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

Abstract

Pseudorabies virus (PRV) is a swine alphaherpesvirus that is closely related to human herpes simplex virus (HSV). Both PRV and HSV express a variety of viral envelope glycoproteins in the plasma membranes of infected cells. Here we show that at least four major PRV glycoproteins (gB, gC, gD, and gE) in the plasma membrane of infected swine kidney cells and monocytes seem to be linked, since monospecific antibody-induced patching of any one of these proteins results in copatching of the others. Further, for all four PRV glycoproteins, monospecific antibody-induced patches were enriched in GM1, a typical marker of lipid raft microdomains, but were excluded for transferrin receptor, a nonraft marker, suggesting that these viral proteins may associate with lipid rafts. However, only gB and, to a lesser extent, gE were found in lipid raft fractions by using detergent floatation assays, indicating that gC and gD do not show strong lipid raft association. Addition of methyl-␤-cyclodextrin (MCD), a cholesterol-depleting agent that is commonly used to disrupt lipid rafts, only slightly reduced copatching efficiency between the different viral proteins, indicating that other factors, perhaps tegument-glycoprotein interactions, may be important for the observed copatching events. On the other hand, MCD strongly reduced polarization of the antibody-induced viral glycoprotein patches to a cap structure, a gE-dependent process that has been described for specific PRV-and HSV-infected cells. Therefore, we hypothesize that efficient gE-mediated capping of antibody-antigen patches may require the lipid raft-associated signal transduction machinery.

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

“…), requiring stronger internalization signals to allow efficient internalization of PRV gB. Furthermore, recent data indicate that some PRV cell surface glycoproteins, including gB, may be linked and segregate in lipid raft-like microdomains during later stages of infection (14). Although recent studies indicate that at least some raft-associated proteins may be internalized via clathrin-dependent pathways, lipid rafts most likely do not constitute very efficient platforms for clathrin-mediated internalization (1,27,30,31,42), perhaps also requiring stronger internalization signals for gB internalization at later time points in infection.…”

“…At least two hypotheses can be put forward that may explain this. First, recent data indicate that several of the major PRV cell surface glycoproteins (gB, gC, gD, and gE) are linked and therefore most likely are present in close proximity to each other (14). The exact nature of this link is still putative but may rely on the numerous protein-protein interactions that are believed to exist between viral glycoproteins and tegument proteins (26).…”

Comparison of the Pseudorabies Virus Us9 Protein with Homologs from Other Veterinary and Human Alphaherpesviruses

“…Together, these data support the notion that PRV Us9 is the critical component for efficient axonal sorting of virus structural proteins though all three proteins are required to achieve maximal sorting efficiency. It is also important that PRV Us9 and gE/gI are enriched in lipid raft microdomains and that localization of Us9 to lipid rafts is critical for its function (10,19,27). Thus, Us9 and gE/gI may be working together, within a specialized domain of vesicular membranes, to recruit axonal sorting machinery.…”

Optimal Replication of Human Cytomegalovirus Correlates with Endocytosis of Glycoprotein gpUL132

“…Likely mechanisms could include complex formation of gpUL132 with additional HCMV-specific glycoproteins which undergo endocytosis. Linkage of different herpesvirus envelope glycoproteins in the plasma membrane, association with lipid rafts, and subsequent internalization have been demonstrated (15). Also, internalization of gI of PRV has been shown to be dependent on gE, a protein that is endocytosed by its own signal while gI is incapable of endocytosis due to the lack of the appropriate internalization signals (54).…”