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As a member of the Alphaherpesvirinae, Equine herpesvirus type 1 (EHV-1) is a prevalent pathogen that causes health risk to horse populations worldwide. To establish persistent infection, EHV-1 has evolved an elegant system to escape from recognition and elimination by host immune responses. Usually in mammalian cells, viral infection is a danger signal and activates antigen presentation mediated by major histocompatibility complex class I (MHC-I). On the cell surface, MHC-I molecules carrying viral antigenic peptides recruit cytotoxic T lymphocytes (CTLs), which in turn kill the virus-infected cells and thus restrain the systemic circulation of virus. However, we recently found that pUL56, a viral component of EHV-1, functions as a major player to effectively prevent MHC-I presentation on the cell surface in vitro and affect a broad range of immune responses in vivo. These facts invoke a question about the molecular basis of pUL56.
Using different pharmacological inhibitors, the endo-lysosomal pathway was determined to be responsible for internalization and degradation of cell surface MHC-I upon EHV-I infection, which concurred with the early expression of pUL56. Further study revealed that dynamin and tyrosine kinase were required for pUL56-induced MHC-I endocytosis, but this endocytic process was not affected by inhibition of clathrin or caveolin-1, which primarily constitutes clathrin- and raft/caveolae-mediated endocytosis pathway, respectively. Moreover, significant restoration of cell surface MHC-I was observed in the presence of the ubiquitin-activating enzyme E1 inhibitor PYR41, suggesting that ubiquitination plays a dominant role in induction of MHC-I downregulation. Apart from MHC-I, pUL56 also triggered reduced expression of other cell surface molecules, including CD46 and CD63.
It is noted that pUL56 by itself is unable to induce MHC-I downregulation in transfected cells, indicating that interaction of another viral protein with pUL56 might be required for this process. To search for the elusive pUL56 interactor, we screened a single gene knockout library of EHV-1 mutants and identified pUL43, encoded by ORF17 gene, as a novel protein that modulates antigen presentation by MHC-I. Specifically, downregulation of cell surface MHC-I was significantly attenuated when cells were infected with the virus mutant containing a stop codon in place of ORF17 gene, which highlights the important role of pUL43 in regulating presentation of MHC-I to the cell surface. pUL43 is evolutionarily conserved throughout the Alphaherpesvirinae subfamily and contains 10 putative transmembrane (TM) domains. Despite mild influence on cell-to-cell spread, EHV-1 pUL43 proved dispensable for virus replication and release. Expression of pUL43 was detectable from 2 h post infection (hp.i.) and increased until 8 h p.i.; afterwards, the pUL43 protein was degraded in lysosomes. Indirect immunofluorescence analysis revealed that pUL43 co-localized with the Golgi apparatus and required the TM domains at the C-terminus to maintain the formation of vesicles. Furthermore, deletion of its hydrophilic region at the N-terminus (amino acid residues 3-40) compensated the low levels of surface MHC-I expression in infected cells. Intriguingly, co-transfection of pUL43 and pUL56 dramatically prevented surface retention of MHC-I, which depended on the PPxY motifs in the cytoplasmic domain of pUL56.
In summary, this dissertation addressed two major concerns: (i) The mechanism of pUL56, by which EHV-1 infection promotes the internalization of cell surface MHC-I molecules via the dynamin-dependent endocytic pathway; (ii) Identification and characterization of pUL43 as a novel MHC-I modulator that acts in concert with pUL56 for regulation of cell surface MHC-I expression. These findings advance our understanding of the intricate strategy that EHV-1 manipulates to circumvent the CTL-mediated immunity and meanwhile shed light on the possible optimization of vaccines against EHV-1 infection.