As an immune evasion mechanism, cytomegaloviruses (CMVs) have evolved proteins that interfere with cell surface trafficking of MHC class-I (MHC-I) molecules to tone down recognition by antiviral CD8 T cells. This interference can affect the trafficking of recently peptide-loaded MHC-I from the endoplasmic reticulum to the cell surface, thus modulating the presentation of viral peptides, as well as the recycling of pre-existing cell surface MHC-I, resulting in reduction of the level of overall MHC-I cell surface expression. Murine cytomegalovirus (mCMV) was paradigmatic in that it led to the discovery of this immune evasion strategy of CMVs. Members of its m02-m16 gene family code for type-I transmembrane glycoproteins, proven or predicted, most of which carry cargo sorting motifs in their cytoplasmic, C-terminal tail. For the m06 gene product m06 (gp48), the cargo has been identified as being MHC-I, which is linked by m06 to cellular adapter proteins AP-1A and AP-3A through the dileucine motif EPLARLL. Both APs are involved in trans-Golgi network (TGN) cargo sorting and, based on transfection studies, their engagement by the dileucine motif was proposed to be absolutely required to prevent MHC-I exposure at the cell surface. Here, we have tested this prediction in an infection system with the herein newly described recombinant virus mCMV-m06AA, in which the dileucine motif is destroyed by replacing EPLARLL with EPLARAA. This mutation has a phenotype in that the transition of m06-MHC-I complexes from early endosomes (EE) to late endosomes (LE)/lysosomes for degradation is blocked. Consistent with the binding of the MHC-I α-chain to the luminal domain of m06, the m06-mediated disposal of MHC-I did not require the β2m chain of mature MHC-I. Unexpectedly, however, disconnecting MHC-I cargo from AP-1A/3A by the motif mutation in m06 had no notable rescuing impact on overall cell surface MHC-I, though it resulted in some improvement of the presentation of viral antigenic peptides by recently peptide-loaded MHC-I. Thus, the current view on the mechanism by which m06 mediates immune evasion needs to be revised. While the cargo sorting motif is critically involved in the disposal of m06-bound MHC-I in the endosomal/lysosomal pathway at the stage of EE to LE transition, this motif-mediated disposal is not the critical step by which m06 causes immune evasion. We rather propose that engagement of AP-1A/3A by the cargo sorting motif in m06 routes the m06-MHC-I complexes into the endosomal pathway and thereby detracts them from the constitutive cell surface transport.
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This work was supported by the Deutsche Forschungsgemeinschaft (DFG), SFB490, individual project E4 “Antigen presentation under the influence of murine cytomegalovirus immune evasion genes“ (A.F.), SFB1292, individual project TP11 “Viral evasion of innate and adaptive immune cells and inbetweeners” (M.J.R. and N.A.W.L.) and DFG FL 696/2-1 and FL 696/3-1 (S.M. and L.F.). The authors appreciate the skilled technical contributions made by Angélique Renzaho (molecular cloning) and Kirsten Freitag (cytofluorometric analysis), Institute for Virology, and Fatima Boukhallouk (biochemistry), Institute for Medical Microbiology and Hygiene. For expert help and advice with CLSM analysis we thank Dr. D. Strand, ‘Confocal Laser Scanning Microscope Core Facility’ of the Research Center for Immunotherapy (FZI) at the University Medical Center of the Johannes Gutenberg-University Mainz. For helping us by critical discussion of the interpretation of data and by finalizing the cargo sorting sketches, special thanks goes to Pero Lucin, Department of Physiology and Immunology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia.
Conflict of interest
The authors declare that they have no conflict of interest.
Animal experiments were approved according to German federal law §8 Abs. 1 TierSchG by the ethics committee of the Landesuntersuchungsamt Rheinland-Pfalz, permission number 177-07/G 14-1-015.
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This article is part of the Special Issue on Immunological Imprinting during Chronic Viral Infection.
Edited by: Stipan Jonjic.
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Fink, A., Mikuličić, S., Blaum, F. et al. Function of the cargo sorting dileucine motif in a cytomegalovirus immune evasion protein. Med Microbiol Immunol 208, 531–542 (2019). https://doi.org/10.1007/s00430-019-00604-x
- Adapter proteins (AP-1, AP-3)
- Antigen presentation
- Cargo sorting
- Dileucine motif
- Early endosomes (EE)
- Endosomal pathway
- Endosomal recycling compartment (ERC)
- Late endosomes (LE)
- Immune evasion
- MHC class-I
- MHC class-I trafficking
- m06 protein
- Trans-Golgi network (TGN)