The toll-like receptor (TLR)-7 has been shown to sense the retroviral infection. However, a surrogate sensor has been implicated. We examined whether retrovirus serves as a TLR3 ligand in human cells by utilizing cell lines LNCaP and PC-3 lacking TLR7, and the xenotropic murine leukemia virus-relamoted virus (XMRV) insensitive to human tripartite motif-containing (TRIM) 5, a newly characterized pattern recognition receptor (PRR). A dominant-negative TLR3 or a chemical inhibitor of TLR3 attenuated the XMRV-induced IP-10/CXCL10 expression, a marker of TLR3 response. These data clearly indicated that retroviral infection exemplified by XMRV activates the TLR3 signal in human cells. (C) 2012 Elsevier Inc. All rights reserved.