A Tyrosine-Based Motif in the Cytoplasmic Tail of Pseudorabies Virus Glycoprotein B Is Important for both Antibody-Induced Internalization of Viral Glycoproteins and Efficient Cell-to-Cell Spread

Abstract

Pseudorabies virus (PRV), a swine alphaherpesvirus, is capable of causing viremia in vaccinated animals. Two mechanisms that may help PRV avoid recognition by the host immune system during this viremia are direct cell-to-cell spread in tissue and antibody-induced internalization of viral cell surface glycoproteins in PRV-infected blood monocytes, the carrier cells of the virus in the blood. PRV glycoprotein B (gB) is crucial during both processes. Here we show that mutating a tyrosine residue located in a YXX⌽ motif in the gB cytoplasmic tail results in decreased efficiency of cell-to-cell spread and a strong reduction in antibody-induced internalization of viral cell surface glycoproteins. Mutating the dileucine motif in the gB tail led to an increased cell-to-cell spread of the virus and the formation of large syncytia.Pseudorabies virus (PRV) is a swine alphaherpesvirus which, like most alphaherpesviruses, has evolved in several ways to subvert the immune system of its host. One noteworthy example of PRV immune modulation is the ability to replicate in the respiratory tracts of vaccinated animals. A viremia often results, giving rise to striking PRV symptoms, including abortion (24, 39). This viremia in vaccinated pigs requires cell-to-cell spread of PRV in tissue and transport of virus via infected monocytes in the blood (23,24,25,39).Mechanisms used by PRV, as well as by the prototypical alphaherpesvirus herpes simplex virus (HSV), to avoid recognition and destruction by the immune system include strategies to downregulate major histocompatibility complex class I-dependent antigen presentation in infected cells (3, 33; for a review, see reference 40), direct cell-to-cell spread of the virus, binding of complement factors via viral glycoprotein C (gC), Fc receptor activity of viral glycoprotein complex gE-gI, and, for PRV, the recently described antibody-induced internalization of viral cell surface proteins in PRV-infected blood monocytes, the natural carrier cells of the virus in vaccinated animals (9,10,14,15,17,18).For PRV, two of these mechanisms are mediated by viral glycoprotein gB: (i) the antibody-induced internalization of viral cell surface glycoproteins (a rapid and massive internalization of the majority of plasma membrane-anchored viral proteins upon aggregation of these proteins caused by the addition of PRV-specific antibodies, a process that likely results in inefficient antibody-dependent lysis of PRV-infected monocytes) and (ii) the direct cell-to-cell spread of the virus (10,29,31,38).PRV gB is a type I membrane glycoprotein of 913 amino acids (aa), consisting of an extracellular domain, a transmembrane region, and a 93-aa cytoplasmic C-terminal tail. At least three putative endocytosis motifs located within the cytoplasmic tail of gB are conserved throughout the alphaherpesvirus family. Two are tyrosine-based YXX⌽ sequences (where Y stands for tyrosine, X stands for any amino acid, and ⌽ represents a bulky, hydrophobic group) and one is a dileucine (LL) motif. YXX⌽ and LL motifs in the c...

Copatching and Lipid Raft Association of Different Viral Glycoproteins Expressed on the Surfaces of Pseudorabies Virus-Infected Cells

“…In both PRV-and HSV-infected cells, efficient capping has been shown to depend on the presence of viral protein gE, whereas internalization has been demonstrated to be initiated mainly by viral proteins gB and gD and to a lesser extent gE (8,10,39). Exactly how these viral proteins initiate the redistribution processes is not fully understood, but it has been shown that tyrosine-based amino acid motifs in the cytoplasmic tails of PRV gB and gE are of crucial importance for internalization and capping, respectively (7,9). In addition, PRV gE-mediated capping has been suggested to require specific tyrosine kinase signaling (9).…”

Virion-Incorporated Glycoprotein B Mediates Transneuronal Spread of Pseudorabies Virus

“…PRV GS443 expresses VP26-green fluorescent protein (GFP) in a PRV Becker background (42). PRV HF22 is a gB-null strain that carries a kanamycin resistance cassette in the gB locus in a PRV Becker background (16). PRV 232 is a gB-null strain expressing VP26-GFP in a PRV Becker background; it was generated for this study by crossing PRV HF22 and PRV GS443 in gB-complementing cells.…”

“…In polarized epithelial cells, gB is targeted to the basolateral surface, presumably via interactions of its cytoplasmic tail with adaptor protein 1B. The basolateral sorting of gB is hypothesized to enhance the efficiency of direct cell-to-cell spread of the virus (16).…”

“…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).…”

Cell-surface expression of a mutated Epstein–Barr virus glycoprotein B allows fusion independent of other viral proteins

“…The gB C terminus is one of the longest of the herpesvirus-encoded glycoproteins and contains important cellular-sorting signals and regulates virally induced membrane fusion (28). The role of the gB tail in regulating herpesvirus-induced membrane fusion is most apparent because many mutations that modulate fusion are located in the gB C terminus (23,24,(29)(30)(31)(32)(33)(34)(35)(36)(37)(38). For EBV, the C terminus also contains domains that are important for cell fusion and the cellular localization of gB (8,21).…”