Stable MLV-VL30 dicistronic retroviral vectors with a VL30 or MoMLV sequence promoting both packaging of genomic RNA and expression of the 3' cistron.

The Friend murine leukemia virus (MLV) and VL30 5' leader sequences were recently found to possess an internal ribosomal entry signal (IRES) and promote cap-independent translation of a downstream cistron. The use of an IRES to generate a dicistronic message provides a way to express two exogenous proteins efficiently and stably within cells. In the present study, we used the VL30 and Moloney (Mo)MLV 5' leader sequences with both RNA translation (IRES) and packaging (E/DLS) functions to construct dicistronic retroviral vectors designed to express human placental alkaline phosphatase (plap) and neomycin phosphotransferase (neo). Vectors containing the VL30 (positions 205-794) and MoMLV (positions 210-1,035) 5' sequences between two cistrons were able to direct synthesis of exogenous proteins and were produced at a high titer, indicating that the IRES and packaging elements were functional in such dicistronic retroviral vectors. In addition, long-term selection for neo expression did not impair plap gene activity. In general, no major genetic rearrangement of the integrated recombinant provirus was observed with the dicistronic constructs upon prolonged culture of the infected cells.