Murine leukemia viruses encode a unique form of Gag polyprotein, gPr80gag or glyco-gag. Translation of this protein is initiated from full-length viral mRNA at an upstream initiation site in the same reading frame as Pr65(gag), the precursor for internal structural (Gag) proteins. Whereas gPr80gag is evolutionarily conserved among gammaretroviruses, its mechanism of action has been unclear, although it facilitates virus production at a late assembly or release step. Here, it is shown that gPr80gag facilitates release of Moloney murine leukemia virus (M-MuLV) from cells along an IFN-sensitive pathway. In particular, gPr80gag-facilitated release occurs through lipid rafts, because gPr80gag-negative M-MuLV has a lower cholesterol content, is less sensitive to inhibition of release by the cholesterol-depleting agent MbetaCD, and there is less Pr65gag associated with detergent-resistant membranes in mutant-infected cells. gPr80gag can also facilitate the release of HIV-1-based vector particles from human 293T cells.