Scanning Mutagenesis of Human Cytomegalovirus Glycoprotein gH/gL

Abstract

The core, conserved function of the herpesvirus gH/gL is to promote gB-mediated membrane fusion during entry, although the mechanism is poorly understood. The human cytomegalovirus (HCMV) gH/gL can exist as either the gH/gL/gO trimer or the gH/gL/UL128/UL130/UL131 (gH/gL/UL128-131) pentamer. One model suggests that gH/gL/gO provides the core fusion role during entry into all cells within the broad tropism of HCMV, whereas gH/gL/UL128-131 acts at an earlier stage, by a distinct receptor-binding mechanism to enhance infection of select cell types, such as epithelial cells, endothelial cells, and monocytes/macrophages. To further study the distinct functions of these complexes, mutants with individual charged cluster-to-alanine (CCTA) mutations of gH and gL were combined to generate a library of 80 mutant gH/gL heterodimers. The majority of the mutant gH/gL complexes were unable to facilitate gB-mediated membrane fusion in transient-expression cell-cell fusion experiments. In contrast, these mutants supported the formation of gH/gL/UL128-131 complexes that could block HCMV infection in receptor interference experiments. These results suggest that receptor interactions with gH/gL/UL128-131 involve surfaces contained on the UL128-131 proteins but not on gH/gL. gH/gL/UL128-131 receptor interference could be blocked with anti-gH antibodies, suggesting that interference is a cell surface phenomenon and that anti-gH antibodies can block gH/gL/UL128-131 in a manner that is distinct from that for gH/gL/gO.

Importance: Interest in the gH/gL complexes of HCMV (especially gH/gL/UL128-131) as vaccine targets has far outpaced our understanding of the mechanism by which they facilitate entry and contribute to broad cellular tropism. For Epstein-Barr virus (EBV), gH/gL and gH/gL/gp42 are both capable of promoting gB fusion for entry into epithelial cells and B cells, respectively. In contrast, HCMV gH/gL/gO appears to be the sole fusion cofactor that promotes gB fusion activity, whereas gH/gL/UL128-131 expands cell tropism through a distinct yet unknown mechanism. This study suggests that the surfaces of HCMV gH/gL are critical for promoting gB fusion but are dispensable for gH/gL/UL128-131 receptor interaction. This underscores the importance of gH/gL/gO in HCMV entry into all cell types and reaffirms the complex as a candidate target for vaccine development. The two functionally distinct forms of gH/gL present in HCMV make for a useful model with which to study the fundamental mechanisms by which herpesvirus gH/gL regulates gB fusion.

FIG 1

CCTA scanning mutagenesis of HCMV gH/gL. Schematic representation of HCMV gH (A) and gL (B) indicating the starting residue of each mutated charged cluster. The designation of gH domains D-I to D-IV was based on homology with the domain designations of EBV gH described by Matsuura et al. (26). Also indicated are the predicted signal peptides (sp), the transmembrane (tm) region of gH, the linear epitope of the neutralizing anti-gH MAb AP86 (33), and cysteine 144 of gL, which was reported by Ciferri et al. to form a disulfide linkage with either gO or UL128 (27). N-term, N terminus; aa, amino acids.

FIG 2

Effect of mutation on cell-cell fusion induced by gB and gH/gL. (A) ARPE-19 cells were transduced with gB and wild-type (WT) or mutant gH/gL in either the presence or absence of MAb 14-4b. Syncytium formation was analyzed by confocal fluorescence microscopy to detect Hoechst 33342-stained nuclei (blue) and wheat germ agglutinin-stained plasma membranes (green). (B) The results of cell-cell fusion assays for all mutant gH and gL combinations. +, gH/gL mutant combinations that resulted in syncytium formation comparable to that for wild-type gH/gL; −, mutant combinations that were impaired for fusion in at least three independent experiments.

FIG 3

Heterodimerization of gH and gL mutants. Human glioblastoma (U373) cells were transduced with the indicated combination of Ad vectors expressing wild-type (A) or mutant (B to D) gH and gL. Cell extracts were immunoprecipitated (IP) with MAb 14-4b or AP86, separated by reducing SDS-PAGE, and identified by immunoblotting with anti-gH or anti-gL antibodies. Mass standards (in kilodaltons) are indicated to the left of each panel.

FIG 4

Cell surface expression of wild-type and mutant gH/gL complexes. ARPE-19 epithelial cells expressing wild-type or mutant gH/gL complexes were analyzed for surface expression by CELISA using anti-gH MAb AP86. Shown are the numbers of relative light units (RLU) for individual gH mutants (A), individual gL mutants (B), and representative combinations (C). Black and white bars, mutants that tested positive and negative for cell-cell fusion, respectively; horizontal dashed lines, the threshold for surface expression, as defined by the surface detection of gH expressed without gL. Experiments were performed in triplicate, and error bars represent standard errors.

FIG 5

Effect of gH/gL mutation on assembly of gH/gL/UL128-131. (A) ARPE-19 epithelial cells expressing wild-type or mutant gH/gL heterodimers alone or together with UL128-131 proteins were inoculated with HCMV, and infection was assessed by immunofluorescence detection of immediate early gene expression. (B) Relative HCMV infection was calculated as the percentage of HCMV-infected cells when cells expressed wild-type or mutant gH/gL plus UL128-131 divided by the percentage of HCMV-infected cells when cells expressed only the wild-type or mutant gH/gL dimer. Black bars, gH/gL mutants that were positive for promoting gB-mediated cell-cell fusion; white bars, mutants that were negative for fusion. Representative mutant gH/gL/UL128-131 complexes were tested in triplicate, and error bars represent standard errors.

FIG 6

Coimmunoprecipitation and surface expression of H.671/gL/UL128-131. (A) ARPE-19 cells expressing either wild-type gH/gL/UL128-131 or H.671/gL/UL128-131 (or H.671/gL/UL128-131 lacking UL130) were immunoprecipitated with the anti-gH antibody AP86. Samples were separated by SDS-PAGE and analyzed by immunoblotting. Mass markers (in kilodaltons) and protein labels are displayed on the left and right, respectively. H.wt and L.wt, wild-type gH and gL, respectively. (B) Surface expression of wild-type gH/gL and H.671/L when expressed with UL128-131, measured by CELISA. Bars display the amount of surface gH as a percentage of the total amount of gH. Horizontal dashed line, the threshold for surface expression, as defined by the surface detection of gH expressed without gL, Error bars represent standard errors from three experiments.

FIG 7

Effect of anti-gH antibodies on receptor interference. (A to C) ARPE-19 cells expressing either the gH/gL/UL128-131 or H.671/gL/UL128-131 complex were inoculated with HCMV, and infection was assessed by immunofluorescence detection of immediate early gene expression. (D to L) Replicate wells were incubated with isotype control IgG (D to F), 14-4b (G to I), or AP86 (J to L) for 24 h prior to HCMV infection. Experiments were performed in triplicate, and the percentage of immediate early gene-positive cells is displayed in the bottom-right corner for each condition.

FIG 8

Model of gH/gL/UL128-131 receptor interference and blocking by anti-gH antibodies. Step 1, gH/gL/UL128-131 expressed from Ad vectors in epithelial cell is transported to cell surfaces; step 2, interactions with putative receptor molecules render cells resistant to infection by wild-type HCMV; step 3, the addition of anti-gH antibodies to the culture medium prevents interaction between gH/gL/UL128-131 and putative receptors; step 4, cells bearing free putative gH/gL/UL1281-131 receptors are susceptible to HCMV infection.

FIG 9

Homology model of HCMV gH. HCMV gH (residues 199 to 724; strain TR) was computationally analyzed using the solved structure of EBV gH as a homolog template in MODELLER. (A) Ribbon representation of the predicted HCMV gH structure, with domain assignments corresponding to those of EBV gH. Cyan, domain II; magenta, domain III; orange, domain IV. (B) Space-filling representation of the HCMV gH structure model with the locations of CCTA mutagenesis highlighted in green.