The Rous Sarcoma Virus Env Glycoprotein Contains a Highly Conserved Motif Homologous to Tyrosine-Based Endocytosis Signals and Displays an Unusual Internalization Phenotype

Numerous studies have contributed to an increasingly complex picture of the spatial and biochemical organization of the intracellular targeting pathways that include endocytosis (see reference 65 for an overview). Plasma membrane proteins fall into three basic categories regarding their internalization. The first class of glycoproteins undergoes rapid endocytosis via clathrin-coated vesicles, either constitutively, like the low-density lipoprotein (LDL) and the transferrin (Tf) receptors (8), or after activation by ligand binding as for the epidermal growth factor receptor (9, 10). Clustering of proteins into areas of coated-pit formation occurs by interaction of specific amino acid motifs within their cytoplasmic domains with adapter proteins such as AP-2 (4, 5, 42, 43). Two main endocytosis signals have been identified. One contains a critical tyrosine in the context of a YXXO motif in which O represents a bulky, aliphatic residue and for which a consensus sequence can otherwise only be broadly defined. Such a motif can act independently of its position, orientation, and structural context (63) and shows a tendency to form a tight turn (2, 12, 17, 52). YXXO motifs are present in the Tf receptor (TfR) and the mannose-6-phosphate receptor (12), as well as in TGN38 (20), and the LDL receptor contains a related NPXY motif (11). The second type of endocytosis signal is a dileucine-containing motif, which, like the YXXO motif, is transferable onto other membrane proteins (23). Constitutive internalization at the rate of membrane bulk uptake (∼1 to 2%/min) (64) is characteristic of the second class of plasma membrane glycoproteins. These proteins are believed to enter clathrin-coated pits by default, since no concentration occurs (63). The third class of glycoproteins are endocytosed at a very low rate and are essentially excluded from coated-pit structures. The influenza virus hemagglutinin (HA) protein is by far the best-characterized example of this group, and its exclusion from endocytic vesicles seems to be governed by sequences in its membrane-spanning domain (MSD) (references 32, 35, and 58 and references therein). Proteins reaching the early-sorting endosome enter either the late endosome/lysosome pathway, ultimately leading to degradation, or the recycling endosome compartment (for a review see reference 41). There is evidence that transport of some proteins into the lysosomal pathway requires a positive signal that is related to either the YXXO endocytosis signals or a dileucine motif (63). Though these motifs may overlap with internalization signals, they are distinct entities and are crucially defined by their positions in the cytoplasmic tail (28, 54, 65). Lack of such sorting information thus would direct a protein into the recycling pathway by default as suggested by studies of TfR and Lamp-1 (25, 26, 39, 54, 65). In polarized cells, apically endocytosed membrane proteins can also be targeted basolaterally, and for several proteins this process is again governed by tyrosine-containing signals, often found to be colinear or overlapping with YXXO endocytosis motifs (7, 19, 38). The specificity of the different intracellular targeting pathways, while involving similar or overlapping signal sequences, appears to be achieved by the interaction of the motifs with different adapter protein classes (see reference 60 for an overview). A comparison of the cytoplasmic C termini of both avian and mammalian retroviral envelope (Env) proteins reveals a remarkable sequence conservation within each group. In particular, the sequences that encompass a motif that is very similar to the YXXO endocytosis motif consensus sequence are highly conserved in both amino acid composition and distance to the MSD (Fig. ​(Fig.1a).1a). Because Env proteins are generally considered to be highly variable, this sequence conservation points to a biologically important role for such motifs in the survival of retroviruses in vivo. Retroviral glycoproteins are crucial for the binding of virions to their cellular receptors, followed by induction of virus-cell membrane fusion and infection. Env proteins are synthesized as precursor molecules that are proteolytically cleaved into the SU (surface) and TM (transmembrane) subunits before delivery to the cell surface, where they are incorporated into budding virions. Both human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) glycoproteins have been shown to be efficiently and constitutively internalized from the plasma membrane through coated pits by virtue of their tyrosine motifs (4, 18, 31, 56, 57). Interestingly, coexpression of HIV-1 Gag, the capsid precursor protein, downregulates Env endocytosis (18), possibly by competing for Env interaction with cellular factors such as AP-2. The Env protein of HIV-1 and those of HIV-2 and SIV are targeted to the basolateral surface in polarized cells (1, 45, 46), and HIV-1 Env redirects virion release to this site (37, 46), with the membrane-proximal tyrosine in its cytoplasmic domain being crucial for polarized targeting (38). FIG. 1 Conserved amino acid motifs in the cytoplasmic domains of retroviral TM proteins are homologous to tyrosine-based intracellular targeting signals. (a) Sequences of the MSD and cytoplasmic domains and the putative boundaries between them for avian and ... The putative endocytosis motif (YXXO) in the glycoproteins of avian leukosis and sarcoma viruses (ALSV) is remarkably conserved both in sequence and in position within the cytoplasmic domain relative to the transmembranal domain. We have previously described a mutant Prague C strain (PrC) Rous sarcoma virus (RSV) Env protein, Env-μ26, in which a L165R mutation in the MSD of TM dramatically decreased steady-state surface expression of Env with rapid internalization from the plasma membrane and lysosomal degradation (13, 27). From this observation arose the initial questions of whether the tyrosine in the Y190RKM motif found in RSV PrC TM glycoproteins contributes to the very rapid endocytosis observed for Env-μ26 and whether the wild-type glycoprotein displays a phenotype similar to those of other (retro)viral Env proteins. To facilitate a quantitative study of the internalization of RSV PrC Env, we utilized Env proteolytic processing mutant Env-S19 (15, 27). The Env precursor, Pr95, is not cleaved to gp85 (SU) and gp37 (TM) in the trans-Golgi network but is delivered to the cell surface in an uncleaved and fully glycosylated form, gp120. The rate of its biosynthesis, intracellular transport, and incorporation into RSV virions, however, is indistinguishable from that of wild-type Env. Of advantage to us was the unique property that Env-S19 present on the plasma membrane or in virions can be specifically cleaved into gp37 and gp85 by addition of trypsin to the medium, thereby distinguishing surface-exposed from intracellular molecules; mutant Env-S19 treated this way regains its biological function, thus rendering virions infectious (15). We show here that a wild-type ALSV Env protein has unusual endocytic properties in that the active YRKM endocytosis signal is silenced by sequences in the MSD. Thus, wild-type Env is endocytosed with kinetics similar to those defining the bulk rate turnover of membranes. This low rate of internalization is significantly reduced further by mutation of the critical tyrosine in the YRKM motif. Mutations in the MSD region of the glycoprotein “activate” recognition of the endocytosis signal. To our knowledge there is no precedent for the unusual phenotype of RSV Env, and we discuss a model of the underlying mechanism.