Characterization of the human cytomegalovirus gH/gL/UL128-131 complex that mediates entry into epithelial and endothelial cells

Abstract

The entry of human cytomegalovirus (HCMV) into biologically relevant epithelial and endothelial cells involves endocytosis followed by low-pH-dependent fusion. This entry pathway is facilitated by the HCMV UL128, UL130, and UL131 proteins, which form one or more complexes with the virion envelope glycoprotein gH/gL. gH/gL/UL128-131 complexes appear to be distinct from the gH/gL/gO complex, which likely facilitates entry into fibroblasts. In order to better understand the assembly and protein-protein interactions of gH/gL/UL128-131 complexes, we generated HCMV mutants lacking UL128-131 proteins and nonreplicating adenovirus vectors expressing gH, gL, UL128, UL130, and UL131. Our results demonstrate that UL128, UL130, and UL131 can each independently assemble onto gH/gL scaffolds. However, the binding of individual UL128-131 proteins onto gH/gL can significantly affect the binding of other proteins; for example, UL128 increased the binding of both UL130 and UL131 to gH/gL. Direct interactions between gH/UL130, UL130/UL131, gL/UL128, and UL128/UL130 were also observed. The export of gH/gL complexes from the endoplasmic reticulum (ER) to the Golgi apparatus and cell surface was dramatically increased when all of UL128, UL130, and UL131 were coexpressed with gH/gL (with or without gO expression). Incorporation of gH/gL complexes into the virion envelope requires transport beyond the ER. Thus, we concluded that UL128, UL130, and UL131 must all bind simultaneously onto gH/gL for the production of complexes that can function in entry into epithelial and endothelial cells.

FIG. 1.

Expression of gH, gL, UL128, UL130, and UL131 proteins by wild-type and mutant HCMV. Human fibroblasts were infected with HCMV mutant TRΔ4 (lacking UL128, UL130, and UL131) or TRΔ131 (lacking UL131 only) or wild-type (wt) TR at 0.1 PFU/cell; cells were harvested after 10 to 16 days and sonicated; larger cellular debris was removed by centrifugation at 6,000 × g; and then virus particles and microsomes were pelleted through a 20% sorbitol cushion. These preparations were enriched for viral proteins but also contained approximately 70% of the cellular calnexin, an ER protein. Proteins were analyzed by immunoblotting with anti-major capsid protein (MCP) MAb 28-4 or with rabbit antipeptide antibodies directed against gH, gL, UL128, UL130, or UL131.

FIG. 2.

Characterization of gH/gL/UL128/UL130/UL131 complexes in cells infected with wild-type and mutant HCMV. Human fibroblasts were infected with mutant TRΔ4 or TRΔ131, or wild-type (wt) TR. Virion/microsome preparations (as described for Fig. 1) were solubilized in 0.5% NP-40 lysis buffer, and gH/gL complexes were immunoprecipitated (IP) using anti-gH MAb 14-4b followed by immunoblotting with rabbit antipeptide antibodies specific for gH, gL, UL130, or UL131 or with anti-UL128 MAb 4B10. The bracket and arrow indicate slower- and faster-migrating forms of UL130, respectively.

FIG. 3.

endo H treatment of gH and UL130 in extracts of mutant and wild-type HCMV-infected cells. Human fibroblasts were infected with mutant TRΔ4 or TRΔ131, or wild-type (wt) TR. gH complexes were immunoprecipitated (IP) from extracts of virion/microsome preparations (as described for Fig. 1) using anti-gH MAb 14-4b. Precipitated proteins were treated with endo H (+) or incubated in buffer alone (−) and then analyzed by immunoblotting using antipeptide antibodies directed against gH (A) or UL130 (B). Arrowheads indicate endo H-resistant forms of gH and UL130, arrows indicate endo H-sensitive forms, and the asterisk marks a faster-migrating form UL130 precipitated with gH complexes from cells infected with TRΔ131 not treated with endo H.

FIG. 4.

Expression of gH, gL, UL128, UL130, and UL131 by nonreplicating Ad vectors. U373 cells were infected with Ad vectors and labeled with [³⁵S]methionine-cysteine. Cell extracts were immunoprecipitated with rabbit antipeptide antibodies specific for gH, gL, UL128, UL130, or UL131 and analyzed by SDS-PAGE under reducing conditions. Immunoprecipitation was performed in the presence (+) or absence (−) of the synthetic peptide used to generate each antibody.

FIG. 5.

gH/gL/UL128/UL130/UL131 complexes formed by Ad vector expression. U373 cells were infected with Ad vectors expressing different combinations of gH, gL, UL128, UL130, and UL131. Extracts made using 0.5% NP-40 lysis buffer were analyzed directly by immunoblotting (A, C, E, and G) or by immunoprecipitation (IP) with anti-gH MAb 14-4b followed by immunoblotting with anti-gL, -UL130, or -UL131 rabbit antipeptide antibodies or anti-UL128 MAb 4B10 (B, D, F, and H). The numbers below the panels represent the relative signal intensities of gL, UL128, UL130, and UL131 detected in gH/gL complexes normalized to the total amounts of these proteins in expressed in cells (measured by direct immunoblotting in panels A, C, E, and G).

FIG. 6.

Coexpression of all of UL128, UL130, and UL131 increases ER-to-Golgi apparatus export of gH/gL complexes. U373 cells were infected with Ad vectors expressing different combinations of gH/gL and UL128-131 proteins. Cells were pulse-labeled with [³⁵S]methionine-cysteine for 15 min (P), and radioactivity was chased for 60 or 180 min. gH was immunoprecipitated with MAb 14-4b, treated with endo H, and separated by SDS-PAGE under reducing conditions. Arrows and arrowheads indicate endo H-resistant and -sensitive forms of gH, respectively.

FIG. 7.

Construction and expression of sgH. (A) Schematic representation of the HCMV gH protein. Indicated are transmembrane (TM) and cytoplasmic (CT) coding (amino acids 719 to 743) sequences. In sgH, both the TM and CT domains were replaced with a 27-amino-acid sequence derived from HSV type 1 gD that is recognized by MAb DL6. wt, wild type. (B) sgH was expressed in U373 cells and radiolabeled with [³⁵S]methionine-cysteine, and cell extract was analyzed by immunoprecipitation with rabbit (Rbt) anti-gH peptide sera with and without the gH peptide anti-gH MAb 14-4b or MAb DL6.

FIG. 8.

Secretion of sgH coexpressed with gL, UL128, UL130, and UL131 proteins. U373 cells were infected with Ad vectors expressing sgH together with different combinations of gL, UL128, UL130, and UL131. Cells were pulse-labeled (P) with [³⁵S]methionine-cysteine for 15 min, and radioactivity was chased for 30, 60, or 180 min. sgH was immunoprecipitated from cell extracts or culture supernatants by using MAb DL6 and analyzed by SDS-PAGE under reducing conditions. The amount of sgH detected in culture supernatants under each condition was determined and normalized to the value obtained when all five proteins were expressed (panel B).

FIG. 9.

Coimmunoprecipitation analysis of interactions between pairs of gH, gL, UL128, UL130, and UL131 proteins. Pairs of HCMV proteins (indicated in the text above the panels) were expressed from Ad vectors in U373 cells. The cells were labeled with [³⁵S]methionine-cysteine, cell extracts were made using 0.5% NP-40 lysis buffer, and then HCMV proteins were immunoprecipitated with appropriate antibodies (as indicated at the tops of the panels) or with an irrelevant antibody (anti-UL77) followed by SDS-PAGE under reducing or nonreducing conditions.

FIG. 10.

Model for the protein-protein interactions that make up the gH/gL/UL128/UL130/UL131 complex. The HCMV gH protein is predicted to be inserted in the virion envelope and interacts with gL through disulfide linkage(s) (indicated by straight dark lines). Interactions between UL130 and UL131 also involve a disulfide bond(s). UL128 interacts with both UL130 and gL through noncovalent interactions (indicated by cross-hatching), and a surface created by both gH and gL is required for UL131 binding (indicated by dark dots).