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. 2006 Feb;80(3):1191-203.
doi: 10.1128/JVI.80.3.1191-1203.2006.

Infection-dependent nuclear localization of US17, a member of the US12 family of human cytomegalovirus-encoded seven-transmembrane proteins

Subhendu Das  1 , Yelenna Skomorovska-Prokvolit, Fu-Zhang Wang, Philip E Pellett
Affiliations

Infection-dependent nuclear localization of US17, a member of the US12 family of human cytomegalovirus-encoded seven-transmembrane proteins

Subhendu Das et al. J Virol. 2006 Feb.

Abstract

The human cytomegalovirus (HCMV) US12 gene family is a group of predicted seven-transmembrane, G-protein-coupled receptor-related proteins, about which little is known. Specific rabbit polyclonal antibodies detected US17 and US18 beginning 54 and 36 h after infection, respectively, with expression of both proteins dependent on viral DNA synthesis. While US14 and US18 are expressed exclusively in the cytoplasm, we unexpectedly found abundant expression of US17 in both the cytoplasm and nucleoplasm. N- and C-terminally tagged versions of US17 were readily detected in the cytoplasm of transfected mammalian cells, but not in nuclei, suggesting that nuclear localization involves other viral proteins or an infection-triggered cellular process. There was no specific colocalization between US17 and other nuclear expressed HCMV-encoded proteins (IE-2, DNA polymerase processivity factor, and pp28/UL99). To determine whether the observed nuclear localization might be the product of a process by which a soluble C-terminal segment of the full-length protein is expressed, we constructed a recombinant virus that incorporates a synthetic epitope at its N terminus, which in conjunction with the antipeptide antibody that targets its predicted cytoplasmic C-terminal segment, enables simultaneous independent detection of both termini. In cells infected with the recombinant, the US17 N and C termini had limited colocalization, with the N-terminal segment not detected in nuclei, supporting the segmentation hypothesis. Consistent with this, a fragment with an apparent molecular size of 10 kDa was detected by immunoblotting. We have identified the first viral example of a seven-transmembrane protein that is either segmented or expressed in nuclei. Further study will be required to learn the mechanism by which this occurs and the function of the nuclear localizing segment. This likely represents yet another mechanism by which a virus has hijacked or modified cellular regulatory pathways for its benefit.

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Figures

FIG. 1.
FIG. 1.
Generation and specificity of antibodies to US17 and US18. (A) Antipeptide antibodies were generated that target the indicated N-terminal and C-terminal domains of US18 and US17, respectively. aa, amino acids. (B) Use of siRNA to assess antibody specificity. IE-2 and US17 expression were inhibited by treatment with specific siRNAs (as indicated to the right). Cells in the scrambled siRNA column were treated with a nonspecific siRNA, which had no effect on IE-2 or US17 expression. IE-2 was stained red, nuclei were stained blue with DAPI, and US17 was stained green with a rabbit polyclonal antibody directed against the C-terminal segment. Nonconfocal images were obtained at ×20 magnification. (C) Lack of reactivity of US17 antiserum with cells infected with HCMV deleted for US17. HLF infected with an HCMV BAC recombinant from which the US17 gene was deleted were reacted with a monoclonal antibody against the IE-2 protein and rabbit antibodies against either US17 (top pair of panels) or US18 (bottom pair of panels). The BAC recombinant expresses GFP, so infected cells are dually positive for GFP and green fluorescence from the IE-2 staining. The anti-rabbit immunoglobulin G secondary antibody provided the red fluorescence. The right panels show green and red fluorescence merged, and the left panels show red fluorescence alone. Nonconfocal images were obtained at ×20 magnification.
FIG. 2.
FIG. 2.
Time course of US17 (A) and US18 (B) expression during infection. HLF were infected at a MOI of 0.01 with HCMV(AD169) and then fixed and stained at the indicated times after infection (hours after infection [hpi]). Nuclei were stained with DAPI (blue), HCMV IE-2 protein was detected with a mouse monoclonal antibody (red), and US17 and US18 were detected with rabbit antibodies (green). Nonconfocal images were obtained at ×20 magnification.
FIG. 3.
FIG. 3.
Dependence of US17 expression on viral DNA synthesis. Cells were stained for the presence of US17 and US18 (green), and representative viral immediate-early (IE-2), early (UL44), and late (UL99) gene products (red) 96 h after infection with HCMV(AD169) at an MOI of 0.01 in the absence (−) and presence (+) of PAA (100 μg/ml). Nonconfocal images were obtained at ×20 magnification.
FIG. 4.
FIG. 4.
Intracellular distribution of US14, US17, and US18. Rabbit polyclonal antibodies against the indicated N- and C-terminal peptide sequences were used to examine HLF 72 h after infection of HLF at an MOI of 0.01 with HCMV(AD169). Nuclei were stained with DAPI (blue), HCMV IE-2 protein was detected with a mouse monoclonal antibody (red), and US14, US17, and US18 were detected with rabbit antibodies (green). Confocal images were obtained at a magnification of ×63.
FIG. 5.
FIG. 5.
Time course of nuclear versus cytoplasmic distribution of US17. (A) HLF infected with HCMV(AD169) were monitored for US17 expression using the rabbit antipeptide antibody. hr p.i., hours postinfection; C-term, C-terminal segment. (B) Total fluorescence intensities of cytoplasmic and nuclear US17 were measured in 5 to 13 infected cells at each time point using ImagePro 5 software (Media Cybernetics, Inc., Silver Spring, MD), and graphed as the means ± standard deviations (error bars). Confocal images of 1.0-μm thickness (the best plane) were obtained at a magnification of ×63 and were captured using the same photomultiplier tube voltage for each color throughout the time course. hpi, hours postinfection.
FIG. 6.
FIG. 6.
Dependence of US17 nuclear localization on infection. The indicated mammalian cells were transfected with constructs in which US14, US17, and US18 are fused at their N terminus to the FLAG (A) or V5 (B) epitope or at their C terminus to GFP (C). Labeling was with the indicated antibodies and stains. Confocal images were obtained 24 h after transfection at a magnification of ×40 at 2.0× zoom, and the nonconfocal images were obtained at ×20 magnification. PI, propidium iodide.
FIG. 7.
FIG. 7.
Localization of US17 relative to other viral proteins that localize to infected-cell nuclei. (A) US17 and UL99. Panels a and b show examples of UL99 that are either predominantly nuclear or cytoplasmically located; the images shown are from the same slide. (B) US17 and UL44. Confocal images were obtained at a magnification of ×63, 72 h after infection of HLF at an MOI of 0.01 with HCMV(AD169).
FIG. 8.
FIG. 8.
Construction of HCMV BAC incorporating a synthetic epitope at the N terminus of US17. As described in Materials and Methods, a segment of DNA was obtained; this DNA segment contained an ampicillin resistance gene flanked by sequences harboring LoxP sites, plus a V5 synthetic epitope for insertion at the N terminus of US17, and flanking sequences to guide recombinational insertion into the parental HCMV BAC. The V5 epitope was inserted as a segment of 14 amino acids between the US17 initiation codon and the second US17 residue. The locations and contents of the synthetic oligonucleotides used to generate the insertion cassette (Oligo 1 and Oligo 2) are indicated in the diagram. The final construct harbors a 34-bp LoxP site just upstream of the US17 translation initiation codon and the V5 epitope in frame with US17, immediately downstream of the initiation codon.
FIG.9.
FIG.9.
Differential localization of US17 N and C termini in infected cells. (A) Schematic representation of a model in which the N and C termini of US17 are expressed as part of the same primary translation product and subsequently processed to N- and C-terminal fragments, of which only the C-terminal fragment is able to migrate to the nucleus. The N- and C-terminal fragments, seven transmembrane segments (green boxes), V5 epitope (red boxes), and rabbit antipeptide antibody (blue boxes) are shown. (B) HLF infected with a recombinant HCMV that expresses the V5 epitope at its N terminus. Ninety-six hours after infection, cells were stained as indicated in Materials and Methods. The field shown represents a fairly common pattern; the red ring from the V5 epitope staining outlines a cytoplasmic virus assembly compartment. (C) Immunoblot of HCMV-infected HLF probed with the rabbit antipeptide antibody against the US17 C-terminal segment. Immunoblots of uninfected (U) and infected (I) (multiplicity of infection of 0.5 for 96 h) HLF were prepared as described in Materials and Methods. The best extractions were obtained when all processing of infected cells was done at room temperature, and specimens were not boiled prior to electrophoresis. The sizes of the protein standards and the calculated sizes of the US17 segments are shown at the sides of the blot.
FIG. 10.
FIG. 10.
Major hypotheses for US17 segmentation. mRNA species are shown as arrows, the US17 open reading frame is shown as a triangle, with the protein beneath (the boxes are the transmembrane segments). The various possibilities are not mutually exclusive. IRES, internal ribosome entry site.
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