ABSTRACT: BACKGROUND: 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. FINDINGS: 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 T levels. Additional HRP-2 KD in LEDGF/p75-depleted cells reduces integration frequency in transcription units and shifts the integration distribution towards random. CONCLUSIONS: 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.