A short isoform of human cytomegalovirus US3 functions as a dominant negative inhibitor of the full-length form

Abstract

Human cytomegalovirus encodes four unique short (US) region proteins, each of which is independently sufficient for causing the down-regulation of major histocompatibility complex (MHC) class I molecules on the cell surface. This down-regulation enables infected cells to evade recognition by cytotoxic T lymphocytes (CTLs) but makes them vulnerable to lysis by natural killer (NK) cells, which lyse those cells that lack MHC class I molecules. The 22-kDa US3 glycoprotein is able to down-regulate the surface expression of MHC class I molecules by dual mechanisms: direct endoplasmic reticulum retention by physical association and/or tapasin inhibition. The alternative splicing of the US3 gene generates two additional products, including 17-kDa and 3.5-kDa truncated isoforms; however, the functional significance of these isoforms during viral infection is unknown. Here, we describe a novel mode of self-regulation of US3 function that uses the endogenously produced truncated isoform. The truncated isoform itself neither binds to MHC class I molecules nor prevents the full-length US3 from interacting with MHC class I molecules. Instead, the truncated isoform associates with tapasin and competes with full-length US3 for binding to tapasin; thus, it suppresses the action of US3 that causes the disruption of the function of tapasin. Our results indicate that the truncated isoform of the US3 locus acts as a dominant negative regulator of full-length US3 activity. These data reflect the manner in which the virus has developed temporal survival strategies during viral infection against immune surveillance involving both CTLs and NK cells.

FIG. 1.

Effect of the individual US3 gene products on cell surface expression of MHC class I molecules. (A) Schematic representation of US3 variants. Open regions represent shared amino acids (a.a.), and gray and black regions represent individually specific amino acids. (B and C) A total of 1 × 10⁷ K562/B44 (B) and K562/B27 (C) cells were transfected with cDNAs encoding unspliced full-length (US3), singly spliced (SS), or doubly spliced (DS) US3 isoforms. After 48 h of transfection, the cells were incubated with anti-class I MAb W6/32 and then stained with FITC-conjugated anti-mouse IgG Ab, followed by flow cytometry analysis. Fluorescence-activated cell sorter histograms display the surface expression levels of tapasin-dependent HLA-B4402 (B) and tapasin-independent HLA-B2705 (C) alleles in mock (MO) (thin line) and US3 (thick line) transfectants. Isotype-control IgG-stained cells were used as a negative control. (D) Expression levels of ectopic US3 and SS in K562/B44 and K562/B27 cells were determined by metabolic labeling for 30 min and immunoprecipitation with polyclonal anti-US3 sera. TM, transmembrane; CT, cytosolic tail.

FIG. 2.

Effect of coexpression of US3 variants on the cell surface expression and intracellular transport of MHC class I molecules. HeLa cells were cotransfected with the combinations of plasmids indicated and analyzed by flow cytometry (A) and endo H digestion (B). The total amount of transfected plasmids was equalized by an empty vector. (A) Cells were stained with MAb W6/32 and FITC-conjugated anti-mouse secondary Ab, and a total of 10,000 gated events were collected by FACScalibur and analyzed using CellQuest software. (B) The transfectants were radiolabeled for 10 min, chased for the indicated times, and lysed in 1% NP-40 lysis buffer. The lysates were immunoprecipitated with W6/32 MAb to analyze endo H sensitivity of MHC class I molecules (upper panel) or anti-US3 sera to confirm the expression of transfected plasmids (bottom panel). For endo H digestion, the samples were equally separated and treated with either control buffer (−) or endo H (+). The proportion of endo H-resistant proteins was estimated by measuring the density of the endo H-resistant (r) and endo H-sensitive (s) bands with a densitometer and calculating the ratio of the total protein (+ [s]). MO, mock; IP, immunoprecipitation.

FIG. 3.

Effect of the truncated isoform on the half-life, endo H sensitivity, and class I association of full-length US3. The plasmids expressing the indicated US3 variant cDNAs were cotransfected into HeLa cells. After 48 h of transfection, the cells were ³⁵S labeled for 30 min, chased for the indicated times, and lysed. (A and B) NP-40-solubilized supernatants were immunoprecipitated with polyclonal anti-US3 (α-US3) sera (A), followed by endo H digestion (B). (C) Each radiolabeled transfectant was lysed in 1% digitonin and divided into two equal parts. The interactions between US3 proteins and MHC class I molecules were assessed by coimmunoprecipitation using the indicated Abs. Immunopellets were washed four times with 0.1% digitonin, and the bead-bound proteins were separated by SDS-PAGE and visualized by autoradiography.

FIG. 4.

Effect of the truncated isoform on the association of full-length US3 with tapasin and on full-length US3-induced subversion of TAP/tapasin complexes. HeLa (A, B, and D) and 293T (C) cells were transfected as indicated. After 48 h posttransfection, the cells were harvested and analyzed. (A, B, and C) Digitonin-solubilized extracts of the transfectants were coprecipitated with anti-US3 (α-US3) (A), anti-GFP (α-GFP) (B), and anti-tapasin (α-TPN) (C) Abs, and the immunopellets were resolved by SDS-PAGE followed by immunoblot (IB) using the indicated Abs. Aliquots of cell lysates were analyzed by immunoblot to confirm the expression of transfected plasmids and a loading control (bottom panels). (D) Digitonin lysates were subject to coprecipitation using anti-TAP1 serum, followed by immunoblot using anti-tapasin Ab. Aliquots of lysates were analyzed by immunoblot using anti-tapasin Ab to serve as the loading control. A part of transfectants was also prepared for metabolic labeling and immunoprecipitation (IP) using anti-US3 Ab to confirm the expression of transfected plasmids. TPN, tapasin; sTPN, soluble tapasin.

FIG. 5.

Suppression of US3-mediated inhibition of tapasin function by the truncated isoform. (A) HeLa transfectants were radiolabeled with [³⁵S]methionine/cysteine for 10 min and lysed in 1% Triton X-100 lysis buffer. Equal aliquots of lysis supernatants were incubated at 4°C, 37°C, and 50°C for 60 min and then immunoprecipitated with MAb W6/32. (B) The indicated transfectants were biotinylated as described in Materials and Methods, and experiments identical to those shown in panel A were performed. W6/32-reactive class I heavy chains were visualized by immunoblot using horseradish peroxidase-conjugated streptavidin. For each temperature, the intensity of the class I heavy-chain band was quantified by phosphorimaging and displayed in the graph. (C) Expressions of transfected plasmids in panel A were confirmed by immunoprecipitation using anti-US3 sera.