Insight into the Mechanism of Human Herpesvirus 7 U21-mediated Diversion of Class I MHC Molecules to Lysosomes

Abstract

The U21 open reading frame from human herpesvirus-7 encodes a membrane protein that associates with and redirects class I MHC molecules to the lysosomal compartment. The mechanism by which U21 accomplishes this trafficking excursion is unknown. Here we have examined the contribution of localization, glycosylation, domain structure, and the absence of substrate class I MHC molecules on the ability of U21 to traffic to lysosomes. Our results suggest the existence of a cellular protein necessary for U21-mediated rerouting of class I MHC molecules.Human herpesvirus-6 and -7 (HHV-6 and -7)4 are ␤-herpesviruses most closely related to human cytomegalovirus. HHV-6 and -7 possess almost entirely collinear genomes and share many biological properties; both viruses infect T-lymphocytes, although they can infect other cell types as well, and both can cause high fever and exanthem subitum (roseola), although HHV-6 is the most common cause of this childhood disease (1, 2). Almost all of the population is seropositive for both viruses. Infection with HHV-6 occurs by the age of 2, whereas HHV-7 infection usually occurs slightly later (3). The cells infected with HHV-6 and -7 exhibit cytomegaly and are prone to syncytium formation, features reminiscent of those seen in human cytomegalovirus infection.Like all other herpesviruses, HHV-6 and -7 remain latent or establish persistent infections. To do so, they must avoid detection and elimination by the immune system. Viral immune evasion strategies include restriction of viral gene expression, infection at immunoprivileged sites, obstruction of antiviral cytokine function, and interference with antigen presentation (for review see Ref. 4). Notably, all of the herpesviruses thus far examined employ the latter strategy of interfering with viral antigen presentation to cytotoxic T lymphocytes. Some herpesviral proteins interfere with proteolysis of antigens or peptide transport into the ER (5-7). Others retain class I molecules in the ER, mediate their destruction through ER-associated degradation, enhance the internalization of class I molecules, or divert class I molecules to lysosomes for degradation (8 -17). Judging from the number and molecular diversity of these strategies, the removal of class I MHC-peptide complexes from the cell surface must be evolutionarily advantageous to these viruses as a means of escaping immune detection and thriving in a host.Because so many of the viral immunoevasins affect trafficking or stability of class I MHC molecules, in previous work, we took a biochemical approach to examine the maturation and stability of class I molecules in HHV-7-infected T cells (11). We found that class I MHC stability was indeed decreased in HHV-7-infected T cells and that a 55-kDa viral glycoprotein coimmunoprecipitated with class I MHC molecules. We identified this associated protein as the product of the HHV-7 U21 open reading frame. HHV-7 U21 lacks homology to any other gene product or domain, with the exception of the U21 encoded by HHV-6. U21 is a type I membrane...

Human Herpesvirus 7 U21 Tetramerizes To Associate with Class I Major Histocompatibility Complex Molecules

“…1b). This truncated soluble SBPHA-tagged U21 molecule, U21N SBPHA , retained its functionality when expressed in U373 astrocytoma cells; soluble U21N SBPHA could bind to and reroute class I MHC molecules to lysosomes (5). The U21N SBPHA that did not traffic to lysosomes was secreted, which may suggest the existence of a saturable binding partner for U21; soluble U21 expressed in excess of such a binding partner is secreted.…”

“…We have shown previously that U21 binds to and reroutes class I MHC molecules to lysosomes, but neither the requirements for binding of U21 to class I MHC molecules nor the mechanism of U21-mediated lysosomal trafficking are well understood (3,5). It is unclear, for example, whether the binding of U21 to class I MHC molecules is direct or whether their association might rely upon a third, cellular protein.…”

“…Since our AUC experiments and native gel analyses were performed in solution with purified soluble U21, we next sought to determine whether U21 oligomers could be the operative binding unit in vivo as well. We have often observed a higher ratio of U21 to class I MHC molecules in immunoprecipitations of class I MHC molecules, regardless of whether immunoprecipitations were performed from metabolically labeled lysates of HHV-7-infected cells or U21-expressing cells (3,5). Until now, however, we were hesitant to interpret these data as a reflection of the true in vivo ratio of U21: class I MHC molecules in a complex, since the efficiency of metabolic labeling could have differed between the two proteins, and since the immunoprecipitations contained a mixture of radiolabeled and nonradiolabeled class I molecules.…”

“…We tested this hypothesis by expressing a U21 molecule lacking its cytoplasmic tail in U373 cells. We found, to our surprise, that the cytoplasmic tail of U21 was not necessary for the ability of U21 to divert class I molecules to lysosomes (2); the tailless U21 molecule could divert class I MHC molecules to lysosomes, as could a soluble version of U21, lacking both its transmembrane domain and cytoplasmic tail (2,5). Thus, the lumenal domain of U21 is not only responsible for associating with class I molecules, but it also contains the infor-mation necessary to induce rerouting of class I MHC molecules to the lysosomal compartment.…”

“…The HA11 MAb recognizes the influenza hemagglutinin (HA) epitope (Covance, San Diego, CA). The MCW62 (U21N) polyclonal antibody was raised against the putative ␣1␣2 domain of HHV-7 U21, and the MCW50 (U21C) polyclonal antibody was raised against the cytoplasmic tail of U21 (5). Horseradish peroxidase (HRP)-conjugated goat anti-mouse and goat anti-rabbit secondary MAbs were used for immunoblotting (Bio-Rad, Hercules, CA).…”