Major Histocompatibility Complex Class I Downregulation Induced by Equine Herpesvirus Type 1 pUL56 Is through Dynamin-Dependent Endocytosis

Abstract

Here, we report that cell surface MHC-I in EHV-1-infected cells is internalized and degraded in the lysosomal compartment in a pUL56-dependent fashion. pUL56-induced MHC-I endocytosis required dynamin and tyrosine kinase but was independent of clathrin and caveolin-1, the main constituents of the clathrin-and raft/caveola-mediated endocytosis pathways, respectively. Downregulation of cell surface MHC-I was significantly inhibited by the ubiquitin-activating enzyme E1 inhibitor PYR41, indicating that ubiquitination is essential for the process. Finally, we show that downregulation is not specific for MHC-I and that other molecules, including CD46 and CD63, are also removed from the cell surface in a pUL56-dependent fashion. IMPORTANCEWe show that alphaherpesvirus induces MHC-I downregulation through endocytosis, which is mediated by pUL56. The dynamin-dependent endocytic pathway is responsible for MHC-I internalization in infected cells. Furthermore, we discovered that this endocytic process can be disrupted by the inhibiting ubiquitin-activating E1 enzyme, which is indispensable for ubiquitination. Finally, pUL56 action extends to a number of cell surface molecules that are significant for host immunity. Therefore, the protein may exert a more general immunomodulatory effect.

Equine Herpesvirus 1 Multiply Inserted Transmembrane Protein pUL43 Cooperates with pUL56 in Downregulation of Cell Surface Major Histocompatibility Complex Class I

“…It is conceivable that pUL43 is modified similarly and acts as an adaptor for MHC-I endocytosis where pUL56 is the recruiter for Nedd4. This interpretation is supported at least by the colocalization of pUL56 and MHC-I in the Golgi and endosomal vesicles during EHV-1 infection (15).…”

“…In our previous studies, the pUL56 homologue of EHV-1 was identified as a novel viral protein that modulates the presentation of MHC-I molecules at the cell surface by accelerating dynamindependent endocytosis (15). However, pUL56 alone is not suffi- cient to induce the downregulation of MHC-I, as it did so only in the context of viral infection, implying that either a direct or indirect interaction of a viral protein(s) with pUL56 is required for MHC-I depletion.…”

“…Cell lysates were extracted with radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris [pH 7.5], 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 1 mM EDTA) containing a protease inhibitor cocktail (Roche) and Benzonase (Novagen). Samples were separated by SDS-12% polyacrylamide gel electrophoresis (PAGE), as described previously (15). After fractionation, proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Carl Roth).…”

“…EHV-1 pUL49.5, a small type I transmembrane protein that interacts with viral glycoprotein M (gM) (14), inhibits the formation of peptide-loaded MHC-I molecules by preventing ATP binding to TAP (10). pUL56, a type II transmembrane protein, enhances the internalization of MHC-I through dynamin-dependent endocytosis (15). In our previous studies, we reported that pUL56 induced cell surface MHC-I reduction solely in the context of infection and that MHC-I levels were not completely restored when cells were infected with a mutant virus lacking both pUL49.5 and pUL56 (11).…”

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“…Apart from the pUL49.5 homologues, the EHV-1 pUL56 was shown to be involved in downregulation of cell surface MHC-I [3]. Detailed studies revealed that early in infection the expression of pUL56 correlates with internalization of cell surface MHC-I through dynamindependent endocytosis, a process that requires activation of the cellular ubiquitination cascade [4]. Knowing that pUL56 suppresses the MHC-I presentation only during viral infection and not by itself, we speculated that there is at least one viral interactor of pUL56.…”