Optimal infectivity of HIV-1 virions requires synthesis of the HIV-1 regulatory protein Nef in some producer cells but not others. A survey of 18 lymphoid cell lines found that Nef was dispensable in three, each of which harbored gammaretroviruses. Nef-dependent cell lines were rendered Nef-independent by a cell-free supernatant from the independent lines or by transfection of cloned murine leukemia virus (MLV). Analysis of MLV deletion mutations identified glycosylated gag (glycogag) as the factor that rescues Nef-defective HIV-1 virions. Glycogag was also demonstrated to be required for the infectivity of MLV virions produced in lymphoid cells. Direct comparison of Nef and glycogag revealed identical dependence for activity on Env-pseudotype and producer cell type. The two proteins colocalize within cells, and both increase the yield of viral cDNA in target cells. The functional similarity of Nef and glycogag is a compelling example of convergent evolution in which two structurally unrelated proteins provide a function necessary for virion infectivity in lymphoid cells.