Moloney murine leukemia virus (MMuLV) Gag protein contains three identified late (L) domains, PPPY, YPAL, and PSAP, which are thought to interact with the endosomal sorting machinery to assist budding. We created single and combined L-domain mutants in all permutations and tested the resulting clones for budding and replication. Budding and replication of all viruses with mutated PPPY were greatly reduced; however, the basal replication level was retained, demonstrated by the slow spread of the viruses in culture. Mutations in PSAP or YPAL did not affect budding or spreading, demonstrating that these two motifs are dispensable for efficient MMuLV replication. Furthermore, the basal budding level was maintained following inhibition of endosomal sorting machinery, emphasizing that the basal budding of MMuLV is independent of this machinery.