Inhibition of MHC class I-restricted antigen presentation by gamma 2-herpesviruses

Abstract

The gamma-herpesviruses, in contrast to the alpha- and beta-herpesviruses, are not known to inhibit antigen presentation to CD8(+) cytotoxic T lymphocytes (CTLs) during lytic cycle replication. However, murine gamma-herpesvirus 68 causes a chronic lytic infection in CD4(+) T cell-deficient mice despite the persistence of a substantial CTL response, suggesting that CTL evasion occurs. Here we show that, distinct from host protein synthesis shutoff, gamma-herpesvirus 68 down-regulates surface MHC class I expression on lytically infected fibroblasts and inhibits their recognition by antigen-specific CTLs. The viral K3 gene, encoding a zinc-finger-containing protein, dramatically reduced the half-life of nascent class I molecules and the level of surface MHC class I expression and was by itself sufficient to block antigen presentation. The homologous K3 and K5 genes of the related Kaposi's sarcoma-associated virus also inhibited antigen presentation and decreased cell surface expression of HLA class I antigens. Thus it appears that an immune evasion strategy shared by at least two gamma-herpesviruses allows continued lytic infection in the face of strong CTL immunity.

Figure 1

Inhibition of CTL-induced ⁵¹Cr release by γHV-68. MEF-1 cells transduced with retrovirus from pMSCV-GFP derivatives were uninfected (□, ■) or infected overnight with γHV-68 (○, ●) before being used as targets in 6-h ⁵¹Cr release assays. E:T, effector-to-target cell ratio. Target cells transduced with pMSCV-GFP alone are shown as triangles. For filled symbols, the peptide cognate for the particular CTL line was added (1 μM) at the time of infection. The pMSCV-GFP derivatives were pMSCV-γHV-68 ORF 61 (amino acids 1–740)-GFP (A), pMSCV-influenza nucleoprotein (amino acids 1–403)-GFP (B), and pMSCV-ovalbumin (amino acids 1–325)-GFP (C). The effectors used were CTL lines specific for the TSINFVKI epitope of the ORF 61 (A), the ASNENMETM epitope of the influenza nucleoprotein (B), and the SIINFEKL epitope of ovalbumin (C).

Figure 2

Inhibition of T cell hybridoma recognition by γHV-68. (A) MEF-1 cells transduced either with pMSCV-ovalbumin-GFP retrovirus (MEF-OVA) or with pMSCV-GFP alone (MEF) were infected or not with γHV-68 (10 pfu per cell) and exposed or not to 1 μM SIINFEKL peptide as indicated. Six hours later, the MEF-1 cells were washed and mixed with B3Z hybridoma cells that recognize the SIINFEKL epitope of ovalbumin. β-Galactosidase production by B3Z in response to SIINFEKL presentation was then assayed with o-nitrophenyl galactopyranoside. (B) MEF-1 cells transduced with pMSCV-M-TSINFVKI-ASNENMETM-SIINFEKL-GFP polytope retrovirus (MEF-P) or with pMSCV-GFP alone (MEF) were infected or not with γHV-68 (10 pfu per cell). Six hours later, the MEF and MEF-P cells were washed three times and mixed in a 1:1 ratio as indicated before overnight culture with B3Z cells and assay for β-galactosidase production.

Figure 3

Inhibition of MHC class I expression by γHV-68. To generate BALB/c-3T3-K^b cells, BALB/c-3T3 cells were transduced with retrovirus derived from pMSCV-H-2K^b-NEO. To generate BHK-21-K^b cells, equivalent retrovirus was used to infect the GP + E86 retroviral packaging line, and tunicamycin-treated (26) BHK-21 cells were then cultured with supernatant from the transduced packaging line. After selection, all transduced cells were infected with γHV-68 (10 pfu per cell) or left uninfected and then stained for surface MHC class I expression (filled histograms) or with secondary antibody alone (open histograms) after overnight culture.

Figure 4

The effect of phosphonoacetic acid treatment on viral host shutoff and MHC class I down-regulation. H-2^b MEFs were either left uninfected or infected overnight with MHV-68 (10 pfu per cell) and exposed or not to 100 μg/ml phosphonoacetic acid (PAA), starting 1 h before infection. (A) Cells were assayed for total cellular protein synthesis by [³⁵S]methionine labeling, followed by SDS/PAGE of total protein extracts. (B) Cells from parallel cultures were stained for surface MHC class I expression, using anti-H-2K^b (filled curves), or were stained with secondary antibody alone (open curves). Equivalent results of the H-2K^b staining were obtained by using anti-H-2D^b.

Figure 5

Identification of a γHV-68 gene that blocks antigen presentation. (A) The HindIII-I fragment of γHV-68 is shown, together with the viral ORFs it contains (10) and the extents of the other viral fragments used for transfection. In each case, H-2K^b-SIINFEKL antigen presentation was assayed, using β-galactosidase production by the B3Z hybridoma. (B) L929-H-2K^b cells were transfected with pCDNA3-SIINFEKL polytope (1 μg), pCDNA3-ORF 50 (1 μg), and a genomic fragment as indicated (1 μg), either in pUC-119 or in pSV40-ZEO (Invitrogen). HI, PP, XH, EH, and BH refer to the γHV-68 genomic DNA fragments indicated in A. For “no epitope,” cells were transfected with pCDNA3-ORF 50 (1 μg) plus empty pCDNA3 vector (2 μg). For “no inhibitor,” cells were transfected with pCDNA3-polytope (1 μg), pCDNA3-ORF 50 (1 μg), and empty pCDNA3 vector (1 μg). As a positive control, cells were transfected with pCDNA3-polytope (1 μg), pCDNA3-ORF 50 (1 μg), and pCDNA3 containing the m152 inhibitory gene of murine cytomegalovirus (1 μg). (C) L929-H-2K^b cells were transfected with pCDNA3-polytope (1 μg) plus the K3 genomic ORF (K3) or the homologous KSHV K3 (KK3) or K5 (KK5) ORFs, each expressed in PCDNA3 (1 μg). Because all expression was driven by the cytomegalovirus immediate-early promoter in pCDNA3 for this experiment, pCDNA3-ORF 50 was not included in the transfection.

Figure 6

Down-regulation of surface MHC class I expression by γHV-68 K3 and its KSHV homologs. The γHV-68 K3, the KSHV K3, and the KSHV K5 ORFs were cloned into pMSCV-GFP and expressed by retroviral transduction, as indicated in human 293 cells stably transfected with murine H-2K^b and murine L929 cells stably transfected with human HLA-A2. GFP⁺ transduced cells were then mixed with GFP⁻ untransduced cells to compare MHC class I expression, in each case using a phycoerythrin-conjugated goat anti-mouse IgG secondary antibody.

Figure 7

Short half-life of MHC class I in cells expressing the γHV-68 K3. Murine RMA cells and RMA cells transduced with pMSCV-γHV-68 K3-NEO retrovirus (RMA-K3) were pulse-labeled for 15 min with [³⁵S]methionine and then chased for the time indicated. H-2D^b molecules were immunoprecipitated with 28.14.8 antibody plus protein A-Sepharose, then digested (+) or not (−) with endoglycosidase H. Control samples were pulse-labeled for 15 min and immunoprecipitated at time point 0 without specific antibody. The bands observed correspond to endoglycosidase H-resistant (R), sensitive (S), and digested (D) H-2D^b.