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

Abstract

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.

FIG. 1.

Radiolabeling and endo H resistance of US7, US8, US9, and US10. HEC-1A cells were grown until confluent and then were infected with Ad vectors expressing US7, US8, US9, or US10 and, in the case of US7, US9, and US10, simultaneously with Adtettrans. After 20 h, the cells were incubated in medium lacking methionine and cysteine for 1 h and then were radiolabeled with [³⁵S]methionine-cysteine for 1 h. The cells were immediately lysed (Pulse) or were incubated for an additional 1, 2, or 4 h in medium containing excess unlabeled methionine and cysteine (1, 2, and 4 hr Ch., respectively), and then cell extracts were made. US7, US8, US9, and US10 were immunoprecipitated using rabbit polyclonal antibodies and digested (+) or not digested (−) with endo H. A 30-kDa marker protein is indicated.

FIG. 2.

Confocal immunofluorescence microscopy of cells expressing US7, US8, US9, or US10 and stained for ER and cell junction markers. HEC-1A cells plated on plastic microscope slides were infected with Ad vectors expressing US7, US8, US9, or US10 and simultaneously with Adtettrans. After 24 h, the cells were fixed, permeabilized, and stained simultaneously with antibodies specific for US7, US8, US9, or US10 and with antibodies specific for calnexin or protein disulfide isomerase (PDI), markers for the ER, or β-catenin, a marker for cell junctions. In every case, the green fluorescence emanates from the cellular marker and the red fluorescence is from US7 to US10.

FIG. 3.

Confocal immunofluorescence microscopy of cells expressing US7, US8, US9, or US10, stained for Golgi and TGN markers. HEC-1A cells were infected with recombinant Ad vectors as in Fig. 2. The cells were fixed, permeabilized, and stained simultaneously with antibodies specific for US7, US8, US9, or US10 and with antibodies specific for GM130 or p115, markers for the Golgi apparatus, or TGN-46, a marker for the TGN. The green fluorescence emanates from the cellular marker, and the red fluorescence is from US7-US10.

FIG. 4.

Production of infectious virus by ARPE-19 cells infected with HCMV recombinants lacking US9 or other US2 to US11 genes. ARPE-19 cells were plated in plastic dishes, treated with neuraminidase, and then infected with HCMV recombinants: RV134 (intergenic insert US9/US10), RV61 (ΔUS9), RV80 (ΔUS8, ΔUS9), RV35 (ΔUS6-10), RV47 (ΔUS2, ΔUS3), or wild-type HCMV AD169 in duplicate. After 1.5 to 12 days, the cells and cell culture media were harvested together and sonicated briefly, and infectious virus titers were determined using human fibroblasts.