Inhibition of HLA-DR assembly, transport, and loading by human cytomegalovirus glycoprotein US3: a novel mechanism for evading major histocompatibility complex class II antigen presentation.

Human cytomegalovirus (HCMV) establishes persistent lifelong infections and replicates slowly. To withstand robust immunity, HCMV utilizes numerous immune evasion strategies. The HCMV gene cassette encoding US2 to US11 encodes four homologous glycoproteins, US2, US3, US6, and US11, that inhibit the major histocompatibility complex class I (MHC-I) antigen presentation pathway, probably inhibiting recognition by CD8(+) T lymphocytes. US2 also inhibits the MHC-II antigen presentation pathway, causing degradation of human leukocyte antigen (HLA)-DR-alpha and -DM-alpha and preventing recognition by CD4(+) T cells. We investigated the effects of seven of the US2 to US11 glycoproteins on the MHC-II pathway. Each of the glycoproteins was expressed by using replication-defective adenovirus vectors. In addition to US2, US3 inhibited recognition of antigen by CD4(+) T cells by a novel mechanism. US3 bound to class II alpha/beta complexes in the endoplasmic reticulum (ER), reducing their association with Ii. Class II molecules moved normally from the ER to the Golgi apparatus in US3-expressing cells but were not sorted efficiently to the class II loading compartment. As a consequence, formation of peptide-loaded class II complexes was reduced. We concluded that US3 and US2 can collaborate to inhibit class II-mediated presentation of endogenous HCMV antigens to CD4(+) T cells, allowing virus-infected cells to resist recognition by CD4(+) T cells.

Human cytomegalovirus US7, US8, US9, and US10 are cytoplasmic glycoproteins, not found at cell surfaces, and US9 does not mediate cell-to-cell spread.

Human cytomegalovirus (HCMV) expresses a large number of membrane proteins with unknown functions. One class of these membrane proteins apparently acts to allow HCMV to escape detection by the immune system. The best characterized of these are the glycoproteins encoded within the US2 to US11 region of the HCMV genome that mediate resistance to CD8(+) and CD4(+) T cells. US2, US3, US6, and US11 block various aspects of the major histocompatibility complex (MHC) class I and class II antigen presentation pathways, functioning in cytoplasmic membranes to cause retention, degradation, or mislocalization of MHC proteins. Distantly homologous genes in this region, US7, US8, US9, and US10, are not well characterized. Here, we report expression of the glycoproteins encoded by US7 to US10 by using replication-defective adenovirus (Ad) vectors. US7, US9, and US10 remained sensitive to endoglycosidase H and were exclusively or largely present in the endoplasmic reticulum (ER) as determined by confocal microscopy. US8 reached the Golgi apparatus and trans-Golgi network and was more quickly degraded. Previous studies suggested that US9 could localize to cell junctions and mediate cell-to-cell spread in ARPE-19 retinal epithelial cells. We found no evidence of US9 at cell junctions of HEC-1A epithelial cells. HCMV recombinants lacking US9 produced smaller plaques on ARPE-19 cell monolayers but also exhibited defects in virus replication compared with wild-type HCMV in these cells. Other HCMV recombinants constructed in a similar fashion that were able to express US9 also produced small plaques and some of these exhibited defects in production of infectious progeny in ARPE-19 cells. Thus, there was no correlation between defects in cell-to-cell spread (plaque size) and loss of expression of US9, and it is possible that US9(-) mutants produce smaller plaques because they produce fewer progeny. Together, our results do not support the hypothesis that US9 plays a direct role in HCMV cell-to-cell spread.