RNA polymerase II inhibitors dissociate antigenic peptide generation from normal viral protein synthesis: a role for nuclear translation in defective ribosomal product synthesis?

Abstract

Following viral infection, cells rapidly present peptides from newly synthesized viral proteins on MHC class I molecules, likely from rapidly degraded forms of nascent proteins. The nature of these defective ribosomal products (DRiPs) remains largely undefined. Using inhibitors of RNA polymerase II that block influenza A virus neuraminidase (NA) mRNA export from the nucleus and inhibit cytoplasmic NA translation, we demonstrate a surprising disconnect between levels of NA translation and generation of SIINFEKL peptide genetically inserted into the NA stalk. A 33-fold reduction in NA expression is accompanied by only a 5-fold reduction in K(b)-SIINFEKL complex cell-surface expression, resulting in a net 6-fold increase in the overall efficiency of Ag presentation. Although the proteasome inhibitor MG132 completely blocked K(b)-SIINFEKL complex generation, we were unable to biochemically detect a MG132-dependent cohort of NA DRiPs relevant for Ag processing, suggesting that a minute population of DRiPs is a highly efficient source of antigenic peptides. These data support the idea that Ag processing uses compartmentalized translation, perhaps even in the nucleus itself, to increase the efficiency of the generation of class I peptide ligands.

FIGURE 1

Influenza NA expression is inhibited by DRB treatment. A, L-K^b cells were infected with insOVA for 30 min and then cultured for 1 h before the addition of DRB. After five additional hours in culture, cells were analyzed for cell-surface NA expression by flow cytometry. B, Cells were infected as in A, and DRB was added to infected cells at the indicated times. Cells were collected every hour and stored at 4°C until the completion of the experiment and analyzed for cell surface NA. The mean fluorescence intensity (MFI) of the cell population is reported on the y-axis. C, mRNA from cytosolic and nuclear cell fractions was subjected to reverse transcription, followed by real-time PCR. Levels of NA mRNA in each sample were normalized to GAPDH mRNA levels to control for differences in sample recovery. D, Cells were infected and treated with DRB at the indicated times. Six hours p.i., total-cell lysates were prepared and analyzed by SDS-PAGE and Western blot analysis for NA, HA, NP, and cellular p97. E, The relative amount of NA present in cells is plotted for each DRB treatment.

FIGURE 2

MHC class I Ag presentation is partially diminished by DRB treatment of infected cells. A, L-K^b cells were infected with PR8 or insOVA for 30 min and cultured for 6 h; cells were collected each hour for analysis of cell-surface NA (black, left axis) and K^b-SIINFEKL (red trace, right axis). B, L-K^b cells infected with insOVA were treated with DRB, CHX, or BFA 1 h p.i and cultured for 6 h. Cells were collected at indicated times for analysis, as in A.

FIGURE 3

DRB-insensitive Ag presentation is dependent on the timing of treatment. A, L-K^b and HeLa-K^b cells were infected and cultured as in Fig. 1. DRB was added 1 or 2.5 h p.i. At indicated times, cells were collected and analyzed by flow cytometry for cell-surface NA (black trace) and K^b-SIINFEKL (red trace), and the relative amounts of each cell-surface molecule was plotted as a percentage of maximum expression (obtained from untreated cells at 6 h p.i). B, At 6 h p.i., the amount of cell-surface NA and K^b-SIINFEKL (K^b-S) on infected cells treated with DRB 1 h p.i was determined on L-K^b and HeLa-K^b cells and plotted as the percentage of the maximum level obtained in untreated cells. The relative ratio of K^b-SIINFEKL/NA for DRB-treated cells was compared with untreated cells and plotted for each cell type.

FIGURE 4

AMD, but not LMB, prevents NA synthesis. A, L-K^b cells were infected with insOVA for 30 min and cultured for 6 h. At 1 h p.i, cells were left untreated or were treated with AMD. Cell-surface NA (black trace, left axis) and K^b-SIINFEKL (red trace, right axis) were determined by flow cytometry, as in Fig. 2. B, Same as A, except cells were treated with LMB instead of AMD.

FIGURE 5

DRB treatment does not prevent NA maturation or degradation. A, Western blot samples were prepared from DRB-treated, PR8-infected cells in the absence of DTT, along with lysates of untreated PR8-infected cells diluted as indicated. NA dimer (~98 kDa) and low levels of NA monomer (~50 kDa) were detected by Western blot. B, The NA signal from diluted PR8 samples was plotted, and the line of best fit was calculated and used to accurately measure levels of NA in DRB-treated cells. C, L-K^b cells were infected with PR8 and cultured for 6 h. After 1 h, cells were left untreated, or DRB was added. At 2 h p.i, cells were treated with MG132. Total-cell lysates were prepared and analyzed for NA expression by Western blot analysis, as in Fig. 1. Approximate molecular mass (in kDa) are shown.

FIGURE 6

Cell-surface K^b levels are impacted minimally by DRB and AMD treatment. A, L-K^b cells were mock infected or infected with insOVA IAV, then treated with the indicated drugs, and analyzed for surface K^b by flow cytometry. B, Uninfected L-K^b cells or cells infected with insOVA were washed in a cold citric acid buffer to remove existing peptide–MHC complexes from the cell surface and then were cultured with or without indicated inhibitors for specified times. K^b levels on the cell surface were determined by flow cytometry. The mean fluorescence intensity (MFI) of the population analyzed is plotted on the y-axis.