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.

FIG. 1.

Immunoblot analysis of viral glycoproteins in HCMV TR- and AD169-infected cells and extracellular virus particles. Human fibroblasts (NHDF) were infected with 2 IU per cell of TR or AD169 for 7 days. Extracellular viral particles were purified from clarified culture supernatants by centrifugation though 20% sorbitol cushions (V). Virus particles and infected cells (C) were extracted with 1% Triton X-100 and analyzed by Western blotting using mouse MAb specific for gB (27-156) or calnexin or rabbit polyclonal antibodies specific for gH, gL, TR gO (TR gO254 produced against gO residues 254 to 271), AD169 gO, or UL130. Note that the TR gO254 antibodies do not recognize AD169 gO, and AD169 gO antibodies do not recognize TR gO; thus, separate blots are shown. A longer exposure of the TR gO blot was provided to show the very small quantity of TR gO in virus particles. Numbers below each lane indicate the relative abundance of each protein. Shown are representative results from three independent experiments. Molecular weight markers are shown along the left side of each panel.

FIG. 2.

Immunoblot analysis of glycoproteins expressed by HCMV clinical and laboratory strains. Human fibroblasts (NHDF) were infected for 7 days with 2 IU per cell of the following various HCMV strains: TR, AD169, Merlin (ME), FIX (FX), Toledo (TL), or Towne (TN). Extracellular viral particles purified from culture supernatants by centrifugation through 20% sorbitol (V) and infected cells (C) were analyzed by immunoblotting as shown in Fig. 1. (A) Blots were probed with either of the two anti-peptide antibodies, TR gO254 antibodies or a second TR gO antipeptide serum, denoted TR gO110 (produced against gO residues 110 to 127). (B) Replicate blots of virions and cells derived from TR-, Merlin-, Fix-, Toledo-, or Towne-infected cells were probed with anti-gH, anti-gL, anti-TR gO254, anti-TR gO110, or anti-AD169 gO antibodies. Each lane in each panel was exposed from the same membrane (i.e., the same experiment), although some lanes were removed digitally. Molecular weight markers are shown along the left side of each panel.

FIG. 3.

Pulse-chase analysis of the maturation of N-linked oligosaccharides associated with gO and gH in TR-infected cells versus AD169-infected cells. Human fibroblasts (NHDF) were infected for 4 days with 3 IU/cell of TR (A and C) or AD169 (B and D). Cells were labeled for 15 min with [³⁵S]methionine/cysteine and then label chased for 45, 120, or 240 min. Cell extracts were analyzed by immunoprecipitation (IP) with anti-TR gO254 antibodies (A), anti-AD169 gO antibodies (B), or anti-gH MAb 14-4b (C and D). Immunoprecipitated proteins were either left untreated or treated with endo H or PNGase F. Arrows indicate bands corresponding to gO, gH, and gL, and faster-migrating forms of these proteins after oligosaccharides were removed by endo H or PNGase F (H, F). Molecular weight markers are shown along the left side of each panel.

FIG. 4.

Immunoblot analysis of the maturation of HCMV glycoproteins in virions and cells. TR- and AD169-infected fibroblast extracts and extracts of extracellular particles were prepared using 1% Triton X-100. Extracts were either left untreated or treated with endo H or PNGase F, and then proteins were subjected to gel electrophoresis and immunoblot analysis using rabbit polyclonal antibodies specific for gH, gL, or gO. (A) TR gO254 were used to detect gO; (B) AD169 gO-specific antibodies were used. Molecular weight markers are shown along the left side of each panel.

FIG. 5.

Expression of TR gO by nonreplicating Ad vectors. U373 cells were infected with various Ad vectors for 16 to 24 h and labeled for 2 h with [³⁵S]methionine/cysteine, and then expressed HCMV proteins were immunoprecipitated and analyzed by gel electrophoresis. (A) Cells were infected with 50 PFU/cell of an Ad vector expressing TR gO or with Ad vectors expressing gH, gL, and gO (50 PFU/cell each). gO was immunoprecipitated using rabbit TR gO254 antibodies in the presence or absence of gO 254-271 peptide as a competitive inhibitor. (B) Cells were infected with 50 PFU/cell of either Ad TR gO or an Ad vector containing a codon-optimized TR gO gene, AdTRgO(co). gO was immunoprecipitated using anti-TR gO254 antibodies with or without peptide. The two panels represent samples analyzed on the same gel. (C) Cells were infected with Ad vectors expressing gH and gL or gH, gL, and AdTRgO(co). gH was precipitated using anti-gH MAb 14-4b, gL with rabbit anti-gL antibodies, or gO precipitated with rabbit anti-TR gO254 antibodies. Molecular weight markers are shown along the left side of each panel.

FIG. 6.

Maturation of N-linked oligosaccharides associated with gH and gO expressed by Ad vectors. U373 cells were infected with the indicated combinations of Ad vectors expressing gH, gL, TR gO, UL128, UL130, and UL131. Cells were labeled for 5 min with [³⁵S]methionine/cysteine, and then the label was chased for 0, 60, or 180 min. (A) gH was immunoprecipitated with anti-gH MAb 14-4b; (B) gO was immunoprecipitated using TR gO254 antibodies. Immunoprecipitated proteins were either left untreated or treated with endo H. Arrows indicate proteins corresponding to gH, gO, or endo H-sensitive forms of these proteins (H). Molecular weight markers are shown along the left side of each panel.

FIG. 7.

Assembly of gH/gL/gO complexes expressed by Ad vectors. U373 cells were infected with Ad vectors expressing gH, gL, and TR gO [AdTRgO(co)] for 18 to 24 h and labeled for 5 min with [³⁵S]methionine/cysteine, and then the label was chased for 0, 15, 45, 60, or 75 min. Cell extracts were immunoprecipitated using anti-gH MAb 14-4b, and proteins were either left untreated or treated with PNGase F. Arrows indicate bands corresponding to gH, gO, gL, or PNGase F-sensitive forms of these proteins (F). Molecular weight markers are shown on the left.

FIG. 8.

Stability of gH/gL/gO complexes expressed by Ad vectors. U373 cells were infected with Ad vectors expressing gH, gL, and TR gO [AdTRgO(co)] for 18 to 24 h and labeled with [³⁵S]methionine/cysteine for 10 min, and the label was chased for 1, 3, 6, or 9 h. Cell extracts were immunoprecipitated with anti-gH MAb 14-4b, and then proteins were either left untreated or were treated with endo H or PNGase F. Arrows indicate bands corresponding to gH, gO, gL, or endo H- or PNGase F-sensitive forms of these proteins (H, F). Molecular weight markers are shown on the left.

FIG. 9.

Pulse-chase analysis of HCMV glycoproteins expressed in cells and incorporated into extracellular virus particles. Human fibroblasts (NHDF) were infected with 3 IU/cell of HCMV TR for 4 days. The cells were labeled for 1 h with [³⁵S]methionine/cysteine (500 uCi/ml), and then the label was chased for 3 or 24 h. The following three fractions were prepared: cells (C), virus particles derived from cell culture supernatants by centrifugation through 20% sorbitol cushions (V), and the cell culture supernatants after viruses were pelleted (S). Each fraction was extracted with 1% Triton X-100, and then gH or gO was immunoprecipitated using anti-gH MAb 14-4b (A) or anti-TR gO254 antibodies (B). Immunoprecipitated proteins were left untreated or treated with endo H. Arrows indicate bands corresponding to gH, gO, gL, or endo H-sensitive forms of these proteins (H). Arrowheads mark unidentified proteins that coprecipitated with gH. Molecular weight markers are shown along the left side of each panel.