Internalization of Pseudorabies Virus Glycoprotein B Is Mediated by an Interaction between the YQRL Motif in Its Cytoplasmic Domain and the Clathrin-Associated AP-2 Adaptor Complex

Abstract

The cytoplasmic domain of pseudorabies virus (PRV) glycoprotein B (gB) contains three putative internalization motifs. Previously, we demonstrated that the tyrosine-based YQRL motif at positions 902 to 905, but not the YMSI motif at positions 864 to 867 or the LL doublet at positions 887 and 888, is required for correct functioning of gB during antibody-mediated internalization of PRV cell surface-bound glycoproteins. In the present study, we demonstrate that the YQRL motif is also crucial to allow spontaneous internalization of PRV gB, and thus, that spontaneous and antibody-mediated internalizations of PRV gB occur through closely related mechanisms. Furthermore, we found that PRV gB colocalizes with the cellular clathrin-associated AP-2 adaptor complex and that this colocalization depends on the YQRL motif. In addition, by coimmunoprecipitation assays, we found that during both spontaneous and antibody-dependent internalization, PRV gB physically interacts with AP-2, and that efficient interaction between gB and AP-2 required an intact YQRL motif. Collectively, these findings demonstrate for the first time that during internalization of an alphaherpesvirus envelope protein, i.e., PRV gB, a specific amino acid sequence in the cytoplasmic tail of the protein interacts with AP-2 and may constitute a common AP-2-mediated mechanism of internalization of alphaherpesvirus envelope proteins.Pseudorabies virus (PRV), a swine alphaherpesvirus closely related to the human pathogens herpes simplex virus (HSV) and varicella-zoster virus (VZV), is the causative agent of Aujeszky's disease (4, 24). Its genome encodes at least 11 glycoproteins, which have homologs in other herpesviruses (24). In PRV-infected cells, newly synthesized glycoproteins travel from the endoplasmic reticulum via the Golgi to the plasma membrane (25). These glycoproteins play important roles in the viral life cycle, as well as in the pathogenesis of PRV infections (9, 29).Interestingly, several alphaherpesvirus-encoded cell surfaceassociated envelope glycoproteins have been reported to be internalized, either spontaneously or upon binding of antigenspecific antibodies (12,13,17,32,35,36,44). The biological function of spontaneous internalization in the virus life cycle is not yet fully understood, although some hypothetical roles have been proposed (reviewed in reference 9), such as the possible involvement of internalization in delivering the viral cell surface proteins to a specific compartment, where viral envelopment takes place; in redirecting viral proteins to specific membrane surfaces (such as the apical, lateral, or basal surfaces of polarized cells); or in immune evasion. Antibodydependent internalization of viral cell surface proteins may also be implicated in immune evasion, since it has been shown to decrease the efficiency of antibody-dependent lysis of PRVinfected cells (49).Recently, several groups reported on the amino acid sequence motifs involved in the internalization of different alphaherpesvirus envelope glycoproteins. Two types...

Virion-Incorporated Glycoprotein B Mediates Transneuronal Spread of Pseudorabies Virus

“…Importantly, the association between Us9 and lipid rafts is required for the axonal sorting of viral particles (28). gB also strongly partitions with lipid rafts in PC12 cells and has a large cytoplasmic tail that interacts with cellular adaptor proteins (16,28,48). We therefore hypothesized that gB may participate, in concert with Us9, gE, and gI, in the sorting of virions into axons, possibly by recruiting appropriate cellular effectors via its cytoplasmic tail domain.…”

“…By homology to vesicular stomatitis virus fusion protein G, the ectodomain of gB is predicted to contain fusion loops; indeed, mutation of these regions in HSV-1 gB inhibits its fusion function (20). Mutagenesis of the gB cytoplasmic tail in HSV-1 and PRV revealed its role in the regulation of the fusion function, virion incorporation of gB, and interactions with cellular adaptor proteins (16,32,34,48; summarized in reference 39). Tyrosine motif-mediated interaction of PRV gB with adaptor protein 2 leads to its clathrin-dependent internalization (48).…”

“…Mutagenesis of the gB cytoplasmic tail in HSV-1 and PRV revealed its role in the regulation of the fusion function, virion incorporation of gB, and interactions with cellular adaptor proteins (16,32,34,48; summarized in reference 39). Tyrosine motif-mediated interaction of PRV gB with adaptor protein 2 leads to its clathrin-dependent internalization (48). In polarized epithelial cells, gB is targeted to the basolateral surface, presumably via interactions of its cytoplasmic tail with adaptor protein 1B.…”

Antibody-Induced Internalization of the Human Respiratory Syncytial Virus Fusion Protein

“…A second type of internalization is induced by the interaction of specific antibodies with viral proteins expressed on the surface of infected cells, followed by internalization of antibody-antigen complexes in the cell (25,27,28). Such viral protein internalization may result from cross-linking or depend on specific endocytic motifs in the cytoplasmic or transmembrane domains of glycoproteins, such as common tyrosine-based sorting motifs and dileucine motifs (20,24,29,30).…”

An immunoreceptor tyrosine-based inhibition motif in varicella-zoster virus glycoprotein B regulates cell fusion and skin pathogenesis

“…Whereas syncytia formation is typical of VZV, engineered or spontaneous substitutions in the gBcyt in other herpesviruses lead to syncytial herpesvirus phenotypes (15)(16)(17). The cytoplasmic domains of gB homologs contain α-helices and endocytosis motifs (18)(19)(20). Endocytosis, linked to dileucine and YXXΦ motifs (X, any amino acid; Φ, a large hydrophobic residue), is necessary for gB cellsurface retrieval, trans-Golgi network (TGN) localization, and incorporation into the virion envelope (21).…”