- Previous efforts to develop a mouse model for HIV/AIDS have been impaired by multiple blocks to HIV replication, including barriers to viral entry, proviral transcription, and assembly. Expression of human cofactors in murine cells overcomes early restrictions, but does not lead to the production of infectious HIV particles. Here we show that stable expression of a codon-optimized synthetic HIV-1 Gag–Pol construct (sGP) in murine cell lines results in efficient Gag production and viral-like particle (VLP) release. Stable expression of the sGP construct in murine cells such as NIH3T3 and A9 improved Gag processing resulting in efficient VLP release comparable to that found in human cells. Using highly efficient transient transfection procedures, we increased Gag expression, and were able to produce infectious HIV particles in NIH3T3 cells. However, the infectivity of VLPs produced in murine cells was significantly below that generated in 293T cells. Reduced infectivity of VLPs produced in murine cells correlated with lower HIV reporter RNA levels in these cells. Taken together, improving the expression of HIV-1 Gag–Pol by using the sGP construct overcomes, at least in part, late restrictions in murine cells.