Activity of Friend mink cell focus-forming retrovirus during myelo-erythroid hematopoiesis

Friend mink cell focus-forming (FMCF) viruses are recombinants between the Friend murine leukemia virus (F-MuLV) and endogenous polytropic retroviruses involved in a number of retrovirus-induced malignancies of the myelo-erythroid compartment. To analyze the contribution of the viral cis regulatory elements to the host range determinants within the hematopoietic system, we performed a series of marker gene experiments using both transient transfection and retroviral-mediated stable transduction of indicator cell lines representing distinct developmental stages. According to our data, the U3 region in the long terminal repeat (LTR) of FMCF viruses possesses an enhancer assembly that allows efficient transcription in both early and late myelo-erythroid stem and progenitor cells. Retroviral gene expression, however, is subjected to stage-dependent transcriptional controls during blood cell maturation. We obtained evidence that a repressor element overlapping with the primer binding site in the viral leader region compromises U3-mediated gene expression in a stage-dependent manner, with the strongest restriction observed in the most primitive cells analyzed, FDCP-mix. In addition, our data indicate a second hurdle for retroviral gene expression in early hematopoietic cells that is independent of the primer binding site and most likely related to inefficient utilization of U3-located enhancers. These data shed light on the mechanisms of host range restriction within the hematopoietic system and define a basis for the design of retroviral vectors aimed to overcome transcriptional inefficiency in early hematopoietic cells. Thus, we developed novel retroviral vectors combining FMCF-type U3 regions with a permissive leader from the murine embryonic stem cell virus. These vectors are highly efficient for gene transfer and expression in both early and late myelo-erythroid cells, indicating that they will be of great use for a variety of experimental and therapeutic applications.