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 HSV-1-specific CTL induction in vivo.

<p>Six-week-old female C57BL/6J mice were mock-infected (n=21) or infected with 1 x 10⁶ PFU HSV-1(F) (n=21), YK511 (Us3-K220M) (n=21), YK513 (Us3-repair) (n=21) or YK591 (ΔICP47) (n=10)/footpad. At 4 d post-infection, spleen (A) and popliteal lymph node (B) cells were obtained and stained with MHC-I tetramers specific for the H-2K^b-restricted HSV-1 gB immunodominant epitope (SSIEFARL). Cells were then stained with anti-CD8α and anti-CD3ε antibodies and analyzed by flow cytometry. The percentage of CD8⁺ and CD3⁺ cells from mock-infected mice that were positive for gB-specific MHC-I tetramer was subtracted from the percentage of CD8⁺ and CD3⁺ cells from mice infected with each virus that were also positive for gB-specific MHC-I tetramer. Each data point is the mean ± standard error.</p

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 HSV-1-specific antigen presentation.

<p>B6MEFs (A) and MHC-I-/-MEFs (B) were infected with HSV-1(F), YK511 (Us3-K220M), YK513 (Us3-Repair) or YK591 (ΔICP47) at an MOI of 1 for 12 h and then co-cultured for an additional 12 h with <i>lacZ</i>-inducible CTL hybridoma cells recognizing HSV-1 gB (HSV-2.3.2E2), followed by β-galactosidase assays. Each data point is the mean ± standard error of triplicate samples, and is representative of three independent experiments.</p

Effect of depletion of CD8⁺ T cells or NK cells on YK511 (Us3K220M) replication in vivo.

<p>Six 6-week-old female C57BL/6J mice were mock-depleted or depleted of CD8⁺ T cells (A) or NK1.1⁺ cells (B) and infected with 1 x 10⁶ PFU YK511 (Us3-K220M) or YK513 (Us3-repair)/footpad. At 1 and 4 d post-infection, virus titers in the footpads from the infected mice were determined by standard plaque assays on Vero cells. Each data point is the mean ± standard error of the PFU/gram/footpad.</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

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, 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