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. 2013 Sep;87(17):9680-90.
doi: 10.1128/JVI.01167-13. Epub 2013 Jun 26.

Comparative analysis of gO isoforms reveals that strains of human cytomegalovirus differ in the ratio of gH/gL/gO and gH/gL/UL128-131 in the virion envelope

Momei Zhou  1 , Qin Yu, Anya Wechsler, Brent J Ryckman
Affiliations

Comparative analysis of gO isoforms reveals that strains of human cytomegalovirus differ in the ratio of gH/gL/gO and gH/gL/UL128-131 in the virion envelope

Momei Zhou et al. J Virol. 2013 Sep.

Abstract

Herpesvirus glycoprotein complex gH/gL provides a core entry function through interactions with the fusion protein gB and can also influence tropism through receptor interactions. The Epstein-Barr virus gH/gL and gH/gL/gp42 serve both functions for entry into epithelial and B cells, respectively. Human cytomegalovirus (HCMV) gH/gL can be bound by the UL128-131 proteins or gO. The phenotypes of gO and UL128-131 mutants suggest that gO-gH/gL interactions are necessary for the core entry function on all cell types, whereas the binding of UL128-131 to gH/gL likely relates to a distinct receptor-binding function for entry into some specific cell types (e.g., epithelial) but not others (e.g., fibroblasts and neurons). There are at least eight isoforms of gO that differ by 10 to 30% of amino acids, and previous analysis of two HCMV strains suggested that some isoforms of gO function like chaperones, disassociating during assembly to leave unbound gH/gL in the virion envelope, while others remain bound to gH/gL. For the current report, we analyzed the gH/gL complexes present in the virion envelope of several HCMV strains, each of which encodes a distinct gO isoform. Results indicate that all strains of HCMV contain stable gH/gL/gO trimers and gH/gL/UL128-131 pentamers and little, if any, unbound gH/gL. TR, TB40/e, AD169, and PH virions contained vastly more gH/gL/gO than gH/gL/UL128-131, whereas Merlin virions contained mostly gH/gL/UL128-131, despite abundant unbound gO remaining in the infected cells. Suppression of UL128-131 expression during Merlin replication dramatically shifted the ratio toward gH/gL/gO. These data suggest that Merlin gO is less efficient than other gO isoforms at competing with UL128-131 for binding to gH/gL. Thus, gO diversity may influence the pathogenesis of HCMV through effects on the assembly of the core versus tropism gH/gL complexes.

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Figures

Fig 1
Fig 1
Comparison of amino acid sequences of gO encoded by laboratory strains and clinical isolates of HCMV. (A) Phylogenetic relationships of predicted gO amino acid sequences of clinical HCMV isolates described in Rasmussen et al. (14) and the HCMV BAC clones used in the current studies (bold). Phylogeny was estimated using neighbor-joining with the Jones-Taylor-Thornton codon model implemented in MAFFT, version 7. Horizontal bars represent the expected number of amino acid substitutions per site according to the indicated scale. (B) MAFFT alignment of one representative gO sequence from each of the eight groups indicated in panel A is shown (top). Darker shading indicates sequence conservation. The approximate locations of six conserved cysteine (c) residues are indicated. The middle panel shows Kyte-Doolittle hydrophilicity analysis of TRgO. In the bottom panel are sequences of the synthetic peptides used to raise anti-gO antibodies. Asterisks indicate conserved residues, and shaded boxes highlight the position of a proline in each peptide sequence.
Fig 2
Fig 2
Expression of HCMV gO isoforms by replication-defective adenovirus (Ad) vectors. U373 cells were transduced with Ad vectors expressing gO isoforms (HA tagged) derived from HCMV TR (TRgO), Merlin (MEgO), TB40 (TBgO), AD169 (ADgO), and Towne (TNgO). Cells were labeled for 5 min with [35S]methionine-cysteine, and then the label was chased for 0 or 180 min. Proteins were immunoprecipitated with anti-HA antibodies, left untreated (−), or treated with endo H (H) or PNGase F (F), and separated by SDS-PAGE (8%) under reducing conditions. Arrows and arrowheads mark untreated and deglycosylated forms of gO, respectively. Mass markers (kDa) are shown on the left.
Fig 3
Fig 3
Western blot detection of gO isoforms by antipeptide rabbit sera. Primary human fibroblasts were transduced with Ad vectors expressing the indicated gO isoforms (HA tagged). Cell extracts were analyzed by Western blotting using anti-HA antibodies (A) or antibodies raised against peptides derived from gO of HCMV TR (B), Merlin (C), TB40 (D), or AD169 (E). For detection with antipeptide antibodies (B to E), long and short chemiluminescent detections (top and bottom, respectively) were performed in parallel, allowing comparison of signal intensities. Mass markers (kDa) are shown on the left.
Fig 4
Fig 4
Effects of distinct gO isoforms on the intracellular trafficking of a common isoform of gH/gL. U373 cells were transduced with Ad vectors expressing gH/gL (derived from TR) alone or together (+) with Ad vectors expressing gO isoforms derived from TR (TRgO), Merlin (MEgO), TB40 (TBgO), AD169 (ADgO), and Towne (TNgO). Cells were pulse-labeled with [35S]methionine-cysteine, and then the label was chased for 0 or 180 min. Proteins were immunoprecipitated with anti-gH MAb 14-4b, left untreated (-), or treated with endo H (H) or PNGase F (F), and separated by SDS-PAGE under reducing conditions. Arrows mark untreated or endo H-resistant forms of gH and gL. Arrowheads mark deglycosylated forms of gH and gL. Mass markers (kDa) are shown on the left.
Fig 5
Fig 5
Detection of Golgi-associated glycoforms of gO isoforms during coexpression with gH/gL. U373 cells were transduced with Ad vectors expressing gH/gL (derived from HCMV TR) alone (left panel) or together with Ad vectors expressing gO isoforms derived from TR (TRgO), Merlin (MEgO), TB40 (TBgO), AD169 (ADgO), and Towne (TNgO). Cells were labeled for 5 min with [35S]methionine-cysteine, and then the label was chased for 180 min. Proteins were immunoprecipitated with anti-gH MAb 14-4b, denatured with SDS and reducing agents, and then gO isoforms were immunoprecipitated with anti-HA antibodies, left untreated (−) or treated with endo H (H) or PNGase F (F) as indicated, and analyzed by SDS-PAGE under reducing conditions. Diffuse bands corresponding to glycosylated gO and endo H-resistant gO glycoforms [gO (H)] are indicated with vertical lines. Forms of gO deglycosylated by endo H or PNGase F are indicted by the arrow. Forms of gO deglycosylated by PNGase F only are indicated by the arrowhead. Mass markers (kDa) are shown on the left.
Fig 6
Fig 6
Analysis of disulfide bonds in the interactions between gH/gL and gO isoforms. U373 cells were transduced with Ad vectors expressing gH/gL (derived from TR) alone (−) or together with Ad vectors expressing gO isoforms derived from TR (TRgO), Merlin (MEgO), TB40 (TBgO), AD169 (ADgO), and Towne (TNgO). Cell extracts were separated under nonreducing conditions and analyzed by Western blotting using anti-gL (A) or anti-HA (B) antibodies. The predicted migrations of gH/gL/gO trimers, gH/gL dimers, and gO monomers are indicated to the right of both panels. Mass markers (kDa) are shown on the left.
Fig 7
Fig 7
Comparative analysis of gH/gL/gO complexes from different strains of HCMV. Extracts of HFF cells (C) infected with BAC-derived HCMV TR, Merlin, TB40/e, AD169, or PH or virions (V) collected from the culture supernatant were separated by nonreducing or reducing SDS-PAGE, transferred to PVDF membranes, and probed with anti-gO (A) or anti-gL (B) antibodies. For gO blots, anti-TBgO antibodies were used for TR, TB, and PH samples, anti-MEgO antibodies were used for ME samples, and anti-ADgO antibodies were used for AD samples. Arrowheads mark bands corresponding to the disulfide-linked gH/gL/gO trimer, and vertical lines indicate bands that correspond to gO, gL, or the disulfide-linked gH/gL heterodimer. Mass markers (kDa) are shown on the left.
Fig 8
Fig 8
Suppression of UL128-131 expression enhances incorporation of gH/gL/gO into the Merlin virion envelope. The BAC-derived Merlin clone containing Tet operator sequences in the UL130/131 promoter was used to infect HFF cells or HFF cells expressing the Tet repressor protein (HFFtet). Extracellular virions collected from culture supernatants were analyzed by reducing and/or nonreducing Western blotting using anti-gL (A), anti-gO (B), or anti-UL128, anti-UL130, and UL131 (C) antibodies, as indicated. Mass markers (kDa) are shown on the left.
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