Journal of Virology, February 2010, p. 1221-1236, Vol. 84, No. 3

Immune Evasion Proteins of Murine Cytomegalovirus Preferentially Affect Cell Surface Display of Recently Generated Peptide Presentation Complexes

Institute for Virology, University Medical Center of the Johannes Gutenberg University, Mainz, Germany

Received 2 October 2009/ Accepted 6 November 2009

For recognition of infected cells by CD8 T cells, antigenic peptides are presented at the cell surface, bound to major histocompatibility complex class I (MHC-I) molecules. Downmodulation of cell surface MHC-I molecules is regarded as a hallmark function of cytomegalovirus-encodedimmunoevasins. The molecular mechanisms by which immunoevasins interfere with the MHC-I pathway suggest, however, that this downmodulation may be secondary to an interruption of turnover replenishment and that hindrance of the vesicular transport of recently generated peptide-MHC (pMHC) complexes to the cell surface is the actual function of immunoevasins. Here we have used the model of murine cytomegalovirus (mCMV) infection to provide experimental evidence for this hypothesis. To quantitate pMHC complexes at the cell surface after infection in the presence and absence of immunoevasins, we generated the recombinant viruses mCMV-SIINFEKL and mCMV-m06m152-SIINFEKL, respectively, expressing the K^b-presented peptide SIINFEKL with early-phase kinetics in place of an immunodominant peptide of the viral carrier proteingp36.5/m164. The data revealed 10,000 K^b molecules presenting SIINFEKL in the absence of immunoevasins, which is an occupancy of10% of all cell surface K^b molecules, whereas immunoevasins reduced this number to almost the detection limit. To selectivelyevaluate their effect on preexisting pMHC complexes, cells were exogenously loaded with SIINFEKL peptide shortly after infection with mCMV-SIINFEKA, in which endogenous presentation is prevented by an L174A mutation of the C-terminal MHC-I anchor residue. The data suggest that pMHC complexes present at the cell surface in advance of immunoevasin gene expression are downmodulated due to constitutive turnover in the absence of resupply.