A Single-Amino-Acid Substitution in Herpes Simplex Virus 1 Envelope Glycoprotein B at a Site Required for Binding to the Paired Immunoglobulin-Like Type 2 Receptor α (PILRα) Abrogates PILRα-Dependent Viral Entry and Reduces Pathogenesis ▿

Paired immunoglobulin-like type 2 receptor α (PILRα) is a herpes simplex virus 1 (HSV-1) entry receptor that associates with O-glycans on HSV-1 envelope glycoprotein B (gB). Two threonine residues (Thr-53 and Thr-480) in gB, which are required for the addition of the principal gB O-glycans, are essential for binding to soluble PILRα. However, the role of the two threonines in PILRα-dependent viral entry remains to be elucidated. Therefore, we constructed a recombinant HSV-1 carrying an alanine replacement of gB Thr-53 alone (gB-T53A) or of both gB Thr-53 and Thr-480 (gB-T53/480A) and demonstrated that these mutations abrogated viral entry in CHO cells expressing PILRα. In contrast, the mutations had no effect on viral entry in CHO cells expressing known host cell receptors for HSV-1 gD, viral entry in HL60 cells expressing myelin-associated glycoprotein (MAG) (another HSV-1 gB receptor), viral attachment to heparan sulfate, and viral replication in PILRα-negative cells. These results support the hypothesis that gB Thr-53 and Thr-480 as well as gB O-glycosylation, probably at these sites, are critical for PILRα-dependent viral entry. Interestingly, following corneal inoculation in mice, the gB-T53A and gB-T53/480A mutations significantly reduced viral replication in the cornea, the development of herpes stroma keratitis, and neuroinvasiveness. The abilities of HSV-1 to enter cells in a PILRα-dependent manner and to acquire specific carbohydrates on gB are therefore linked to an increase in viral replication and virulence in the experimental murine model

Transport of misfolded endoplasmic reticulum proteins to the cell surface by MHC class II molecules.

Nascent MHC class II molecules are associated with the invariant chain and are transported to the endolysosomal pathway, where MHC class II molecules acquire peptide antigens. On the other hand, misfolded endoplasmic reticulum (ER) proteins are generally degraded in the cells and are neither expressed on the cell surface nor secreted. Here, we found that MHC class II molecules associate with some misfolded ER proteins via the peptide-binding groove in competition with invariant chain. The misfolded proteins associated with MHC class II molecules are transported intact to the cell surface without processing to peptides. Furthermore, these complexes efficiently stimulate antigen-specific B cells. These findings reveal that MHC class II molecules function as a chaperone for the cell surface expression of misfolded ER proteins. In addition, we suggest that MHC class II molecules present not only peptides but also intact host-cell-derived proteins on the cell surface. These findings provide new insights into the function of MHC class II molecules

Us3 Kinase Encoded by Herpes Simplex Virus 1 Mediates Downregulation of Cell Surface Major Histocompatibility Complex Class I and Evasion of CD8⁺ T Cells

<div><p>Detection and elimination of virus-infected cells by CD8⁺ cytotoxic T lymphocytes (CTLs) depends on recognition of virus-derived peptides presented by major histocompatibility complex class I (MHC-I) molecules on the surface of infected cells. In the present study, we showed that inactivation of the activity of viral kinase Us3 encoded by herpes simplex virus 1 (HSV-1), the etiologic agent of several human diseases and a member of the <i>alphaherpesvirinae</i>, significantly increased cell surface expression of MHC-I, thereby augmenting CTL recognition of infected cells in vitro. Overexpression of Us3 by itself had no effect on cell surface expression of MHC-I and Us3 was not able to phosphorylate MHC-I in vitro, suggesting that Us3 indirectly downregulated cell surface expression of MHC-I in infected cells. We also showed that inactivation of Us3 kinase activity induced significantly more HSV-1-specific CD8⁺ T cells in mice. Interestingly, depletion of CD8⁺ T cells in mice significantly increased replication of a recombinant virus encoding a kinase-dead mutant of Us3, but had no effect on replication of a recombinant virus in which the kinase-dead mutation was repaired. These results indicated that Us3 kinase activity is required for efficient downregulation of cell surface expression of MHC-I and mediates evasion of HSV-1-specific CD8⁺ T cells. Our results also raised the possibility that evasion of HSV-1-specific CD8⁺ T cells by HSV-1 Us3-mediated inhibition of MHC-I antigen presentation might in part contribute to viral replication in vivo.</p> </div

Effect of Us3 kinase activity on susceptibility of HSV-1-infected cells to NK cell recognition.

<p>B6MEFs (A) and MHC-I^-/-MEFs (B) were infected with HSV-1(F), YK511 (Us3-K220M), or YK513 (Us3-Repair) at an MOI of 1 for 12 h and then co-cultured with NK cells isolated from C57BL/6J mouse splenocytes for an additional 24 h. IFN-γ in the co-culture cell supernatants was quantified by ELISA. Each data point is the mean ± standard error of triplicate samples, and is representative of three independent experiments.</p

Effect of Us3 kinase activity on cell surface expression of MHC-I (H-2Kb and H-2Db), gD and gH in HSV-1-infected MEFs from C57BL/6J mice (B6MEFs).

<p>(A and B) Surface expression of H-2Kb (A) and H-2Db (B) in B6MEFs mock-infected or infected with HSV-1(F), YK511 (Us3-K220M) or YK513 (Us3-repair) at an MOI of 3 for 18 h and analyzed and quantitated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072050#pone-0072050-g002" target="_blank">Figure 2B</a>. Each data point is the mean ± standard error of three independent experiments. (C and D) Surface expression of gD (C) and gH (D) in B6MEFs infected with HSV-1(F), YK511 (Us3-K220M) or YK513 (Us3-repair) at an MOI of 3 for 18 h and analyzed and quantitated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072050#pone-0072050-g002" target="_blank">Figure 2B</a>. Each data point is the mean ± standard error of triplicate samples, and is representative of three independent experiments.</p

Schematic diagram of the genome structures of wild type YK304 and the relevant domains of the recombinant viruses used in this study.

<p>Line 1, YK304 genome carrying a bacmid (BAC) in the intergenic region between UL3 and UL4. Line 2, domains encoding the UL40 to UL42 open reading frames. Line 3, UL41 gene encoding vhs. Lines 4-7, recombinant viruses with mutations in the UL41 gene. Line 8, domains encoding the Us11 to Us12 open reading frames and the viral replication origin S (Ori-S). Line 9, recombinant virus with mutation in the Us12 gene encoding ICP47.</p

Effect of Us3 kinase activity on cell surface expression of MHC-I, gD and gH in HSV-1-infected MRC-5 cells.

<p>(A) Cell surface expression of MHC-I in human MRC-5 cells mock-infected (filled purple histogram) or infected with wild-type HSV-1(F) (green line), YK511 (Us3-K220M) (pink line) or YK513 (Us3-repair) (blue line) at an MOI of 3 for 18 h and analyzed by flow cytometry. The data are representative of five independent experiments. (B) Quantitation of cell surface expression of MHC-I in infected MRC-5 cells. The relative mean fluorescence intensity (MFI) for MHC-I expression on the surface of cells infected with the indicated virus is shown as the fluorescence intensity of virus-infected cells relative to that of mock-infected cells. Each data point is the mean ± standard error of five independent experiments. (C and D) Surface expression of gD (C) and gH (D) in MRC-5 cells infected with HSV-1(F), YK511 (Us3-K220M) or YK513 (Us3-repair) at an MOI of 3 for 18 h and analyzed and quantitated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072050#pone-0072050-g002" target="_blank">Figure 2B</a>. Each data point is the mean ± standard error of triplicate samples, and is representative of three independent experiments.</p