Volume 6, Issue 5, Pages 422-432 (November 2009) Two Mechanistically Distinct Immune Evasion Proteins of Cowpox Virus Combine to Avoid Antiviral CD8 T Cells  Minji Byun, Marieke C. Verweij, David J. Pickup, Emmanuel J.H.J. Wiertz, Ted H. Hansen, Wayne M. Yokoyama  Cell Host & Microbe  Volume 6, Issue 5, Pages 422-432 (November 2009) DOI: 10.1016/j.chom.2009.09.012 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 Identification of CPXV12 by Loss-of-Function Screening (A) Schematic diagram of CPXV mutants. Numbers indicate positions in kilobase (kb) corresponding to the wild-type CPXV strain Brighton Red genome. CPXV203 was replaced with GFP in CPXVΔ203 and A518Δ203. (B) MC57G cells infected with indicated CPXV mutants were analyzed for surface Kb or Db expression at 24 hr postinfection. (C) MC57G cells infected with CPXV mutants indicated above each histogram were harvested at 24 hr postinfection, and their surface Kb expression levels were compared to those of CPXVΔ203- or A518Δ203-infected cells. (D) MC57G cells (Kb, Db) and L929 cells (Dk) were infected with CPXVΔ203 or CPXVΔ12Δ203 and analyzed for MHC class I expression at 24 hr postinfection. Cell Host & Microbe 2009 6, 422-432DOI: (10.1016/j.chom.2009.09.012) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 CPXV12 Delays MHC Class I Trafficking (A) Surface expression levels of Kb and Db were measured in C1498 cells transduced with either vector alone or CPXV12. GFP serves as a marker for transduction. (B) Intracellular trafficking of Kb molecules was examined in the presence and absence of CPXV12. C1498 cells transduced with either vector alone or CPXV12 were sorted several times until the GFP-positive population was >95% (data not shown). They were labeled with [35S]-Met/Cys for 10 min and chased for the indicated periods of time. Kb molecules were immunoprecipitated from cell lysates and treated with Endo H prior to SDS-PAGE. The proportion of Endo H-resistant form at each chase time point was plotted. A representative result of three independent experiments is shown. EH-R, Endo H resistant; EH-S, Endo H sensitive. (C) C1498 cells transduced with either vector alone or CPXV12 were sorted several times until the GFP-positive population was >95% (data not shown). MC57G cells were infected with either CPXVΔ12Δ203 or CPXVΔ203 at a moi of 3. Cell lysates were treated with Endo H where indicated, and Kb heavy chain was detected by immunoblotting. Cell Host & Microbe 2009 6, 422-432DOI: (10.1016/j.chom.2009.09.012) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 CPXV12 Inhibits MHC Class I Dissociation from the PLC (A) Digitonin lysates of C1498 cells transduced with either vector alone or CPXV12 (left four lanes) and of MC57G cells infected with either CPXVΔ12Δ203 or CPXVΔ203 (right two lanes) were subjected to immunoprecipitation with anti-HA or anti-TAP1 serum. Precipitated proteins were separated by SDS-PAGE and blotted for indicated proteins. ND, not determined. (B) C1498 cells transduced with either vector alone or CPXV12 were labeled with [35S]-Met/Cys for 10 min and then chased for the indicated periods of time. Cells were lysed with 1% digitonin and subjected to immunoprecipitation with anti-TAP1 serum. From the coprecipitated proteins, Kb was further purified by reimmunoprecipitation with anti-Kb serum. Protein bands corresponding to Kb heavy chain (HC) and β2m are indicated. Asterisk (∗) indicates nonspecific protein. The levels of Kb heavy chain at each time point compared to the initial amounts (set as 100%) are plotted in the bottom graph. Mean ± standard error of mean (SEM) from two independent experiments is shown. Cell Host & Microbe 2009 6, 422-432DOI: (10.1016/j.chom.2009.09.012) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 CPXV12 Impairs Peptide Loading (A) C1498 cells transduced with indicated constructs were labeled with [35S]-Met/Cys for 10 min and chased for 60 min. Cell lysates were incubated with indicated concentrations of peptide VNVDYSKL at 4°C for 30 min and subsequently incubated at 37°C for 30 min. Kb was immunoprecipitated with monoclonal antibody B8-24-3, which recognizes peptide-bound Kb. Band intensities were quantified and plotted as percent of maximum, which represents the amount of Kb before the 37°C incubation (set as 100%). Mean ± SEM from three independent experiments is shown. (B) C1498 cells transduced with vector alone or CPXV12 were incubated with indicated concentrations of SIINFEKL or ASNENMETM peptide in the culture medium for 16 hr at 37°C and analyzed for surface Kb expression by flow cytometry. (C) Peptide transport activity of TAP was assessed in C1498 cells transduced with vector alone, BHV-1 UL49.5, or CPXV12. Translocation of the fluorescently labeled peptide CVNKTERAY was evaluated in the presence or absence of ATP. Peptide transport is expressed as percentage of translocation, relative to the translocation observed in control cells (set as 100%). Statistical significance was determined by Student's t test. (D) Surface levels of Kb and Db were compared between C1498 cells transduced with indicated constructs. Cell Host & Microbe 2009 6, 422-432DOI: (10.1016/j.chom.2009.09.012) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 Evasion of Antiviral CD8 T Cell Responses by CPXV12 and CPXV203 C57BL/6 mice were intranasally infected with 1 × 104 PFU of wtCPXV. Splenocytes were harvested at day 7 postinfection. MC57G cells were mock infected or infected with indicated viruses at a moi of 2 for 18 hr. (A) Surface expression levels of Kb, Db, and poxvirus HA on MC57G cells were analyzed by flow cytometry. (B) Splenocytes were incubated with MC57G cells for 7 hr and analyzed for IFN-γ by intracellular staining followed by flow cytometry. Numbers indicate percentage of IFN-γ+ cells among total CD8+ lymphocytes. A representative result of three similar experiments is shown. (C) Same as in (B), except mean ± SEM from three independent experiments is shown. Statistical significance was determined by Student's t test. ∗, p < 0.05; ns, not significant. (D) Splenocytes were coincubated with 51Cr-labeled MC57G cells for 4 hr. Lysis of MC57G cells was measured by 51Cr-release assay. E/T represents effector (splenocytes) to target (MC57G cells) ratio. Mean ± SEM from three independent experiments is shown. Cell Host & Microbe 2009 6, 422-432DOI: (10.1016/j.chom.2009.09.012) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 CPXV12 and CPXV203 Abrogate CD8 T Cell Immunity against CPXV In Vivo (A) Nine-week-old C57BL/6 mice were infected intranasally with 5 × 104 PFU of the indicated viruses and monitored for survival. Results were pooled from two independent experiments. (B) One-step growth curves for the indicated viruses were obtained in Vero cells. Genome copy numbers were determined by quantitative PCR. Mean ± SEM of triplicates is shown. (C) Nine-week-old C57BL/6 mice were infected intranasally with indicated viruses. Spleens from infected mice were harvested at day 7 or day 10 postinfection, and the viral loads were determined by quantitative PCR. Dotted lines indicate limits of detection. Statistical significance was determined by Student's t test. (D) Nine-week-old C57BL/6 mice were infected intranasally with 1 × 104 PFU of the indicated viruses and monitored for survival. Starting 2 days prior to infection, mice were treated with 1 mg of either isotype-control or CD8-depleting antibody every fourth day throughout the monitoring period. Results were pooled from two independent experiments. Cell Host & Microbe 2009 6, 422-432DOI: (10.1016/j.chom.2009.09.012) Copyright © 2009 Elsevier Inc. Terms and Conditions