Surface expression of MHC I was investigated in ORFV-infected and non-infected Vero cells by flow cytometry using the MHC I specific monoclonal antibody (mAb) W6/32 as described in Methods. As shown in Figure 1a, ORFV infection resulted in a significant decrease of the MHC I surface expression. Twelve hours post infection (hpi) about 80% of MHC I was detectable on the cell surface compared to non-infected cells, which was further reduced to 70% at 24 hpi, and to almost 50% at 36 hpi. These decreases were statistically highly significant as determined by One-way ANOVA analysis (P < 0.001). Reduction of MHC I surface expression was dependent on live, replicating ORFV. Thus, infection of the cells with β-propiolactone-inactivated virus did not change the amount of MHC I expressed on the surface of Vero cells (Figure 1a, inact. ORFV).

To analyze whether expression of early or late ORFV genes might be responsible for the MHC I down-regulation, AraC was used to inhibit viral DNA synthesis and thereby preventing intermediate and late gene expression of ORFV 6. Figure 1b demonstrates that blocking of ORFV intermediate and late gene transcription (+ AraC) did not abolish MHC I down-regulation or affect MHC I surface presentation in non-infected cells. Infection of Vero cells and the effect of AraC were controlled by immunofluorescence studies using the mAb 13 C10, which is directed against the late major envelope protein of ORFV (Figure 1e).

Virus-infected cells were treated with Brefeldin A (BFA) to examine the biological stability of cell surface expressed MHC I molecules. BFA prevents the anterograde MHC I transport from the endoplasmic reticulum (ER) to the Golgi apparatus, and thereby inhibits the supply of newly synthesized MHC I to the cell surface. This experimental design allows the analysis of the half-life of surface expressed pre-existing MHC I by using flow cytometry. BFA-treated, non-infected Vero cells showed a 40 and 60% reduction of surface MHC I after 8 and 20 h incubation, respectively (Figure 1c, ni). In contrast, virus-infected Vero cells showed at the same BFA-incubation time points only a marginal MHC I decrease of 10% and 30% (Figure 1c, ORFV). These results suggest that ORFV infection increases the half-life of the remaining MHC I surface population by affecting surface stability and/or recycling of MHC I molecules. To examine whether early and/or late ORFV gene expression might be responsible for the increase in MHC I surface survival, cells were additionally treated with AraC during ORFV infection and BFA treatment. Figures 1c, d show that the MHC I half-life on the surface of non-infected cells was not altered by AraC. In infected cells the presence of AraC has some neutralizing influence on the ORFV mediated half-life effect on surface MHC I (compare Figure 1c, d). Thus, the ORFV-dependent increase of MHC I surface stability might be partially controlled also by late gene products.

A semi-quantitative RT-PCR was used to determine whether ORFV infection might influence the MHC I mRNA synthesis and thereby reduces MHC I surface expression. The amount of mRNA specific for the housekeeping gene GAPDH was related to the amount of MHC I mRNA at different times after infection. Each PCR product taken at the linear phase of PCR amplification was analyzed by gel densitometry. As shown in Figure 2a, the ratio of MHC I to GAPDH mRNA in non-infected cells ranged between 0.63 and 0.65 (Lanes 2 and 4), which remained almost unaltered 10 or 24 h after ORFV infection (lanes 1 and 3). Thus, the observed decrease of MHC I surface expression cannot be attributed to a prevention or inhibition of MHC I mRNA transcription by ORFV.

Next we analyzed whether and to what extent intracellular maturation of MHC I along the secretory route might be affected by ORFV infection. Endoglycosidase H (Endo H) – cleavage experiments were performed with anti-MHC I immunoprecipitates from detergent extracts of biosynthetically labelled, infected or non-infected Vero cells. Endo H is used to monitor posttranslational modification of glycosylated proteins within the Golgi. The MHC I-attached high mannose oligosaccharides are modified by a series of different ER and Golgi enzymes. Endo H is able to cleave oligosaccharides until the medial Golgi enzyme α-mannosidase II removes two mannose subunits. Since all later carbohydrate structures are Endo H-resistant, the enzyme monitors MHC I maturation within the late secretory route.

As can be seen from the SDS-PAGE analysis in Figure 2b upper panel, 12 h after ORFV infection intracellular MHC I-maturation is comparable in infected and non-infected Vero cells. In both situations we observed an approximately 1:1 signal ratio between Endo H-sensitive and -resistant MHC I molecules (Figure 2b upper panel, compare lanes 1 and 2). An additional minor species (approximately 10% of total MHC I signal) of partially resistant MHC I was also visible in infected cells (Figure 2b, upper panel, lane 2). After 24 and 36 h of infection, the population of Endo H-resistant MHC I was almost unaffected whereas the amount of Endo H-sensitive MHC I decreased by more than half (Figure 2b upper panel, lanes 4 and 6) as determined by densitometric scanning. Most importantly, the latter phenomenon was linked to a simultaneous increase of partially Endo H-resistant MHC I molecules by 45 and 55%, respectively. No such formation of unusual MHC I forms could be observed for non-infected control cells after 24 or 36 h of incubation (Figure 2b, upper panel, compare lanes 1, 3 and 5). The distinct behaviour of MHC I maturation in ORFV-infected cells was also seen in Western blot experiments, in which lysates of infected and non-infected Vero cells were assayed by using a different anti-MHC I antibody (mouse mAb, clone LY5.1, see Figure 2b, lower panel) with apparently higher specificity for the mature forms of MHC I. The two Endo H-resistant and -sensitive protein bands found after immunoprecipitation (Figure 2b, upper panel) or in Western blotting (Figure 2b, lower panel) by the two different anti-MHC I antibodies (W6/32 and LY5.1) most likely represent different allelic MHC I products expressed in Vero cells. Taken together, these findings suggest that ORFV-infection interferes with the functional requirements for proper carbohydrate trimming of MHC I within the cis- and/or medial-Golgi or the transport between the exocytic compartments.

Next, we investigated whether ORFV infection might affect the secretory pathway and Golgi transport of MHC I and thereby prevents intracellular trafficking of newly synthesized MHC I to the cell surface. Therefore, we analyzed infected cells by confocal immunofluorescence after co-staining of intracellular MHC I and Giantin, a main component of the cis- and medial-Golgi. The results in Figure 3a demonstrate that virus infection caused substantial changes in the localization patterns of MHC I and Giantin. Already 10 hpi, MHC I dispersed into the cytoplasm with a punctuated vesicular structure (Figure 3a - panel A) continuing to 24 hpi (Figure 3a - panel G), whereas MHC I in non-infected Vero cells showed a dense and ring-shaped perinuclear staining (Figure. 3a - panels D and J).

In non-infected cells, Giantin-staining was characterized by a compact perinuclear pattern (Figure 3a - panels E and K) that disappeared during ORFV infection and scattered throughout the cytoplasm (Figure 3a - panels B and H). Simultaneously, co-localization between Giantin and MHC I, which was clearly seen in non-infected cells (Figure 3a - panels F and L), was reduced during virus infection (Figure 3a – panels C and I) as verified by calculating the coefficient of co-localization (Pearson value; data not shown). The ORFV-induced Golgi spreading was also found in AraC-treated infected cells (Figure 3a, panels M to R) indicating the involvement of early ORFV gene(s). The ORFV-induced dislodgment of Golgi from its original nucleus-associated location into the cytoplasm could be confirmed by quantitative analysis of the distances between Golgi and nucleus in infected and non-infected cells (Figure 3b). The distance from the centre of the nucleus of each cell to the peripheral fringe of the Golgi was almost duplicated in infected cells, in the presence as well as in the absence of AraC, when compared to non-infected cells, and was highly significant according to T test (P < 0.0001).

The trans-Golgi network (TGN) represents another important constituent of the late secretory route involved in exo- as well as endocytic processes 20. The possible influence of ORFV on the TGN structure was examined with a TGN46-specific antibody. Partial co-localization between TGN46 and MHC I was visible in infected and non-infected cells. Similar to Giantin and MHC I, TGN46 lost its prominent perinuclear distribution after virus infection in favour of a punctuated vesicular pattern within the cytoplasm (Figure 4a, compare panels A and D, B and E), which was also seen in infected cells arrested for early gene expression by AraC (data not shown). Quantitative analysis of the images (Figure 4b) revealed a significantly increased distance (P < 0.0001) between TGN and nucleus (17 to 23 μm) in comparison to non-infected cells (9 to 12 μm). In summary, in virus infected cells Golgi and TGN are structurally dispersed into the cytoplasm and these processes are linked to early gene expression.

Since ORFV-infection leads to a fragmentation of Golgi, we explored the viral influence on Golgi-transport of MHC I. COP-I is a protein complex that coats vesicles transporting polypeptides between different Golgi compartments and from the cis-Golgi back to the ER 21. Therefore, we analyzed intracellular staining of MHC I and COP-I-component β-COP in infected and non-infected cells by fluorescence microscopy. Non-infected Vero cells displayed a characteristic juxtanuclear staining pattern of MHC I (Figure 5a - panels D and J) but only partial intracellular co-labelling of MHC I and β-COP (Figure 5a - panels F and L). In infected Vero cells a prominent perinuclear and vesicular MHC I-staining was observed 10 hpi that, however, dispersed into the cytoplasm after 24 hpi (Figure 5a - panels A and G). In contrast to non-infected cells, MHC I/β-COP co-localization could be seen for both infection time points (Figure 5a - panels C and I) confirmed by Pearson value calculation (data not shown). It must be noted that the non-infected cells were photographed with longer exposure time for the sake of better visualization. Additional AraC experiments showed that this effect is also controlled by early ORFV gene expression (data not shown).

Given expression levels of β-COP were analyzed by Western blot experiments in infected and non-infected cells. Figure 5b demonstrates that the 95 kDa β-COP protein was hardly detectable in cell extracts of non-infected Vero cells, most likely due to the fact that β-COP, like other COP-I components, does not stably exist out of the coatomer complex 22. Nevertheless, 24 h after ORFV infection β-COP was clearly visible with reduced amounts expressed after 36 hpi suggesting that the population of stably assembled COP-I structures is drastically enlarged in infected cells. Comparable protein loading was controlled by β-actin staining (Figure 5b, lower panel). Taken together, our findings provide evidence that the amount of MHC I-containing stable COP-I vesicles increased significantly during the first 24 hours after ORFV infection.

The attenuated ORFV strain D1701-V was propagated and titrated in Vero cells as described 36. Virus inactivation was achieved with 0.05% (v/v) β-Propiolactone (Serva) by incubation at 37 °C for 4 h and maintaining the pH-value of 7.6. After overnight incubation at 4 °C the supernatant was collected by centrifugation and plaque assays proved the successful virus inactivation.

The mouse mAb W6/32 specific for HLA-ABC also recognizing simian MHC I 37 was used for flow cytometry, confocal fluorescence microscopy and immunoprecipitation. LY5.1 is a mAb recognizing MHC class I heavy chains of HLA-ABC (Acris). Antibodies specific for Giantin, TGN46 and β-COP were purchased from Abcam, the antibody against ß-actin from Sigma-Aldrich. The mAb 13 C10 is directed against the 39 K major envelope protein of ORFV 38 and was a generous gift of C. McInnes and P. Nettleton (MRI, Pentlands Science Park, Penicuik, Scotland). As second antibodies we used anti-mouse FITC-conjugated antibody (Dianova), anti-mouse Alexa Fluor 488- and Alexa Fluor 555-conjugated antibodies and anti-rabbit Alexa Fluor 488- and Alexa Fluor 555-conjugated antibodies (Fisher Scientific, Invitrogen) and HRP-conjugated anti-rabbit IgG (Dianova).

Vero cells were infected with a m.o.i. of 1.0, harvested and stained successively with primary antibody and FITC-conjugated secondary antibody for 30 minutes at 4 °C. Brefeldin A (BFA, Sigma-Aldrich) was used in a concentration of 10 μg ml^-1, cytosine arabinoside (AraC, Sigma-Aldrich) was added (40 μg ml^-1) during virus infection. For viable cell determination dead cells were stained with 7-AAD (BD Bioscience) prior to FACS analyses using a FACSCalibur (BD Bioscience) and CellQuest Pro (BD Bioscience).

RNA kit (SurePrep True Total RNA Purification Kit, Fisher Scientific) was used to isolate total RNA from infected (m.o.i. 1.0) and non-infected Vero cells according to the manufacturer's instructions. RNA was treated with DNase (DNA-free, Ambion) and 300 ng were used for RT-PCR. Specific RNA of MHC I and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene was amplified by RT-PCR according to the manufacturer's recommendation (OneStep RT-PCR Kit, Qiagen) in a total volume of 10 μl, using GAPDH-specific primers at an annealing temperature of 64 °C 39 or using MHC I generic primers at an annealing temperature of 62 °C 40. PCR products were taken during the linear phase of amplification, separated by gel electrophoresis and the amplicon DNA band intensities were quantified using GelEval 1.32 software (FrogDance Software).

Vero cells were grown and infected (m.o.i. 0.5) in chamber slides (BD Biosciences) and fixed with 2% (v/v) methanol-free formaldehyde (Pierce, Fisher Scientific) in PBS and permeabilized with 0.2% (v/v) Triton-X100 (Sigma-Aldrich) in PBS. After 30 minutes blocking at room temperature in 5% (v/v) FCS in PBS, all antibody incubations were performed in PBS containing 1% (v/v) FCS for 30 minutes at 37 °C. F-actin was stained with Phalloidin-TRITC (Sigma-Aldrich), nuclei were stained with DAPI (1 μg ml^-1, Sigma-Aldrich) before embedding of slides in Mowiol-DABCO. Confocal microscopy was performed with ApoTome confocal fluorescence microscope (Axiovert 200 M; Zeiss) and arranged with AxioVision Rel. 4.8 (Zeiss). The Pearson coefficient showing degree of colocalization was determined using the program CoLocalizer Express (CoLocalizer).

Cells were starved for one hour in methionine-cysteine free Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 4 mM L-glutamine and 1 mM Na-Pyruvate, followed by incubation for additional 12 h in the presence of 10.5 mCi ml^-1 Trans-³⁵ S-Label (MP Biomedicals). Washed labelled cells were solubilised in PBS containing 1% Triton- X100 (Sigma-Aldrich) on ice for 45 minutes. After centrifugation at 14.000 rpm for 5 minutes the supernatants were used for immunoprecipitations at 4 °C overnight with anti MHC I mAb W6/32, which has been coupled directly to cyanogen bromide-activated sepharose (Amersham Life Sci.). Precipitates were digested with 10 mU of Endo H (Sigma-Aldrich) for 12 h at 37 °C and MHC I was eluted with 2.4 M urea, 2% SDS, 20% Glycerine, 125 mM Tris (pH 6.8) for 5 minutes at 95 °C prior to SDS-PAGE. Following electrophoresis fixed and dried gels were exposed to X-ray films (Kodak).

Non-infected and infected (m.o.i. 1.0) cells were dissolved with 1% (v/v) Triton- X100 (Sigma-Aldrich) in PBS for 30 minutes at 4 °C. SDS-PAGE and Western Blot were performed as reported 41. All antibodies were diluted in 1 x RotiBlock (Roth) and for enhanced chemiluminescence (ECL) the substrate Immobilon Western HRP (Millipore) was used. X-ray films for ECL were purchased from Pierce (Fisher Scientific).

Statistical significances were evaluated by One-way ANOVA analysis (Figure 1) or by the T test (Figures 2 and 3) using GraphPad Prism 5 software (La Jolla).