Coordinated Function of Murine Cytomegalovirus Genes Completely Inhibits CTL Lysis 1 Amelia K. Pinto,* Michael W. Munks,* Ulrich H. Koszinowski, † and Ann B. Hill 2 * Murine CMV (MCMV) encodes three viral genes that interfere with Ag presentation (VIPRs) to CD8 T cells, m04, m06, and m152. Because the functional impact of these genes during normal infection of C57BL/6 mice is surprisingly modest, we wanted to determine whether the VIPRs are equally effective against the entire spectrum of H-2 b -restricted CD8 T cell epitopes. We also wanted to understand how the VIPRs interact at a functional level. To address these questions, we used a panel of MCMV mutants lacking each VIPR in all possible combinations, and CTL specific for 15 H-2 b -restricted MCMV epitopes. Only expression of all three MCMV VIPRs completely inhibited killing by CTL specific for all 15 epitopes, but removal of any one VIPR enabled lysis by at least some CTL. The dominant interaction between the VIPRs was cooperation: m06 increased the inhibition of lysis achieved by either m152 or m04. However, for 1 of 15 epitopes m04 functionally antagonized m152. There was little differential impact of any of the VIPRs on K b vs D b , but a surprising degree of differential impact of the three VIPRs for different epitopes. These epitope-specific differences did not correlate with functional avidity, or with timing of VIPR expression in relation to Ag expression in the virus replication cycle. Although questions remain about the molecular mechanism and in vivo role of these genes, we conclude that the coordinated function of MCMV’s three VIPRs results in a powerful inhibition of lysis of infected cells by CD8 T cells. The Journal of Immunology, 2006, 177: 3225–3234. C ytomegaloviruses are ubiquitous species-specific viruses that persist within a host for its entire life. CMVs encode a group of proteins called VIPRs (viral genes that inhibit Ag presentation to CD8 + T cells) (1). Even though all CMVs encode VIPRs, the VIPRs found in each species have developed specialized mechanisms to interfere with the MHC class I pathway of their particular host. Murine CMV (MCMV) 3 has three VIPRs, m04, m06, and m152, which encode the glycoproteins m04/gp34, m06/gp48, and m152/ gp40. All three VIPRs function to inhibit CD8 T cell recognition of infected cells, but each VIPR employs a unique strategy to accomplish this task. m152 primarily functions by blocking MHC class I transport from the endoplasmic reticulum (ER)-Golgi in- termediate compartment (ERGIC) to the Golgi, resulting in an ac- cumulation of peptide-loaded class I molecules in the ERGIC and a reduction in cell surface class I expression (2– 4). Interestingly, although m152 has a pronounced effect on MHC class I transport, no direct biochemical interaction between m152/gp40 and MHC class I has ever been demonstrated. In contrast, m06/gp48 forms a tight association with MHC class I molecules in the ER. A dileucine motif in the cytoplasmic tail of m06/gp48 targets the MHC class I-m06/gp48 complex to a lysosomal compartment, where both proteins undergo rapid proteolysis (5), causing a dra- matic reduction in cell surface MHC class I expression (6, 7). MCMV’s third VIPR, m04/gp34, is primarily ER resident. A small portion of m04/gp34 forms a stable association with MHC class I molecules in the ER (8, 9). These complexes are exported to the cell surface, where they remain for several hours. However, the exact mechanism by which m04/gp34 inhibits CD8 T cell recog- nition remains to be determined. The fact that multiple VIPRs are encoded by both human CMV (HCMV) and MCMV is intriguing, and the advantage to the virus of this multiplicity is still not clear. For MCMV, the possibility that these genes were redundant was soon excluded: removing any one of MCMV’s three VIPRs enabled recognition of infected cells by at least some CTL clones (10, 11). Evidence has been obtained for cooperativity, i.e., that the combined actions of two or more VIPRs more efficiently inhibited Ag presentation than any VIPR acting alone (10). There is also evidence that different VIPRs may play a greater role in some cell types than others. For example, we observed that m04 appeared to play a more prominent role in mac- rophages than in fibroblasts (11). We also suggested that MCMV’s VIPRs acted in complemen- tary fashion, with efficient action of one VIPR against some MHC class I isoforms being complemented by a more efficient action of another VIPR against other isoforms (10). Metabolic labeling and pulse chase analysis revealed that m152 inhibited the transport of H-2D b more efficiently than H-2K b . Although m04/gp34 copre- cipitated with both K b and D b , we observed that three K b -restricted MCMV-specific CTL clones could lyse cells infected with a MCMV lacking m04 (m04), whereas two D b -restricted clones could not. We thus suggested that a contribution from m04 was required to completely inhibit K b -restricted Ag presentation, be- cause of m152’s relatively weak activity against K b . In contrast, because D b transport was much more efficiently inhibited by m152, *Oregon Health and Science University, Molecular Microbiology and Immunology, Portland, OR 97239; and † Ludwig Maximilians University of Munich, Max von Pet- tenkofer Institute, Munich, Germany Received for publication April 14, 2006. Accepted for publication June 16, 2006. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This research was support by National Institutes of Health (AI47206A and AI50099A; to A.B.H.), American Heart Association Fellowship (0215188Z; to A.K.P.), National Eye Institute training grant (ACAEI0071; to A.K.P.), and Deutsche Forschungsgemeinschaft (SFB 455; to U.H.K.). 2 Address correspondence and reprint requests to Dr. Ann B. Hill, Oregon Health Science University, Department of Microbiology and Immunology, 3181 Southwest Sam Jackson Park Road, Portland, OR 97239; E-mail address: hillan@ohsu.edu 3 Abbreviations used in this paper used in this paper: MCMV, murine CMV; ER, endoplasmic reticulum; ERGIC, ER-Golgi intermediate compartment; HCMV, hu- man CMV; BAC, bacterial artificial chromosome; BMM, primary bone marrow macrophage; wt, wild type; MOI, multiplicity of infection; PAA, phosphonoacetic acid; RT, reverse transcriptase; MFI, mean fluorescence intensity; IE, immediately early. The Journal of Immunology Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 by guest on September 30, 2021. Copyright 2006 Pageant Media Ltd. http://classic.jimmunol.org Downloaded from