The HCMV genome shows an inherent instability during in vitro
. The spontaneous deletion of 13–15 kb from HCMV laboratory strains correlates with reduced virulence in vivo
and increased susceptibility of HCMV-infected cells to NK cell–mediated cytolysis in vitro
. Downregulation of surface HLA class I strips HCMV-infected cells of a key set of NK cell inhibitory ligands and thus renders them potentially vulnerable to NK cell attack. Should activating ligands become dominant, the balance would shift toward priming of NK cell killing. However, HCMV has responded to this strong selective pressure. Multiple NK cell inhibitory functions have been identified in studies with strain AD169: UL16 downregulates activating NK cell ligands MICB and ULBP1 and ULBP2, UL40 upregulates the inhibitory NK cell ligand HLA-E, and UL18 may itself act as an inhibitory NK cell ligand. However, infection with HCMV clinical isolates consistently bestowed substantially more effective protection from NK cell attack than did the highly passaged laboratory strain AD169, indicating that NK cell–evasion functions had been lost from laboratory strains. Here we have identified UL141 as a powerful NK cell evasion function that has a chief function in the enhanced protective effect noted with HCMV clinical isolates. Whether UL141 is the only gene in the UL/b
′ region to inhibit NK cells cannot be concluded from these studies, as we used only a single NK cell line for our initial screening of the UL133–UL150 genes. Treatment of TB40E-Bart-infected targets, which showed disruptions in the UL141, UL144 and UL145 genes, with the D171 mAb to CD155 only partially blocked NK cell killing, indicating either an inefficient blocking of the CD155-CD226 interaction or an added function for either UL144 or UL145 in NK inhibition.
Regardless of this, UL141 was capable of suppressing killing of 67% of all NK cell clones tested, consistent with targeting of a principal NK cell–regulatory pathway by the gene. We calculated this value using a rigorous definition of inhibition (more than 10% reduction in specific target lysis). Insight into the broad NK cell inhibitory effect of UL141 came from the identification of its cellular target, CD155. CD155 is a ligand for the activating receptor CD226, which has been reported as being present on almost all NK cells20
, and was expressed universally on all the NK cell bulk cultures and clones evaluated here (data not shown). UL141 showed specificity for CD155 in that it did not influence the expression of CD112 (nectin-2), another reported ligand for CD226. Furthermore, the UL141-related gene HCMV UL14 had no detectable effect on either CD112 or CD155 (data not shown). The efficiency with which UL141 inhibits NK cell function indicates the interaction of CD226 with CD155 has a major role in the regulation of NK cell function26
Parallels exist between the action of UL141 and the HCMV-encoded NK cell inhibitor UL16. Both are proteins residing in the endoplasmic reticulum that act to promote intracellular retention of NK cell–activating ligands. HCMV contains many gene families thought to be generated through gene duplication, but the lack of sequence similarity between the UL16 and UL141 proteins suggests they may act through different mechanisms. Perhaps the most important functional distinction is that UL16 targets stress-induced NKG2D ligands, whereas UL141 targets a ligand that is constitutively expressed by a wide range of cell types. The function of CD155 as a constitutive activating ligand is likely to stimulate NK cell recognition if surface HLA class I is downregulated, as noted during HCMV infection as well as during other viral infections and cell transformation. UL141 suppressed NK cell function when expressed in either stably transfected cells or with the use of a replication-deficient adenovirus vector, neither of which downregulates HLA class I expression or upregulates CD155. In autologous assays, constitutive CD155 expression may therefore have been sufficient to stimulate NK cell killing of fibroblasts expressing physiological amounts of HLA class I. Such spontaneous killing may be accentuated by in vitro
cell culture. In tissues, heterophilic interactions between membrane-bound CD155 and nectin-3 contribute to cell-cell and cell-matrix adhesion and restrict these proteins to adhesion junctions. During in vitro
culture, virus infection27
or cell transformation, cell-cell and cell-matrix interactions become disrupted, potentially making CD155 available to interact with CD226 resulting in NK cell activation and cytolysis.
The function of CD226 is dependent on its forming a complex with the adhesion molecule LFA-1 on the effector cell. Activation of NK cell effectors through CD226 thus also indicates involvement in effector-target adhesion through the interaction of LFA-1 on effectors with the adhesion molecule ICAM-1 on the HCMV-infected target; it is well established that ICAM-1 is upregulated during HCMV infection27,28
. Furthermore, CD155 interacts with another nectin family member, CD96 (TACTILE), to stimulate adhesion between NK cells and their targets and thus promote NK cell–mediated cytotoxicity21
. Given those observations, we tested whether UL141 affected adhesion between NK cells and their targets but were unable to detect any changes. UL141 therefore seems to act mainly by impeding signaling through the activating receptor CD226 rather than by radically reducing adhesion between NK cell effectors and their targets.
The function of UL141 may yet prove to transcend its function in impeding NK cell recognition, as CD226 is also expressed on T cells, monocytes, megakaryocytes and B cell subsets20,29-31
. The best characterized functions of CD155 are associated with its roles in cell-cell adhesion and cell motility. CD155 interacts with Tctex-1, a subunit of the dynein motor complex linked to endocytosis and retrograde transport31,32
, is found at the leading edges of migrating cells33
and has been linked to fibroblast migration34
and monocyte transendothelial migration35
. UL141 suppression of CD155 function has the potential to affect multiple cell processes and may thus have even more influence on the biological interaction of HCMV and its host.