Coxsackievirus B3 proteins directionally complement each other to downregulate surface major histocompatibility complex class I

Abstract

Picornaviruses carry a small number of proteins with diverse functions that subvert and exploit the host cell. We have previously shown that three coxsackievirus B3 (CVB3) proteins (2B, 2BC, and 3A) target the Golgi complex and inhibit protein transit. Here we investigate these effects in more detail and evaluate the distribution of major histocompatibility complex (MHC) class I molecules, which are critical mediators of the CD8(+) T-cell response. We report that concomitant with viral protein synthesis, MHC class I surface expression is rapidly downregulated during infection. However, this phenomenon may not result solely from inhibition of anterograde trafficking; we propose a new mechanism whereby the CVB3 2B and 2BC proteins upregulate the internalization of MHC class I (and possibly other surface proteins), perhaps by focusing of endocytic vesicles at the Golgi complex. Thus, our findings indicate that CVB3 carries at least three nonstructural proteins that directionally complement one another; 3A disrupts the Golgi complex to inhibit anterograde transport, while 2B and/or 2BC upregulates endocytosis, rapidly removing proteins from the cell surface. Taken together, these effects may render CVB3-infected cells invisible to CD8(+) T cells and untouchable by many antiviral effector molecules. This has important implications for immune evasion by CVB3.

FIG. 1.

CVB3 gene expression correlates with the downregulation of surface MHC class I. All experiments were repeated at least three times, and results are shown for a single representative experiment. (A) HeLa (RW) cells were mock infected or infected with wild-type CVB3 at an MOI of 10. Following virus adsorption, cells were harvested, washed, and incubated in suspension at 37°C with 5% CO₂. At the indicated times postharvest, an aliquot of cells from each culture was stained with mouse anti-human β₂M antibody, fixed, and analyzed by flow cytometry to assess surface MHC class I levels (MFI β₂M). (B) During the experiment depicted in panel A, cell lysates from the CVB3 infection time course were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis to examine the onset of viral protein expression. A rabbit anti-3A antibody detected the mature 3A protein as well as the immediate precursor polypeptide 3AB (indicated to the right of the panel). A longer exposure of this region of the blot is shown below. (C) Comparison of uninfected and infected cell surface MHC class I expression within a single-cell population. HeLa (RW) cells were infected at a low MOI (0.1) with a recombinant CVB3 that expresses EGFP, and surface staining (as in panel A) was carried out to assess MHC class I levels on the surfaces of uninfected (black bars) versus infected (white bars) cells, which were distinguished based on the expression of GFP, which became detectable at ∼3 h postinfection. (D) HeLa (RW) cells were mock infected or infected with CVB3 at an MOI of 10. Following virus adsorption, both cell populations were harvested, washed, and incubated in suspension at 37°C with 5% CO₂. Two μg/mL brefeldin A (BrA) was added to the uninfected population immediately postharvest, and at the indicated times, an aliquot of cells from each population was stained with mouse anti-human β₂M antibody. The cells were then fixed and analyzed by flow cytometry to assess the percentage of cells that were positive for β₂M expression.

FIG. 2.

Confocal microscopy confirms reduced levels of MHC class I on the surfaces of CVB3-infected cells. (A) A recombinant CVB3 carrying a membrane-targeted version of DsRed protein has been described previously by our laboratory (7). HeLa (RW) cells grown in monolayers on cover glass were infected with DsRed(mem) CVB3 at a low MOI (0.1) and fixed at 5 h postinfection. A monoclonal antibody recognizing a common HLA heavy chain epitope (HLA-ABC; clone W6/32) was used to stain unpermeabilized cells for MHC class I analysis by confocal microscopy. z-series images (encompassing the entire plasma membrane region) were acquired and projected using ImageJ, and the mean fluorescence was measured. Grayscale images of MHC class I (stained with fluorescein isothiocyanate), DsRed(mem) protein localization, and DAPI-stained nuclei (blue) are shown (first three panels), along with a merged image (far right panel). The white arrows (left panel) highlight reduced MHC class I on the surfaces of three infected cells that are positive for DsRed(mem) protein expression. (B) Images from 30 to 50 uninfected or infected cells were acquired as described for panel A, and the MFIs of MHC class I staining (with standard errors of the means) were calculated for uninfected and infected cells. The mean MFI for uninfected cells (white bar) was assigned the value 1.0, and the MFI for infected cells (red bar) is shown relative to that value.

FIG. 3.

CVB3 allows trafficking of nascent MHC class I molecules early in infection and then mediates their rapid disappearance. All experiments were repeated at least three times, and the results shown are for a single representative experiment. (A) To ascertain the kinetics of nascent MHC class I trafficking to the cell surface, a citrate-phosphate low-pH wash strategy was employed (34, 37). Before the wash (left panel), flow cytometric analysis indicated that approximately 98% of the cell population stained positive for β₂M (upper right quadrant). Following a citrate-phosphate wash (pH 3.0) for 1 min, preexisting β₂M associated with the MHC class I heavy chain was rendered virtually undetectable within the cell population (right panel, upper left quadrant). (B) HeLa (RW) cells were mock infected or infected with CVB3 at an MOI of 10. Following adsorption, cells were washed with citrate-phosphate buffer as described above. At the indicated time points, the percentage of cells expressing nascent β₂M at the cell surface was measured by flow cytometry and plotted. One sample contained 2 mM guanidine hydrochloride, a known inhibitor of picornavirus RNA synthesis. Two additional uninfected cell populations contained either 2 μg/ml BrA (to halt anterograde secretory transport) or 100 μg/ml cycloheximide (to stop protein synthesis). (C) To analyze anterograde trafficking kinetics at later times postinfection, three acid-washed cell populations were treated exactly as described for panel B (uninfected [black diamonds], infected with CVB3 [yellow triangles], or left uninfected and treated with BrA [red squares]). Three additional populations were CVB3 infected at time zero and washed at 3, 4, or 5 h postinfection (orange, blue, and green plots, respectively).

FIG. 4.

CVB3 2B, 2BC, and 3A affect MHC class I surface expression to different degrees. (A) Dicistronic reporter plasmids (7) expressed the CVB3 2B, 2BC, or 3A protein together with a membrane-targeted version of EGFP [EGFP(mem)], whose translation was driven by the internal ribosome entry site contained within the CVB3 5′-untranslated region (5′UTR). The control construct (not shown) was the empty parental plasmid, expressing internal ribosome entry site-driven EGFP(mem) but lacking an upstream viral protein. (B) EGFP(mem) expressing dicistronic constructs was transfected into HeLa (RW) cell monolayers, and at 16 h posttransfection, MHC class I (HLA-ABC) (red fluorescence, first column) and nuclei (blue fluorescence, third column) were stained. Cells were left unpermeabilized so that only surface MHC class I was revealed. Confocal z-series images were acquired, and z-dimensional profiles, shown below each image, were generated along the x-y axes indicated by the yellow lines (first column). A and B, apical and basal sides of the cell monolayers, respectively. Merged images for each of the four constructs tested are shown in the last column. (C) A series of cells from a minimum of three independent transfection experiments (as depicted in panel A) were analyzed for surface MHC class I expression relative to that of a mock-transfected control. The values shown represent normalization to the control construct. Student's t test (P values are given above bars) was done, comparing 2B, 2BC, or 3A to the control.

FIG. 5.

Differing effects of three CVB3 proteins on levels of surface MHC class I heterodimers, as confirmed by flow cytometry. Cells were transfected with EGFP(mem)-expressing dicistronic constructs (depicted in Fig. 4A) that coexpressed CVB3 2B, 2BC, or 3A or no viral protein (control construct). At 16 h posttransfection, cells were harvested, and their surface β₂M levels were measured by staining with an anti-β₂M antibody conjugated to phycoerythrin. The MFI of the β₂M signal present on the surfaces of GFP-positive cells was measured and graphed. Student's t test was done to compare the control to each of the three constructs, and the P values are depicted above the bars.

FIG. 6.

MHC class I is redistributed to the Golgi region in the presence of CVB3 2BC. (A) EGFP(mem)-expressing dicistronic constructs were transfected into HeLa (RW) cell monolayers, and at 16 h posttransfection, MHC class I (HLA-ABC) (red fluorescence, first column) and nuclei (blue fluorescence, third column) were stained. In contrast to the approach taken in Fig. 4, the cells depicted here were subjected to a permeabilization step prior to HLA-ABC staining to reveal not only surface but also intracellular MHC class I localization. Confocal z-series images were acquired, and z-dimensional profiles, shown below each image, were generated along the x-y axes indicated by the yellow lines. A and B, the apical and basal sides of the cell monolayers, respectively. Merged images for each of the four constructs tested are shown in the last column. (B) To assess the localization of intracellular MHC class I relative to the Golgi complex in cells expressing 2BC, cells transfected with this construct were stained using a rabbit polyclonal antibody to giantin, a major component of the Golgi complex (fourth column; depicted as green fluorescence in the merged image). HLA-ABC staining is depicted in red as described above, and nuclei were revealed by staining with DAPI. The merged image shows colocalization between MHC class I and the Golgi subcompartment.

FIG. 7.

CVB3 2B and 2BC expression focuses endocytic vesicles to the Golgi compartment. (A) Cells were transfected with each EGFP(mem) dicistronic construct, and at 16 h posttransfection, endosome localization was tracked using the lipophilic styryl dye AM4-65, which fluoresces red (first column) and allows the localization of newly endocytosed vesicles. Nuclei (DAPI staining) and EGFP(mem) signals are depicted in columns two and three, respectively. (B) The ImageJ “analyze particles” feature was utilized to determine the total area of AM4-65 fluorescence in relationship to the total area of the cell across a minimum of 50 cells from three independent experiments. These values were graphed and their standard deviations calculated (error bars). Student's t test was done to compare each of the three transfections to the values for the control cell population, and P values are given above the bars.