We reported previously the anti-viral activity named HRF (HIV-1 Resistance Factor) secreted by HIV-1 resistant cells. This work describes the identification of HRF from cell culture supernatant of HRF-producing cells (HRF(+) cells). Employing the proteomics and cell based activity assay we recovered ten peptides sharing 80-93% sequence homology with other eukaryotic DING proteins; discrete amino acid characteristics found in our material suggested that HRF is a new member of DING proteins family and consequently we designated it as X-DING-CD4 (extracellular DING from CD4(+) T cells). The presence of X-DING-CD4 in the extracellular compartment of HRF(+) but not control HRF(-) cells was confirmed by specific anti-X-DING-CD4 antibody. Similar as the un-fractionated HRF(+) cell culture supernatant, the purified X-DING-CD4 blocked transcription of HIV-1 LTR-promoted expression of luciferase gene and replication of HIV-1 in MAGI cells. The X-DING-CD4 -mediated anti-viral activity in MAGI cells could be blocked by specific antibody. (C) 2009 Elsevier Inc. All rights reserved.