Human Cytomegalovirus TR Strain Glycoprotein O Acts as a Chaperone Promoting gH/gL Incorporation into Virions but Is Not Present in Virions

Abstract

Human cytomegalovirus (HCMV) produces the following two gH/gL complexes: gH/gL/gO and gH/gL/ UL128-131. Entry into epithelial and endothelial cells requires gH/gL/UL128-131, and we have provided evidence that gH/gL/UL128-131 binds saturable epithelial cell receptors to mediate entry. HCMV does not require gH/gL/UL128-131 to enter fibroblasts, and laboratory adaptation to fibroblasts results in mutations in the UL128-131 genes, abolishing infection of epithelial and endothelial cells. HCMV gO-null mutants produce very small plaques on fibroblasts yet can spread on endothelial cells. Thus, one prevailing model suggests that gH/gL/gO mediates infection of fibroblasts, while gH/gL/UL128-131 mediates entry into epithelial/endothelial cells. Most biochemical studies of gO have involved the HCMV lab strain AD169, which does not assemble gH/gL/UL128-131 complexes. We examined gO produced by the low-passage clinical HCMV strain TR. Surprisingly, TR gO was not detected in purified extracellular virus particles. In TR-infected cells, gO remained sensitive to endoglycosidase H, suggesting that the protein was not exported from the endoplasmic reticulum (ER). However, TR gO interacted with gH/gL in the ER and promoted export of gH/gL from the ER to the Golgi apparatus. Pulse-chase experiments showed that a fraction of gO remained bound to gH/gL for relatively long periods, but gO eventually dissociated or was degraded and was not found in extracellular virions or secreted from cells. The accompanying report by P. T. Wille et al. (J. Virol., 84:2585-2596, 2010) showed that a TR gO-null mutant failed to incorporate gH/gL into virions and that the mutant was unable to enter fibroblasts and epithelial and endothelial cells. We concluded that gO acts as a molecular chaperone, increasing gH/gL ER export and incorporation into virions. It appears that gO competes with UL128-131 for binding onto gH/gL but is released from gH/gL, so that gH/gL (lacking UL128-131) is incorporated into virions. Thus, our revised model suggests that both gH/gL and gH/gL/UL128-131 are required for entry into epithelial and endothelial cells.

Human Cytomegalovirus Glycoprotein gO Complexes with gH/gL, Promoting Interference with Viral Entry into Human Fibroblasts but Not Entry into Epithelial Cells

“…The loss of UL128-131 appears to be an adaptation to growth in fibroblasts, because viruses without UL128-131 apparently possess a selective advantage when growing in these cells. HCMV also expresses another glycoprotein, gO, that forms distinct complexes with gH/gL that are necessary for HCMV entry into human fibroblasts (14,15,17,19,27,38,40). These observations fit with the notion that all herpesviruses rely on gH/gL complexes for entry into cells (31).…”

“…We had previously shown that with HFFs and MRC-5 cells, it was necessary to use higher doses of Ad vectors expressing gH, gL, UL128, UL130, and UL131 (90 PFU/cell of each vector) in order to match the levels of gH/gL expression to those in ARPE-19 cells (10 PFU/cell) (26). The Ad vector that expresses TRgO(co) was codon optimized and expresses very high levels of gO and was used at 3 PFU/cell based on previous analyses of expression, again matching gO expression to that of gH/gL and UL128-131 (27). Following 24 h of transduction with Ad vectors, cells were infected with HCMV TR (2 PFU/cell) for an additional 48 h, and the cells were then stained for the immediate-early protein IE-86 as described previously (26).…”

“…To test whether the coexpression of gO with gH/gL might increase interference, ARPE-19 epithelial cells, human foreskin fibroblasts (HFFs), and MRC-5 fibroblasts were transduced with Ad vectors expressing gO, gH/gL, gH/gL/gO, gH/gL/UL128-131, gB, or GFP as previously described (26,27). We had previously shown that with HFFs and MRC-5 cells, it was necessary to use higher doses of Ad vectors expressing gH, gL, UL128, UL130, and UL131 (90 PFU/cell of each vector) in order to match the levels of gH/gL expression to those in ARPE-19 cells (10 PFU/cell) (26).…”

“…However, the situation is more complex, and two important sets of observations must be considered. First, gO of clinical strain TR was assembled with gH/gL and promoted endoplasmic reticulum (ER) export, but gO was not incorporated into the envelope of extracellular virions and did not attain resistance to endoglycosidase H (27). In addition, an HCMV TR gO-null mutant produced normal quantities of extracellular virus particles, but these particles exhibited highly reduced (90 to 95%) quantities of gH and gL and were highly defective in entering fibroblasts (40).…”

“…) adenovirus (Ad) vectors that express HCMV TR gH, gL, UL128, UL130, UL131, gB, and gO were described previously (25,27,37). Ad vector stocks were generated by infecting 293 M cells (Microbix) at 0.1 PFU/cell.…”