Intracellular localization of proteasomal degradation of a viral antigen

Abstract

To better understand proteasomal degradation of nuclear proteins and viral antigens we studied mutated forms of influenza virus nucleoprotein (NP) that misfold and are rapidly degraded by proteasomes. In the presence of proteasome inhibitors, mutated NP (dNP) accumulates in highly insoluble ubiquitinated and nonubiquitinated species in nuclear substructures known as promyelocytic leukemia oncogenic domains (PODs) and the microtubule organizing center (MTOC). Immunofluorescence revealed that dNP recruits proteasomes and a selective assortment of molecular chaperones to both locales, and that a similar (though less dramatic) effect is induced by proteasome inhibitors in the absence of dNP expression. Biochemical evidence is consistent with the idea that dNP is delivered to PODs/MTOC in the absence of proteasome inhibitors. Restoring proteasome activity while blocking protein synthesis results in disappearance of dNP from PODs and the MTOC and the generation of a major histocompatibility complex class I-bound peptide derived from dNP but not NP. These findings demonstrate that PODs and the MTOC serve as sites of proteasomal degradation of misfolded dNP and probably cellular proteins as well, and imply that antigenic peptides are generated at one or both of these sites.

Figure 1

Proteasome-dependent production of Ova_257-264 from VV-encoded proteins. L-K^b cells incubated for 90 min in the absence (top) or presence (bottom) of 50 μM LC were infected for 8 h with the indicated rVV in the presence or absence of LC, respectively. Cells were stained with 25-D1.16 mAb and analyzed by cytofluorography.

Figure 3

dNP_pep rescued by zLLL localizes in PODs and the MTOC. rVV-infected 143B cells were incubated for 3 h before the addition of 20 μM zLLL. After 6.5 h, cells were fixed, permeabilized, stained with the indicated antibodies, and imaged with the LCSM. The second row displays cells expressing NP_pep, in the first and third, cells expressing dNP_pep. In the bottom row, dNP_pep expressing cells were first extracted with 1% NP-40 and then fixed with methanol/acetone (80:20) for 15 min at −20°C to enable staining with the γ-tubulin–specific mAb. Arrows point to the MTOC. Gray-scale images on the sides are merged in the middle with the color indicated by the text describing the antibody specificity. Bar, 10 μM.

Figure 2

Proteasome-dependent degradation of dNP_pep. 143B cells were pulse radiolabeled with [³⁵S]Met and chased for up to 120 min at 37°C in the presence or absence of LC. Radioactive proteins soluble in 1% TX100 (sol) or insoluble (ins) were separated by SDS-PAGE and the bands corresponding to dNP_pep or NP_pep located in the dried gel (a) and quantitated (b) after normalization using a VV-encoded protein as an internal standard (indicated as VV).

Figure 4

Fractionation and biochemical characterization of dNP_pep. (A) Extracts from rVV-infected 143B cells incubated with or without 20 μM zLLL were analyzed by Western blotting using anti-NH₂ antibodies. Note that the panel on the left is from a different gel than that on the right, and that the corresponding bands are more compressed. (B) HeLa cells infected with the rVV indicated for 90 min were incubated for an additional 3.5 h in the presence of 40 μM zLLL. Total cell extracts were analyzed by Western blotting using antibodies against the NH₂- or COOH-terminal peptides. Calculated M _r of the lower mobility bands indicated in red. (C) 143B cells expressing dNP_pep (left) or NP_pep (right) were extracted sequentially by NP-40, DNase I digestion, 2 M NaCl, DNase I/RNAase digestion, and then paraformaldehyde fixed, stained with anti-PML (top) or anti-NH₂ antibodies, and analyzed using the LCSM. Gray-scale images on the top and bottom are merged in the middle with the color indicated by the text describing the antibody specificity. The arrow points to the MTOC. (D) 143B cells infected with a control virus (C), NP_pep (N), or dNP_pep (D) were labeled for 5 min with [³⁵S]Met and chased for 40 min. Cells were extracted as above, and acetone precipitates were analyzed by SDS-PAGE and the radiolabeled proteins visualized using a PhosphorImager. Shown are the regions of the gel containing the proteins of interest along with control cellular and VV proteins. The intensity of total and NP-40 lysates was reduced fourfold before the 16–8 bit digital conversion to enable visualization of individual protein bands in all of the fractions. Arrows indicate NP_pep and dNP_pep. Based on PhosphorImager quantitation (and taking into account sample recovery), 2.3-fold more NP_pep is present in total lysates than dNP_pep, whereas 6.8-fold more dNP_pep is recovered in the Empigen BB step.

Figure 5

Effect of dNP expression on intracellular localization of components of the Ub-proteasome pathway. 143B cells infected for 4 h with VV-dNP_pepGFP were incubated for an additional 4 h in the presence of 10 μM zLLL, fixed, permeabilized and stained for cellular components using the antibodies indicated. dNP_pepGFP was located by its autofluorescence. Gray-scale images in each column are merged on the bottom with the color indicated by the text describing the antibody specificity. Arrows point to the MTOC. Bar, 10 μm.

Figure 6

Effect of proteasome inhibitors on the intracellular localization of components of the Ub-proteasome pathway in uninfected cells. 143B cells were incubated for 6 h in the presence of 21 μM zLLL, fixed, permeabilized, and stained for cellular components using the antibodies indicated. Gray-scale images in each column are merged on the bottom with the color indicated by the text describing the antibody specificity. Arrows point to the MTOC. Bar, 10 μm.

Figure 7

Effect of canavanine on intracellular localization of NP and cellular proteins. 143B cells infected for 2 h with VV-NP were incubated for an additional 6 h in the presence of 15 mM canavanine. Cells were fixed, permeabilized, and stained with the antibodies indicated. NP stained by anti-COOH antisera is shown in the right column. Gray-scale images on the left and right are merged in the middle column with the color indicated by the text describing the antibody specificity. Arrows point to the MTOC. Bar, 10 μm.

Figure 8

Proteasome-mediated in situ degradation of dNP_pep and generation of antigenic peptides. 143B cells infected for 4 h with VV-dNP_pepGFP were incubated for an additional 90 min in the presence of 10 μM zLLL in the absence (−90′ zLLL) or presence of protein synthesis inhibitors emetine (25 μM) and cycloheximide (25 μM) (−90′ zLLL EC) and then fixed, or washed, and incubated for 4 h in protein synthesis inhibitors in the absence (240′ EC) or presence of zLLL (240′ zLLL EC) and then fixed. Cells were permeabilized and stained using PBC antiserum. dNP_pepGFP was located by its autofluorescence. Bar, 10 μm.

Figure 9

Antigenic peptides are generated from a pool of dNP_pep accumulated in the presence of proteasome inhibitor. 143B cells were coinfected with a rVV expressing K^b and mouse β₂m and a rVV expressing the protein indicated. Cells were infected for 4 h in the presence of 10 μM zLLL to prevent the generation of K^b–Ova_257-264 complexes, washed, and incubated for 4 h in protein synthesis inhibitors in the presence (bottom left histogram) or absence (bottom right histogram) of zLLL. As controls, cells were incubated for 8 h in the absence of inhibitors (top left histogram) or in the presence of protein synthesis inhibitors (top right histogram). Cells were indirectly stained with the 25-D1.16 mAb to quantitate the number of cell surface K^b-Ova_257-264 complexes.