Freely available: Link. Beihua Dong1, Robert H. Silverman1, Eugene S. Kandel2* 1 Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, United States of America, 2 Department of Molecular Genetics and Virology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, United States of Americ Abstract: Background Murine Leukemia Virus (MLV) is a rodent gammaretrovirus that serves as the backbone for common gene delivery tools designed for experimental and therapeutic applications. Recently, an infectious gammaretrovirus designated XMRV has been identified in prostate cancer patients. The similarity between the MLV and XMRV genomes suggests a possibility that the two viruses may interact when present in the same cell. Methodology/Principal Findings We tested the ability of XMRV to complement replication-deficient MLV vectors upon co-infection of cultured human cells. We observed that XMRV can facilitate the spread of these vectors from infected to uninfected cells. This functional complementation occurred without any gross rearrangements in the vector structure, and the co-infected cells produced as many as 104 infectious vector particles per milliliter of culture medium. Conclusions/Significance The possibility of encountering a helper virus when delivering MLV-based vectors to human cells in vitro and in vivo needs to be considered to ensure the safety of such procedures.