Viral glycoproteins gB and gD of the swine alphaherpesvirus pseudorabies virus (PRV), which is closely related to human herpes simplex virus and varicella-zoster virus, are able to drive internalization of antibodyantigen complexes that may form at the cell surface of infected monocytes, thereby protecting these cells from efficient antibody-mediated lysis. We found earlier that gB relies on an endocytosis motif in its cytoplasmic domain for its function during this internalization process. Here, we report that the PRV gD protein also contains a functional endocytosis motif (YRLL) in its cytoplasmic domain that drives spontaneous endocytosis of gD from the cell surface early in infection and that acts in concert with the endocytosis motif in gB to contribute to efficient internalization of antibody-antigen complexes in PRV-infected monocytes.Alphaherpesviruses have developed numerous strategies to delay or avoid recognition and elimination by different components of the immune system (11,21,45). The swine alphaherpesvirus pseudorabies virus (PRV) especially excels at circumventing antibody-dependent immunity, which allows it to replicate and sometimes spread in pigs that have been vaccinated with an inactivated vaccine (26, 48). PRV-infected blood monocytes play a pivotal role in spread of PRV in the presence of virus-neutralizing antibodies and carry the virus via the blood throughout the body (26).In PRV-infected blood monocytes, like in other PRV-infected cells, newly produced viral envelope proteins are incorporated in the plasma membrane (12,24), thereby rendering the cell recognizable for antibody-dependent immunity (13).However, we found earlier that binding of virus-specific antibodies to viral cell surface proteins in PRV-infected blood monocytes leads to rapid internalization of the antibody-antigen complexes (12), thereby lowering the susceptibility of the infected cell towards antibody-mediated cell lysis (41). This internalization process was found to be clathrin mediated and to depend on two of the PRV proteins at the cell surface, gB and gD (42).Clathrin-mediated endocytosis of cellular transmembrane proteins typically depends on so-called endocytosis motifs in their cytoplasmic domain, most notably YXXL and LL motifs (Y standing for tyrosine, L for leucine, and X for any amino acid). These motifs initiate endocytosis by establishing an interaction with the clathrin-associated AP-2 adaptor complex as a first step in the formation of clathrin-coated vesicles (3,4,20,36).We found that the function of gB in internalization of antibody-antigen complexes from the surface of PRV-infected monocytes depends on a functional tyrosine-based endocytosis motif (YQRL) in its cytoplasmic domain (10), and this motif was found to allow an interaction between gB and the AP-2 complex (43).How PRV gD is involved in internalization of antibodyantigen complexes, on the other hand, is unknown. PRV gD is a type I membrane glycoprotein of 402 amino acids, consisting of an extracellular domain, transmembrane region, and a ...