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Herpesviruses are known to influence expression of major histocompatibility complex (MHC) class I molecules on the surface of infected cells using a variety of mechanisms. Downregulation of MHC class I expression prohibits detection and elimination of infected cells by cytotoxic T lymphocytes. To investigate the effect of rat cyomegalovirus (RCMV) infection on MHC class I expression, we infected immortalized and primary rat fibroblasts with RCMV and monitored surface expression of MHC class I molecules at various time-points postinfection. These experiments revealed a downregulation of MHC class I surface expression by RCMV, a phenomenon that has also been reported for human and murine CMV. However, in contrast to the other cytomegaloviruses, RCMV causes only a temporal downregulation of MHC class I, with a maximal decrease at 12 h postinfection. Unlike murine and human CMV, RCMV does not induce proteolytic degradation of MHC class I molecules. In RCMV-infected cells, the MHC class I molecules are stable, but their exit from the ER is delayed.

In the present study, the human TEB4 is identified as a novel ER (endoplasmic reticulum)-resident ubiquitin ligase. TEB4 has homologues in many species and has a number of remarkable properties. TEB4 contains a conserved RING (really interesting new gene) finger and 13 predicted transmembrane domains. The RING finger of TEB4 and its homologues is situated at the N-terminus and has the unconventional C4HC3 configuration. The N-terminus of TEB4 is located in the cytosol. We show that the isolated TEB4 RING domain catalyses ubiquitin ligation in vitro in a reaction that is ubiquitin Lys48-specific and involves UBC7 (ubiquitin-conjugating enzyme 7). These properties are reminiscent of E3 enzymes, which are involved in ER-associated protein degradation. TEB4 is an ER degradation substrate itself, promoting its own degradation in a RING finger- and proteasome-dependent manner.

Human cytomegalovirus encodes two glycoproteins, US2 and US11, which cause rapid degradation of MHC class I molecules, thus preventing recognition of virus-infected cells by the immune system. This degradation process involves retrograde transport or ‘dislocation’ of MHC class I molecules from the endoplasmic reticulum (ER) to the cytosol, where they are deglycosylated by an N-glycanase and degraded by the proteasome. At present it is unknown whether ubiquitination is required for US2- and US11-mediated dislocation and degradation of MHC class I molecules. Here, we show that in E36ts20 hamster cells, which contain a temperature-sensitive mutation in the E1 ubiquitin-activating enzyme, US11-mediated degradation of MHC class I molecules is strongly impaired at the non-permissive temperature, indicating the necessity for ubiquitination in this process. We next addressed the question of whether ubiquitination is a condition for the retrograde movement of MHC class I molecules from the ER to the cytosol, or whether ubiquitination is merely required for recognition of dislocated MHC class I molecules by the proteasome. In the absence of a functional ubiquitin system, complexes of US11 and MHC class I molecules accumulate in the ER. In this state the membrane topology of MHC class I molecules does not significantly change, as judged from proteinase K digestions. Thus the results indicate that a functional ubiquitin system is essential for dislocation of MHC class I molecules from the ER to the cytosol.