HRP-2 determines HIV-1 integration site selection in LEDGF/p75 depleted cells
© Schrijvers et al.; licensee BioMed Central Ltd. 2012
Received: 9 August 2012
Accepted: 19 September 2012
Published: 9 October 2012
Lens epithelium–derived growth factor (LEDGF/p75) is a cellular co-factor of HIV-1 integrase (IN) that tethers the viral pre-integration complex to the host cell chromatin and determines the genome wide integration site distribution pattern of HIV-1. Recently, we demonstrated that HIV-1 replication was reduced in LEDGF/p75 knockout (KO) cells. LEDGF/p75 KO significantly altered the integration site preference of HIV-1, but the pattern remained distinct from a computationally generated matched random control set (MRC), suggesting the presence of an alternative tethering factor. We previously identified Hepatoma-derived growth factor related protein 2 (HRP-2) as a factor mediating LEDGF/p75-independent HIV-1 replication. However, the role of HRP-2 in HIV-1 integration site selection was not addressed.
We studied the HIV-1 integration site distribution in the presence and absence of LEDGF/p75 and/or HRP-2, and in LEDGF/p75-depleted cells that overexpress HRP-2. We show that HRP-2 functions as a co-factor of HIV-1 IN in LEDGF/p75-depleted cells. Endogenous HRP-2 only weakly supported HIV-1 replication in LEDGF/p75 depleted cells. However, HRP-2 overexpression rescued HIV-1 replication and restored integration in RefSeq genes to wild-type levels. Additional HRP-2 KD in LEDGF/p75-depleted cells reduces integration frequency in transcription units and shifts the integration distribution towards random.
We demonstrate that HRP-2 overexpression can compensate for the absence of LEDGF/p75 and indicate that the residual bias in integration targeting observed in the absence of LEDGF/p75 can be ascribed to HRP-2. Knockdown of HRP-2 upon LEDGF/p75 depletion results in a more random HIV-1 integration pattern. These data therefore reinforce the understanding that LEDGF/p75 is the dominant HIV-1 IN co-factor.
We reported that LEDGF/p75 KO shifts HIV-1 integration away from transcription units , consistent with previous LEDGF/p75 KD experiments [2, 3]. However, even in the absence of LEDGF/p75, HIV-1 significantly favored integration in transcription units when compared to random . This may be explained by an intrinsic feature of the IN protein or the pre-integration complex, or by the presence of one or more additional co-factors that target integration into transcription units. Recently, we reported that HRP-2, the only known cellular protein besides LEDGF/p75 that combines a PWWP-domain and an IBD-like domain [9, 13] (Figure 1), plays a role in LEDGF/p75-independent HIV-1 replication in both human LEDGF/p75 KO and KD cell lines . In this study we examined the contribution of HRP-2 in directing integration site selection of HIV-1.
Previously, we demonstrated that HRP-2 KD in LEDGF/p75-depleted cells additionally hampered HIV-1 replication (see reference  and data not shown). Here, we evaluated the effect of additional HRP-2 KD in LEDGF/p75-depleted cells on HIV-1 integration site distribution. We generated stable HRP-2 KD and LEDGF/p75 KD cells (86% and >90% KD on mRNA level for HRP-2 and LEDGF/p75, respectively), double KD cells (>90% LEDGF/p75 KD and 84% HRP-2 KD on mRNA level) and complemented LEDGF/p75 KD cells with HRP-2 (Figure 2A and data not shown). The respective cell lines were challenged with HIV-fLuc integration sites were amplified and their distribution pattern was characterized.
Integration frequency of HIV in RefSeq genes
% in RefSeq genes
LEDGF/p75 KD HRP-2 KD
LEDGF/p75 KD + HRP-2
LEDGF/p75 KD HRP-2 KD
LEDGF/p75 KD + HRP-2
Taken together, our work underscores the dominant role of LEDGF/p75 over HRP-2 in HIV-1 replication, which can be explained by the lower affinity of HRP-2 for HIV-1 IN compared to LEDGF/p75 [7, 9, 15]. Since HRP-2 overexpression in LEDGF/p75-depleted cells rescues HIV-1 replication, the relative expression levels of LEDGF/p75 and HRP-2 in relevant primary host cells are of importance. Different groups have reported on expression levels of LEDGF/p75 in primary activated and resting T-cells  or in different patient populations [22, 23], yet the levels in other subsets of HIV target-cells as well as the HRP-2 expression levels remain to be determined. Currently, there is no evidence that HRP-2 plays a role in HIV-1 replication in cells expressing WT levels of LEDGF/p75. Both LEDGF/p75 and HRP-2 carry a PWWP domain, recently identified as a chromatin reader recognizing epigenetic marks, such as methylated histone side-chains [6, 24, 25]. Previously, we demonstrated that swapping the PWWP-domain of LEDGF/p75 with that of HRP-2 could rescue lentiviral replication and integration site selection in genes . Here, integration distribution in LEDGF/p75 KD cells overexpressing HRP-2 was comparable to wild-type cells (Figure 3 and Additional file 2 and 3), suggesting only subtle differences between LEDGF/p75 and HRP-2 for the interaction with the local chromatin. Although LEDGF/p75 and HRP-2 double KD shifted integration significantly out of transcription units, integration remained distinct from random in the double KD cells (Table 1). Multiple (mutually non-exclusive) reasons can be put forward to explain this observation. First, in these experiments we employed RNAi and even though the knockdown was potent (>90% and 84% on mRNA level for LEDGF/p75 and HRP-2 respectively), residual LEDGF/p75 and HRP-2 might account for the residual bias. The generation of a human double KO cell line, devoid of both LEDGF/p75 and HRP-2, will provide a more definite answer. Second, this result could suggest the presence of additional cellular co-factor(s) that affect targeting in the absence of LEDGF/p75 and HRP-2. Transcription factor IIS (TFIIS) for example harbors an IBD-like domain [6, 8], although structurally more distantly related to the IBD of LEDGF/p75 and HRP-2, but lacks a PWWP-domain. However, in the presence of LEDGF/p75, HRP-2 does not seem to play a role in HIV replication  or targeting (this work), suggesting alternate IBD containing tethers will probably only play a minor role in HIV replication or targeting in WT conditions, unless expression levels differ strongly, accrediting LEDGF/p75 as principal tether . Third, the bias might reflect specific constraints of the viral IN, the local chromatin environment or the pre-integration complex itself. Although HIV integration favors weak palindromic sequences, our analysis indicated that this preference is irrespective of the presence or absence of LEDGF/p75 and/or HRP-2 ( and data not shown).
In conclusion, our data provide an explanation for why LEDGF/p75 depletion alone does not completely retarget integration distribution towards random, and they fit with previous data that HRP-2 binds the IN dimer with lower affinity [7, 9]. These data also reinforce our understanding that LEDGF/p75 is the dominant cellular co-factor determining HIV-1 integration site selection.
RS and JDR are doctoral fellows of the Flemish Fund for Scientific Research (FWO Vlaanderen), SV is a doctoral fellow of the Iwild-type. JDR is holder of a Mathilde-Krim postdoctoral fellowship (amfAR). Research was funded by grants from the Iwild-type (SBO grant CellCoVir), the FWO and the EU (FP7 THINC). This work was supported by NIH grants AI52845 and AI082020, the University of Pennsylvania Center for AIDS Research, and the Penn Genome Frontiers Institute with a grant with the Pennsylvania Department of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
We thank Paulien Van de Velde and Barbara Van Remoortel for excellent technical assistance.
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