Regulation of the cell surface expression of classical and non-classical MHC proteins by the human cytomegalovirus UL40 and rhesus cytomegalovirus rh67 proteins

The signal sequences of the human cytomegalovirus (CMV) UL40 protein, and its rhesus CMV counterpart, Rh67, contain a peptide (VMAPRT\[L/V\]\[F/I/L/V\]L, VL9) that can be presented by Major Histocompatibility Complex (MHC) antigen E. The CMV VL9 peptides replace VL9 peptides derived from classical MHC (Ia) signal sequences, which are lost when CMV disrupts MHC Ia expression, as well as antigen processing and presentation. This allows infected cells to maintain surface expression of MHC-E and escape killing by NK cells expressing the inhibitory NKG2A/CD94 receptor. We demonstrate that processing of the Rh67 VL9 peptide mirrors that of UL40, despite the lack of sequence conservation elsewhere in the two proteins. As previously shown for UL40, up-regulation of MHC-E expression by Rh67 only requires its signal sequence, with sequences upstream of VL9 critical for conferring independence from TAP, the Transporter Associated with Antigen Processing. Additionally, we show that processing of both VL9 peptides depends on cleavage of the signal sequences by the host protease Signal Peptide Peptidase. Notably, our results also reveal that the mature UL40 and Rh67 proteins contribute to CMV immune evasion by down-regulating surface expression of MHC Ia. Unexpectedly, while the Rh67 VL9 peptide is resistant to the effects of the Rh67 protein, the UL40 protein is able to counteract up-regulation of MHC-E expression mediated by its own VL9 peptide. This suggests differences in the mechanisms by which the two VL9 peptides up-regulate MHC-E, which may have implications for translating a RhCMV-vectored SIV vaccine to HIV-1 using HCMV as a vector. IMPORTANCE Cell surface MHC-E expression, which requires a peptide (VMAPRT\[L/V\]\[F/I/L/V\]L, “VL9”) from the signal sequences of other MHC class I proteins, prevents cells from being killed by CD94/NKG2A-expressing Natural Killer cells. In cells infected with human CMV, the endogenous VL9 peptide is replaced by one from the signal sequence of the HCMV UL40 protein. We show that processing of the VL9 peptide of Rh67, the rhesus CMV equivalent of UL40, mirrors that of UL40, despite a lack of sequence homology between the two proteins. Of note, we also show that the mature UL40 and Rh67 proteins, which have no previously described function, contribute to CMV immune evasion by reducing classical MHC class I surface expression. Importantly, the mature UL40 protein, but not the mature Rh67 protein, can decrease the up-regulation of MHC-E mediated by its signal sequence VL9 peptide, which may have implications for HCMV as a vaccine vector. ### Competing Interest Statement The authors have declared no competing interest.