Several retroviruses downmodulate the cell surface expression of envelope (Env) proteins through peptide sequences located in the cytoplasmic tail of the transmembrane (TM) subunit. We investigated whether cell surface expression of a chimeric protein containing the cytoplasmic domain of the TM protein (CTM) of bovine leukemia virus (BLV) was regulated by two membrane-proximal dileucine motifs or by tyrosine Y487 or Y498 in YXXL motifs. A chimeric protein composed of the extracellular and membrane-spanning portions of human CD8-␣ plus a wild-type (wt) BLV CTM was detectable on the surface of only 40% of the cells in which it was transiently expressed. Replacement of either dileucine pair with alanines increased the level of surface display of chimeric proteins. Nearly all cells became surface positive when both dileucine motifs were altered simultaneously and when either an N-terminal segment containing both dileucine motifs or a C-terminal segment containing all YXXL motifs was deleted. In contrast, replacement of Y487 or Y498 with alanine or phenylalanine enabled only small increases in surface display compared with wt levels. Chimeric proteins had similar stabilities but were downmodulated from the cell surface at three different rates. Point mutants segregated into each of the three groups of proteins categorized according to these different rates. Interestingly, Y487 mutants were downmodulated less efficiently than Y498 mutants, which behaved like wt. CD8-CTM chimeric proteins were phosphorylated on serine residues, but the native BLV Env protein was not phosphorylated either in transfected cells or in a lymphoid cell line constitutively producing BLV. Thus, both dileucine and YXXL motifs within the BLV CTM contribute to downmodulation of a protein containing this domain. Interactions with other proteins may influence surface exposure of Env protein complexes in virus-infected cells, assisting in viral evasion of adaptive immunity.The cytoplasmic domains of several retroviral envelope (Env) proteins control the level of Env present on the surface of infected cells. A low level of surface expression is mediated by intracellular retention of Env proteins as well as their rapid endocytosis from the plasma membrane (22,51,54). The existence of mechanisms to ensure low-level cell surface display of Env proteins points to a feature of the retroviral life cycle that most probably evolved to reduce clearance of virus-producing cells by the immune system (21, 22, 35). The main target for antibody recognition of retroviral Env protein complexes is the surface (SU) protein subunit, which is anchored onto the membranes of infected cells and virions by the membrane-spanning domain of the transmembrane (TM) subunit of an Env monomer (26). A labile disulfide bond links the SU and TM subunits of bovine leukemia virus (BLV) (30), a deltaretrovirus. Upon binding of oligomers of retroviral SU to cellular receptors, the TM subunits mediate virus-cell fusion (26). In parallel with other retroviral TM proteins, the extracellular...