Inhibition by woodchuck hepatitis virus of class I major histocompatibility complex presentation on hepatocytes is mediated by virus envelope pre-S2 protein and can be reversed by treatment with gamma interferon

Abstract

Presentation of class I major histocompatibility complex (MHC) is severely down-regulated on hepatocytes in chronic hepatitis caused by woodchuck hepatitis virus (WHV). To determine which of the viral proteins mediates class I MHC antigen suppression, cultured normal woodchuck hepatocytes were transfected with the complete WHV genome, sequences encoding individual virus proteins, or whole virus genomes in which transcription of selected proteins was disabled by site-specific mutagenesis. It was found that hepatocyte presentation of class I MHC antigen was significantly inhibited following transfection with complete WHV genome or with viral subgenomic fragments encoding envelope pre-S2 protein or pre-S1 protein, which naturally encompasses pre-S2 amino acid sequence. In contrast, hepatocytes transfected with WHV X gene alone demonstrated a profound enhancement in the class I antigen display, whereas those expressing virus major S protein or nucleocapsid (core) protein were not different from control hepatocytes. Analysis of the mutated WHV sequences confirmed that the envelope pre-S2 protein was responsible for inhibition of the class I MHC antigen display. Interestingly, treatment with recombinant woodchuck gamma interferon (rwIFN-gamma) restored the inhibited presentation of the class I antigen. Moreover, the class I antigen suppression was not associated with down-regulation of hepatocyte genes for class I MHC heavy chain, beta(2)-microglobulin, transporters associated with antigen processing, and proteasome subunits. These findings indicate that the defective presentation of class I MHC antigen on hepatocytes transcribing WHV is a consequence of posttranscriptional suppression exerted by virus pre-S2 protein and that this hindrance can be fully reversed by IFN-gamma.

Figures

FIG. 1.

Schematic presentations of p-WHV containing ∼1.1 times the length of the WHV genome and expression vectors carrying WHV sequences encoding individual virus proteins which were generated and examined during the course of this study. The nucleotide 5′-end and 3′-end positions for each WHV ORF and for the p-WHV construct, as well as the location of the endogenous EcoRI site within p-WHV, are marked. Relative positions for endogenous WHV promoters (Pro) and an enhancer (Enh) are indicated along the p-WHV sketch, while the location of the pC-DNA3.1-derived cytomegalovirus promoter (CMV Pro) is indicated at the beginning of each WHV sequence insert. All numbers denote the nucleotide positions according to the WHV/tm3 genome sequence (GenBank accession number AY334075 [24]).

FIG. 2.

Schematic presentation of the strategy applied for the generation of p-WHV mutated sequences by introducing premature stop codons. Designated names of the constructs and the introduced nucleotide modifications are identified on the left side of the panel. Relative locations of the stop codons inserted into WHV ORFs (open arrows) are marked by crosses. Detailed information on the site-specific mutagenesis leading to creation of each mutant is shown under each ORF sketch. The bold numbers at the 5′ and 3′ ends of the outlined sequences show nucleotide positions in relation to the EcoRI site in the WHV/tm3 genome (see the legend to Fig. 1). Bold and blue letters in the nucleotide sequences stand for introduced nucleotides, and the underlined three-nucleotide sequences represent the stop codons created. The aa sequences of interest and their relative positions (numbers after aa) within respective viral proteins are shown above the nucleotide sequences, while the aa sequences naturally overlapping the sequence of interest are depicted under the nucleotide sequences to indicate that no aa alterations were introduced. The asterisk marks termination of amino acid translation.

FIG. 3.

Expression of WHV genes in WCM-260 hepatocytes transfected with p-WHV. WCM-260 hepatocytes transiently transfected (after 48 to 72 h) (A) or stably transfected (after 2 to 3 months in the presence of G418) (B) with p-WHV were analyzed for expression of viral mRNAs by RT-PCR using primer pairs specific for S, C, and X genes. The amplified products were identified by Southern blot hybridization by probing with complete rWHV DNA. Total RNA isolated from the liver of a woodchuck with chronic WHV hepatitis was examined each time as a positive control. RNA samples not transcribed to cDNA (RT −) were analyzed in parallel with those subjected to RT reaction (RT +) to exclude possible contamination with DNA. The molecular sizes (bp) of the detected PCR products are marked on the right side of the panel. In panel C, WHV pre-S1/pre-S2 and core proteins were visualized by immunostaining in WCM-260 hepatocytes transiently transfected with p-WHV. Following exposure of the cells to anti-WHV pre-S and anti-WHc antibodies, secondary antibodies conjugated with Cy5 or FITC were applied to detect WHV pre-S (left) and WHcAg (center), respectively. The overlaid image (right) is shown to illustrate pre-S1/pre-S2 and WHcAg colocalization. Bars, 20 μM.

FIG. 4.

Validation of the expression of the plasmid constructs carrying WHV subgenomic sequences encoding individual virus proteins. WCM-260 hepatocytes transiently transfected (A) or stably transfected (B) with p-WHpS1, p-WHpS2, p-WHmS, p-WHc, or p-WHx were analyzed for the presence of relevant mRNA by RT-PCR using primer pairs specific for the pre-S1 (S1), pre-S2 (S2), or mS region of the S gene or for the C or X gene. The amplicons were identified by Southern blot hybridization by probing with rWHV DNA. For other details, see the legend to Fig. 3.

FIG. 5.

Modulation of the class I MHC antigen display on WCM-260 hepatocytes stably transfected with p-WHV or with plasmid constructs encoding individual WHV proteins. (A) The class I antigen presentation on WCM-260 hepatocytes stably transfected with p-WHV, p-WHpS1, p-WHpS2, p-WHmS, p-WHc, or p-WHx was assessed by flow cytometry by comparing the means (horizontal short lines) of MFI values obtained from three independent experiments. The data are presented as the percentages of MFI where the MFI values given by WCM-260 hepatocytes stably transfected with empty pC-DNA3.1 vector (e) were taken as 100%. P values were determined as indicated in Materials and Methods. (B) A representative example of flow cytometry histograms illustrating the display of the class I antigen on WCM-260 hepatocytes from one of three experiments shown in panel A. The levels of the class I antigen on the hepatocytes were determined prior to (IFN-γ −) and after (IFN-γ +) treatment with rwIFN-γ, as described in Materials and Methods. The data are shown as overlaid histograms representing the cells transfected with plasmids carrying WHV sequences (open symbols) and the cells transfected with empty vector (filled symbols).

FIG. 6.

Determination of the competence of p-WHV mutants with the disabled expression of selected structural or nonstructural proteins for the encoding of virus envelope and core proteins. WCM-260 hepatocytes transiently transfected with p-ΔWHpS1, p-ΔWHmS, p-ΔWHc, p-ΔWHx, or p-ΔWHp were probed with anti-WHV pre-S or with anti-WHc by immunostaining, as described in Materials and Methods and in the legend to Fig. 3. Bars, 20 μM.

FIG. 7.

The class I MHC antigen display on WCM-260 hepatocytes transfected with p-WHV mutants in which expression of selected viral proteins was disabled by the introduction of appropriate premature stop codons. (A) Hepatocytes stably transfected with p-ΔWHpS1, p-ΔWHmS, p-ΔWHc, p-ΔWHx, or p-ΔWHp or with p-WHV or empty vector (e) as controls were assessed for class I antigen surface presentation by flow cytometry, as described in the legend to Fig. 5 and in Materials and Methods. (B) A representative example of flow cytometry histograms demonstrating the class I antigen display on WCM-260 hepatocytes transfected with different p-WHV mutants from one of three experiments depicted in panel A prior to (IFN-γ −) and after (IFN-γ +) treatment with rwIFN-γ. For further details, see the legend to Fig. 5 and Materials and Methods.

FIG. 8.

The display of class I MHC antigen on the surface of WCM-260 hepatocytes transfected with different WHV expression constructs following treatment of the cells with IFN-γ. Hepatocytes stably transfected with p-WHV, p-WHpS1, p-WHpS2, p-WHmS, p-WHc, or p-WHx (A) or with p-ΔWHpS1, p-ΔWHmS, p-ΔWHc, p-ΔWHx, or p-ΔWHp and control cells transfected with empty pC-DNA3.1 vector (e) or with p-WHV (B) were treated with rwIFN-γ prior to staining for class I antigen with B1b.B9 MAb, as outlined in Materials and Methods. It is of note that the treatment with rwIFN-γ led to the unified augmentation in the class I antigen display to approximately the same level on hepatocytes transfected with different WHV DNA constructs and on the control cells. The data should be interpreted in the context of those shown in Fig. 5 and 7.

FIG. 9.

Quantitation of the class I MHC- and proteasome-linked gene expression in hepatocytes transfected with p-WHV and with constructs encoding individual WHV proteins and either left untreated or subjected to treatment with IFN-γ. The data were generated in three independent experiments by real-time RT-PCR. They are presented as the mean gene copy numbers per reaction, which were calculated using serial 10-fold dilutions of internal plasmid standards and normalized against housekeeping woodchuck β-actin gene cDNA level. The open bars represent hepatocytes not treated with rwIFN-γ, while the stratified bars represent those treated with the cytokine. h.c., heavy chain.