Solid lines, staining of vector-only cells; dashed lines, staining of the mK3-expressing cells

Solid lines, staining of vector-only cells; dashed lines, staining of the mK3-expressing cells. and to determine the requirements for substrate recognition by mK3. Our findings indicate that mK3 interacts with TAP1 and -2 via their C-terminal domains and with class I molecules via their N-terminal domains. Furthermore, by BMS 599626 (AC480) orienting the RING-CH domain of mK3 appropriately BMS 599626 (AC480) with respect to class I, mK3 binding to TAP/tapasin, rather than the presence of unique sequences in class I, appears to be the primary determinant of substrate specificity. Many viruses have developed elaborate mechanisms to evade immune detection (11,26,40,46). These mechanisms are typically specific for a given type of virus and are highly host adapted (10). Thus, viruses have clearly evolved under the selective pressure of the host immune system to develop counter strategies to prevent their elimination. Given the importance of CD8+T cells in immune surveillance against many viral infections, it is not surprising that viruses have evolved genes whose products function to block the expression of class I molecules. Recently, a novel family of viral and cellular proteins (termed here the K3 family) has been identified and found to possess E3 ubiquitin (Ub) ligase activity. Several members of this family have been shown to target class I molecules and/or T-cell costimulation molecules for Ub-dependent degradation (3,15,21). E3 Ub ligase activity is conferred to members of the K3 family by a consensus N-terminal sequence encoding a special type of RING (for really interesting BMS 599626 (AC480) new gene) finger motif, known as the RING-CH type of zinc finger (38), characterized by a cysteine residue in the fourth zinc-coordinating position and a histidine residue in the fifth. Alternatively, this motif has been classified as a subclass of the plant homeodomain (PHD)/leukemia-associated protein (LAP) finger (6). Although proteins in this family are structurally and functionally similar, their target specificities and sites of ubiquitination and degradation are distinct. Understanding how disparate members of the K3 family target different proteins at different subcellular sites is an area of intense investigation that promises to define the role of ubiquitination in regulating intracellular transport and endoplasmic reticulum (ER)-associated degradation pathways (9). Belonging to this family, the mK3 protein encoded by gamma-2 herpesvirus 68 (HV68) consists of a conserved RING-CH finger domain in its N terminus followed by two closely spaced transmembrane (TM) segments and a C-terminal tail. Investigation of the topology of mK3 showed that it is a type III ER protein, with both N- and C-terminal domains projecting into the cytoplasm and a short segment between the two TM regions in the lumen of the ER (3). As with other members of the K3 family, mK3’s RING-CH domain is critical for cysteine-dependent E3 Ub ligase activity that mediates the rapid destruction of major histocompatibility complex (MHC) class I proteins (3). However, the mechanisms underlying the mK3-induced MHC class I degradation are different from those in even its closest homologs, the kK3 and kK5 proteins. The kK3 and kK5 proteins encoded by Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8, target surface class I molecules via accelerated endocytosis, resulting in their Ub-mediated degradation in the lysosome (15). In addition, kK5 is known to also target B7.2 and ICAM-1 molecules (36), and the TM regions of both kK5 and its substrates are critical for targeted degradation, presumably by mediating protein-protein interactions (7,33). Recent studies by Stevenson et al. and Yu et al. have shown that, in contrast to kK3 and kK5, mK3 induces the rapid turnover of nascent, ER-resident class I molecules mainly through the ubiquitination-proteasome pathway (36,47). Furthermore, Stevenson et SLC2A1 al. demonstrated that mice infected with an mK3-deficient virus had a reduced number of latently infected spleen cells and an increased number of virus-specific CD8 T cells compared with mice infected with wild-type (wt) virus (37). These findings established the physiologic relevance of mK3 in immune evasion of CD8 T cells. More recently, Lybarger et al. reported that two class I assembly-specific proteins (TAP and tapasin) are required for mK3 stabilization and for mK3-mediated class I downregulation (20). In the presence of mK3, TAP/tapasin-associated class I heavy chains (H chains) were ubiquitinated while mutants of class I incapable of TAP/tapasin interaction were not ubiquitinated and, therefore, not rapidly degraded. The association of mK3 with TAP/tapasin was found to be class I independent, suggesting that mK3 can associate with TAP/tapasin in a substrate-independent manner. Thus, the.