interferes with its function as a molecular tether but also results in an allosteric inhibition of IN activity. In fact, inhibition of in vitro
IN activity in the absence of LEDGF/p75 by potent LEDGINs has been reported 
. The allosteric mode of inhibition by LEDGINs can as well explain inhibition of HIV-1 replication in LEDGF/p75 KO HRP-2 KD cells 
. In vivo
both mechanisms are intrinsically coupled. LEDGINs compete with LEDGF/p75 as a molecular tether and at the same time interfere with integrase activities probably by affecting conformational flexibility in the intasome. Whereas transdominant inhibition of HIV-1 replication by IBD overexpression 
presumably also acts through this dual mechanism 
, RNAi-mediated depletion of LEDGF/p75 likely only affects tethering and/or targeting. We should however be cautious to translate the results in KO cells to human patients. Since no individuals without functional LEDGF/p75 expression have been documented, LEDGINs will always have to compete with LEDGF/p75 for the IN binding pocket to inhibit integration.
Somatic KO cell lines are cumbersome to generate. This is why few studies used this technology to study the role of cellular cofactors in virus replication. Previously, the role of cyclophilin A in HIV replication was confirmed in a human somatic KO cell line 
as well as the roles of CBF1
in Epstein-Barr virus replication. Our work supports the value of generating human KO cell lines for cofactor validation and drug discovery in general.