Optimal replication of human cytomegalovirus correlates with endocytosis of glycoprotein gpUL132

Abstract

Envelopment of a herpesvirus particle is a complex process of which much is still to be learned. We previously identified the glycoprotein gpUL132 of human cytomegalovirus (HCMV) as an envelope component of the virion. In its carboxy-terminal portion, gpUL132 contains at least four motifs for sorting of transmembrane proteins to endosomes; among them are one dileucine-based signal and three tyrosine-based signals of the YXXØ and NPXY (where X stands for any amino acid, and Ø stands for any bulky hydrophobic amino acid) types. To investigate the role of each of these trafficking signals in intracellular localization and viral replication, we constructed a panel of expression plasmids and recombinant viruses in which the signals were rendered nonfunctional by mutagenesis. In transfected cells wild-type gpUL132 was mainly associated with the trans-Golgi network. Consecutive mutation of the trafficking signals resulted in increasing fractions of the protein localized at the cell surface, with gpUL132 mutated in all four trafficking motifs predominantly associated with the plasma membrane. Concomitant with increased surface expression, endocytosis of mutant gpUL132 was reduced, with a gpUL132 expressing all four motifs in mutated form being almost completely impaired in endocytosis. The replication of recombinant viruses harboring mutations in single trafficking motifs was comparable to replication of wild-type virus. In contrast, viruses containing mutations in three or four of the trafficking signals showed pronounced deficits in replication with a reduction of approximately 100-fold. Moreover, recombinant viruses expressing gpUL132 with three or four trafficking motifs mutated failed to incorporate the mutant protein into the virus particle. These results demonstrate a role of endocytosis of an HCMV envelope glycoprotein for incorporation into the virion and optimal virus replication.

FIG. 1.

Hydrophilicity profile, primary amino acid sequence, and summary of gpUL132 mutants that were used in this study. (A) Computer prediction of the hydrophilicity (HPhilic) of the gpUL132 polypeptide. The potential signal and membrane anchor sequences are indicated by boxes. HPhobic, hydrophobicity. (B) Primary amino acid sequence of gpUL132. The sorting signals which are relevant for this work are underlined and printed in bold. The potential signal and membrane anchor sequences are indicated by a shaded background. An arrow indicates the insertion site for the HA tag. (C) Cartoon of plasmids and recombinant viruses used in this study. The complete UL132 ORF is represented as an open bar. The individual sorting motifs and the respective mutant motifs are indicated and numbered from 1 to 4. The amino acid sequence YVSYDEL, which theoretically harbors the two sorting motifs YVSV₂₃₁ and YDEL₂₃₅, was treated as a single motif (motif 3). The positions of the amino-terminal HA tag and the carboxy-terminal myc tag (gray box) are indicated.

FIG. 2.

Intracellular localization of wt gpUL132 and gpUL132 mutant proteins. HeLa cells were transfected with the indicated plasmids, and protein localization was assayed 48 h later by indirect immunofluorescence in permeabilized cells. gpUL132 was detected using as primary antibody an anti-myc antibody, and the TGN was detected by a polyclonal sheep serum against TGN46. Appropriate secondary antibodies were used to visualize binding of the primary antibody. The appearance of yellow in the merged pictures indicates colocalization of signals.

FIG. 3.

Flow cytometry of wt gpUL132 and gpUL132 mutant protein after transient expression. HeLa cells were transfected with the indicated plasmids and subjected to flow cytometry analysis 48 h later. (A) Intact HeLa cells were stained with PE-conjugated anti-HA antibody and analyzed for cell surface expression of gpUL132 protein (top). Cells were analyzed in samples that were permeabilized before the addition of the anti-HA antibody (bottom). The percentage of cells staining for gpUL132 is indicated in the upper-right quadrant of each graph. SSC, side scatter. (B) Percentage of total gpUL132 detected on the surface of transfected cells. Values are the mean and range of three independent experiments.

FIG. 4.

Endocytosis of wt gpUL132 and gpUL132 mutant proteins in transfected cells. (A to C) HeLa cells were transfected with the indicated plasmids and 48 h later incubated with anti-HA antibody at 4°C. To allow internalization of the gpUL132-IgG complex, cells were shifted to 37°C for the indicated times. Cells were fixed with paraformaldehyde, permeabilized, and incubated with FITC-conjugated anti-mouse antibody. (D and E) HeLa cells were cotransfected with gpUL132- and GGA-1-GFP-expressing plasmids as indicated. gpUL132 was labeled at the cell surface at 4°C with an anti-HA antibody and allowed to internalize for 20 min at 37°C. Following fixation and permeabilization of the cells, gpUL132 was detected using a Cy3-conjugated anti-HA antibody and GGA-1 by GFP fluorescence.

FIG. 5.

Replication of UL132 mutant recombinant viruses. Fibroblasts were seeded in six-well dishes and infected with the indicated recombinant viruses. At the indicated days postinfection, supernatants from the infected cultures were harvested, and infectious virus was titrated using indirect immunofluorescence with an antibody directed against IE1. IU, infectious units. The experiment was repeated three times, and a representative result is shown.

FIG. 6.

Flow cytometry of gpUL132 mutant proteins in infected cells. Fibroblasts were infected with the indicated recombinant viruses and subjected to flow cytometry 92 h later. Cells were analyzed for surface-exposed and total gpUL132, and the fraction of surface-exposed gpUL132 is shown. Data are the mean and range of five independent experiments.

FIG. 7.

Endocytosis of wt gpUL132 and gpUL132 mutant proteins in infected cells. Fibroblasts were infected with the indicated recombinant viruses for 120 h. Cells were fixed with paraformaldehyde and permeabilized before addition of the antibodies (perm). The intracellular localization of gpUL132 was detected with an anti-HA antibody. The TGN was stained using a polyclonal anti-TGN46 sheep serum. To analyze surface expression of gpUL132, cells were incubated with an anti-HA antibody at 4°C for 60 min, fixed with paraformaldehyde, and permeabilized (0 min). Following incubation with a sheep polyclonal anti-TGN46 polyclonal antibody, binding of the first antibodies was developed using appropriate secondary antibodies. Following incubation of cells at 4°C for 60 min with anti-HA antibody, gpUL132-antibody complexes were allowed to internalize for 20 min at 37°C (20 min). Thereafter, gpUL132 and TGN were stained as above.

FIG. 8.

Immunoblot analysis of recombinant viruses. Lysates of gradient-purified extracellular virus particles from the indicated recombinant virus strains were used for immunoblot analysis using either an anti-HA-specific antibody (A) or an anti-UL132 rabbit serum (B) for detection of gpUL132. The two viral forms of gpUL132 (50) are indicated by arrows. Following detection of gpUL132, the blots were stripped and developed with an antibody specific for the major capsid protein of HCMV (anti-MCP).

FIG. 9.

Trafficking and virion incorporation of a TrkB/gpUL132 chimeric protein. (A) Cartoon illustrating the domains of the chimeric proteins. (B) Antibody internalization endocytosis assay of chimeric forms of TrkB molecules. HF cells, grown on 13-mm coverslips, were electroporated with the respective chimeric proteins as described in the text and infected with HCMV. On day 5 postinfection, cells were cooled to 4°C and incubated with an anti-myc MAb for 2 h. The cell cultures were then washed several times with warm medium, and individual coverslips were harvested at the indicated time points. After fixation in paraformaldehyde, the coverslips were reacted with an anti-IE1 MAb to identify infected cells and then developed with FITC-conjugated anti-mouse IgG1 (green) to detect internalized anti-myc antibodies and with TRITC-conjugated anti-mouse IgG2b (red) to detect anti-IE1 antibodies. (C) Virion incorporation of the TrkB/gpUL132 chimeric proteins. HF cells were electroporated with expression plasmids encoding the respective chimeric protein and then infected with HCMV 24 h later. Cells were harvested, and supernatant virus was collected by centrifugation. Viral proteins were solubilized and analyzed by immunoblotting using an anti-myc MAb to detect the chimeric protein and an anti-pp65 MAb to detect viral pp65.