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. 1996 Nov 1;225(1):111-25.
doi: 10.1006/viro.1996.0579.

Identification of the human cytomegalovirus G protein-coupled receptor homologue encoded by UL33 in infected cells and enveloped virus particles

B J Margulies  1 , H Browne, W Gibson
Affiliations

Identification of the human cytomegalovirus G protein-coupled receptor homologue encoded by UL33 in infected cells and enveloped virus particles

B J Margulies et al. Virology. .

Abstract

Human cytomegalovirus (HCMV), strain AD169, contains four genes (US27, US28, UL33, and UL78) that encode putative homologues of cellular G protein-coupled receptors (GCRs). GCRs transduce extracellular signals to alter intracellular processes, and there is evidence that HCMV may elicit such changes at early times following infection. The US27, US28, and UL33 genes are transcribed during infection, and the US28 gene product has been found to be a functional receptor for the beta-chemokine class of immune modulators. The US27, UL33, and UL78 gene products have not been described and we have concentrated on identifying the UL33 protein because it is the most highly conserved of the GCR homologues among the human beta and gamma herpesviruses. We report here cloning UL33 into a recombinant baculovirus (rBV) and expressing it in insect cells; constructing a mutant HCMV with a disrupted UL33 gene; and identifying the UL33 protein in HCMV-infected cells and virus particles. Our results demonstrate that the UL33 protein (i) is expressed as a approximately 36-kDa, heat-aggregatable protein in rBV-infected cells, (ii) is modified heterogeneously by asparagine-linked glycosylation and expressed as a > or = 58-kDa glycoprotein that is present in the region of the cytoplasmic inclusions in HCMV-infected fibroblasts, (iii) is present in virions and two other enveloped virus particles, and (iv) is not essential for growth of HCMV in human foreskin fibroblast cultures.

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Figures

FIG. 1.
FIG. 1.
Expression of recombinant pUL33. Western immunoassay shows proteins produced by Sf9 cells that were infected with wild-type baculovirus (BV; lanes 1, 4, 7, and 10), rBV-33 (lanes 2, 5, 8, and 11), or rBV-Tag33 (lanes 3, 6, 9, and 12) and harvested 3 days after infection. Whole cell lysates were prepared in solubilizing buffer and not heated (lanes 1–3, 7–9) or heated in a boiling water bath (lanes 4–6, 10–12) prior to SDS–PAGE. Western immunoassays were done with the anti-33 antiserum or anti-Tag antibody, as indicated. Circles are shown next to the Tag33 protein, detected by both anti-33 and anti-Tag. Arrow indicates GCR multimers. Molecular weight (in kDa) was estimated from molecular weight markers included during SDS–PAGE (Mark 12; Novex, San Diego, CA) that were visualized by staining that portion of the Immobilon-P with Coomassie brilliant blue (CBB) immediately following electrotransfer.
FIG. 2.
FIG. 2.
Recombinant pUL33 expression in rBV-infected Sf9 cells shown by indirect immunofluorescence. Bound primary antibodies, anti-33 or anti-Tag, were detected by indirect immunofluorescence after incubation with either GAR-LRSC (for rabbit anti-33) or GAM-FITC (for mouse anti-Tag) as secondary reagents. See text for abbreviations. (A) rBV-33-infected cells assayed with rabbit anti-33, as primary, and GAR-LRSC, as secondary, antibodies. (B) BV-infected cells assayed with rabbit anti-33, as primary, and GAR-LRSC, as secondary, antibodies. (C and D) rBV-Tag33-infected cells assayed with both rabbit anti-33 and mouse anti-Tag, as primary reagents and with both GAR-LRSC and GAM-FITC, as secondary reagents. (E) rBV-infected cells assayed with anti-Tag antibody, as primary reagent, and GAM-FITC, as secondary antibody. LRSC fluorescence is shown in A–C, FITC fluorescence in D and E. FIG. 3. GCR33 localizes to the region of the HCMV-infected cell cytoplasmic inclusion. HFFs were infected with wild-type (strain AD169) HCMV (WT; A, D–K, M, and N), HCMV Δ33 (Δ33; B and L), or not infected (Mock; C) and processed for indirect immunofluorescence. Primary reagents used were rabbit F(ab)2 anti-33 (A–C, E, and F), nonspecific rabbit F(ab)2 (D), nonspecific rabbit serum (G and H), nonspecific rabbit Fc (L), a mixture of nonspecific rabbit Fc and rabbit F(ab)2 anti-33 (J and K), C1 peptide-competed rabbit F(ab)2 anti-33 (M), or UL33 peptide-competed rabbit F(ab)2 anti-33 (N). Secondary reagents used were GFARF-LRSC (A–D, M, and N), GFARFc-FITC (L), or a mix of both (E–K). LRSC fluorescence is shown in A–E, G, K, M, and N, FITC fluorescence in F, H, J, and L.
FIG. 4.
FIG. 4.
HCMV-infected HFF cells express glycosylated GCR33. HFF cells were infected with WT HCMV (WT-Inf.; lanes 2, 5, and 8), HCMV Δ33 (Δ33-Inf.; lanes 3, 6, and 9), or not infected (Mock; lanes 1, 4, and 7). Total cell membranes (Membranes) or whole cells (Cells) were isolated 8 days after infection and incubated with or without PNGase F (+/− PNGase). Membrane protein (20 μg) or whole cell protein (15 μg) was subjected to SDS–PAGE and analyzed by Western immunoassay; the Membranes blot was probed with rabbit anti-33, and the cells blot was probed with an antiserum to the basic phosphoprotein (BPP, protein product of HCMV UL32 ORF) (Greis et al., 1994). The asterisked bar represents a molecular weight range of ≈58 to ≈100 kDa; molecular weight (in kDa) was estimated from molecular weight markers included during SDS–PAGE that were visualized by staining that portion of the Immobilon-P with CBB immediately following electrotransfer.
FIG. 5.
FIG. 5.
Peptide comparisons show similarities between rBV (pUL33)- and HCMV-infected cell (GCR33) UL33 proteins. Samples of rBV-33-infected Sf9 cell proteins (lane 1) and membrane preparations from WT HCMV-infected HFF cells without (lane 2) or with (lane 3) PNGase F treatment were subjected to first-dimension SDS–PAGE in a single 12% polyacrylamide gel. The region of each sample spanning the size range of GCR33 and pUL33 was excised from the gel, treated with NCS to cleave the proteins in situ, and the resulting peptides were compared by SDS–PAGE in an 18% DATD-crosslinked polyacrylamide gel by electrophoresis perpendicular to the original direction of migration (i.e., top to bottom), all as described under Materials and Methods. Peptides in the second-dimension gel were detected by Western immunoassay with the anti-33 antiserum (to the carboxyl end of UL33 protein). Immunoreactive peptides are labeled 1 to 5. Circles indicate peptides 4 and 5 in lanes 1 and 2.
FIG. 6.
FIG. 6.
HCMV enveloped particles contain glycosylated GCR33. Cell membranes were isolated from noninfected (Mock; lanes 1 and 2) or WT HCMV-infected (WT; lanes 3 and 4) HFF cells. WT HCMV (WT; lanes 5, 6, 9, 10, 13, 14, 17, 19, and 21) or HCMV Δ33 (Δ33; lanes 7, 8, 11, 12, 15, 16, 18, 20, and 22) enveloped extracellular virus particles were used in these experiments; NIEPs (lanes 5–8, 17, and 18), virions (lanes 9–12, 19, and 20), or dense bodies (lanes 13–16, 21, and 22) were purified from infected cells, treated or not treated with PNGase F (+/− PNGase), and subjected to SDS–PAGE, followed by Western immunoassay with rabbit anti-33. Circles (lanes 5, 9, and 13) indicate a broad, immunoreactive band of material in WT NIEPs, virions, and dense bodies. Another set of samples, not treated with PNGase F (lanes 17–22), was heated in a boiling water bath prior to SDS–PAGE in the same gel and then subjected to Western immunoassay with a mixture of anti-miCP (to minor capsid protein) and anti-LM (to abundant lower matrix tegument protein). The positions of the minor capsid protein (miCP) and lower matrix protein (LM) are indicated; molecular weight (in kDa) was estimated from molecular weight markers included during SDS–PAGE that were visualized by staining that portion of the Immobilon-P with CBB immediately following electrotransfer.
FIG. 7.
FIG. 7.
Size comparison of different forms of the protein encoded by UL33. Sf9 cells infected with WT baculovirus (BV; lane 1), rBV-33 (lane 2), or rBV-N33 (lanes 3, 4, and 5) were harvested 3 days after infection; membranes from noninfected (Mock; lanes 6 and 7) or WT HCMV-infected (WT-Inf.; lanes 8 and 9) HFFs were prepared and treated or not treated with PNGase F (+/− PNGase). Lane 4 (rBV-N33 1:1) is a twofold dilution, and lane 3 (rBV-N33 1:3) a fourfold dilution, of the material in lane 5. All samples were then analyzed by SDS–PAGE and Western immunoassay with the anti-33 antiserum. Circles indicate the three forms of the protein encoded by UL33. The asterisked bar indicates a broad molecular weight range of glycosylated GCR33 and aggregates of GCR33 that are visible near the top of the gel; molecular weights (in kDa) were estimated from molecular weight markers included during SDS–PAGE that were visualized by staining that portion of the Immobilon-P with CBB immediately following electrotransfer.
FIG. 8.
FIG. 8.
The UL33 ORF is dispensable for growth in cell culture. Monolayers of MRC5 cells were infected with WT HCMV or Δ33 viruses at a multiplicity of infection of 5 and washed three times with medium containing 10% FCS after adsorption. Supernatant virus was collected at 16, 24, 48, 72, 96, and 144 hr after infection and was assayed by plaquing on a fresh monolayer of MRC5 cells (Browne et al., 1992; Kaye et al., 1992). Duplicate assays were performed from duplicate infections for each time point. Shown here is a one-step growth curve; standard errors were not greater than 6% between the two sets of measurements that were averaged.
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