Article Figures & Data
Figures
- FIG. 1.
Expression of HCMV glycoproteins US2 to US11 by Ad vectors. Replication-defective (E1−) Ad viruses carrying the US2, US3, US7, US8, US9, US10, and US11 genes were constructed. His16 cells were coinfected for 18 h with each of these Ad vectors and Adtet-trans where appropriate at 100 and 20 PFU/cell, respectively. The infected cells were labeled with [35S]methionine-cysteine for 1 h, and each of the respective glycoproteins US2 to US11 were immunoprecipitated with rabbit polyclonal antibodies before electrophoresis and autoradiography. Molecular masses of marker proteins are indicated at the right.
- FIG. 2.
Inhibition of antigen presentation to CD4+ T cells. His16 cells were infected with Ad vectors expressing various US2 to US11 glycoproteins and Adtet-trans at 50 and 10, 100 and 20, or 150 and 30 PFU/cell, respectively, for 18 h. Infected cells were incubated with the Mtb39 antigen and Mtb39-specific TbH9 CD4+ T cells for an additional 18 h. IFN-γ produced by the T cells was measured by ELISA. The amounts of IFN-γ were normalized to that produced by T cells incubated with antigen-exposed His16 cells infected with Adtet-trans. (A) Experiment in which US2, US3, US7, US8, US9, US10, and US11 were compared; (B) experiment involving US2 and US3. In both experiments, IFN-γ production is in arbitrary units based on a value of 100 obtained when CD4+ T cells were mixed with His16 cells infected with 150 PFU of the control Ad vector, Adtet-trans/cell.
- FIG. 3.
Effects of US3 on class I and class II synthesis and maturation. His16 cells were left uninfected (UN; A to C) or were infected with AdtetUS3 and Adtet-trans at 100 and 20 or 200 and 40 (A and C) or 150 and 30 PFU/cell (B), respectively, for 18 h. Infected cells were labeled with [35S]methionine-cysteine in a pulse-chase format. DR-α and DR-β (A), Ii (B), and MHC-I HC (C) were immunoprecipitated from cell extracts with MAb DA6.147, HB10A, and PIN.1, and rabbit anti-HC serum, respectively. (D) His16 cells were infected with Adtet-trans alone at 120 PFU/cell, AdtetUS2 and Adtet-trans at 100 and 20 PFU/cell, respectively, or AdtetUS3 and Adtet-trans at 100 and 20 PFU/cell, respectively. The cells were labeled for 12 min, the label was chased for 120 min, and then DM-α and DM-β were immunoprecipitated with MAbs 5C1 and MaP.DMB/C, respectively. For endo H treatment, precipitated proteins were divided in half and treated with endo H (+) or not treated (−). The proteins were subjected to SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. r, endo H-resistant species of DM; s, endo H-susceptible species of DM.
- FIG. 4.
US3 binds HLA-DR and inhibits association of Ii with the α/β heterodimer. His16 cells were infected with 120 PFU of Adtet-trans/cell or 100 and 20 PFU of AdtetUS3 and Adtet-trans/cell, respectively. After 18 h, the cells were labeled in a pulse (30 min)-chase (60 [A and B] and 120 min [C]) format. Digitonin cell extracts were subjected to primary immunoprecipitation, followed by denaturation in SDS and reprecipitation. (A) Primary precipitation with a control mouse MAb or anti-DR-α MAb (DA6.147) followed by secondary precipitation with rabbit anti-US3 or anti-DR-α. (B) Primary and secondary precipitations with rabbit antisera to MHC-I HC and US3. (C) Primary precipitation with anti-DR-α antibody DA6.147 followed by secondary precipitation of US3 with rabbit antiserum or Ii with MAb PIN.1. (D) Primary precipitation of DR-α with MAb DA6.147 or of Ii with PIN.1 followed by secondary precipitation of US3, DR-α, DR-β, and Ii with rabbit antisera to US3 or MAbs DA6.147, HB10A, and PIN.1, respectively.
- FIG. 5.
US3 localizes to the Golgi apparatus. His16 cells were infected with 150 and 30 PFU of AdtetUS3 and Adtet-trans/cell, respectively. After 18 h, the cells were fixed and permeabilized and incubated simultaneously with rabbit antibodies to US3 (red) and mouse antibodies to cellular markers (green) PDI (A), p115 (B), TGN46 (C), or LAMP-1 (D). Primary antibodies were visualized with secondary fluorescent antibodies by using a laser scanning confocal microscope.
- FIG. 6.
Effects of US3 on intracellular pools of class II proteins. Neo6 cells were infected with Adtet-trans alone (180 PFU/cell; A and B) or with AdtetUS3 and Adtet-trans (150 and 30 PFU/cell, respectively; C and D). After 18 h, the cells were fixed and permeabilized and incubated simultaneously with rabbit antibodies to class II (red) and mouse antibodies to cellular markers (green) p115 (A and C) and LAMP-1 (B and D), as well as rat antibodies to US3 (blue; C and D). The intracellular proteins were visualized as in Fig. 5.
- FIG. 7.
Effects of US3 on cell surface class II molecules. His16 cells were infected with Adtet-trans alone (120 PFU/cell) or with AdtetUS3 and Adtet-trans (100 and 20 PFU/cell, respectively) for 18 h. Class II molecules on the surfaces of infected cells were stained by incubating suspended cells with L243 and then with fluorescein isothiocyanate-conjugated goat anti-mouse IgG. The results were assessed by flow cytometry. The designation of the histograms are as follows: shaded, secondary antibody control; dotted line, Adtet-trans-infected cells; thin line, AdtetUS2-infected cells; thick line, AdtetUS3-infected cells.
- FIG. 8.
US3 inhibits formation of SDS-stable class II complexes. His16 (A and B) and Neo6 (C) cells were left uninfected or were coinfected with Ad vectors expressing various US2 to US11 glycoproteins and Adtet-trans at 100 and 20 PFU/cell, respectively (A and B) or the indicated amounts of virus (C). After 18 (A and B) or 30 h (C), the cells were labeled in a pulse (60 min; P)-chase (3 or 9 h) format. Class II complexes were immunoprecipitated from cell extracts with MAb HB10A, incubated with 2% SDS at room temperature for 30 min, and subjected to electrophoresis. (A) Visualization of DR-α/β complexes in His16 cells after the pulse or a 3- or 9-h chase. (B) Quantification of DR-α/β complexes in His16 cells after a 9-h chase period (from panel A). (C) Quantification of DR-α/β complexes in Neo6 cells infected with AdtetUS3 or AdtetUS9, following a 9-h chase period (in a separate experiment).
- FIG. 9.
US3 reduces accumulation of DR-α/β complexes in lysosomal compartments. (A) Uninfected Neo6 cells were fractionated with Percoll gradients, and fractions were subjected to electrophoresis and Western blotting for LAMP-1, PDI, and DR-α. For DR-α, cells were infected either with 120 PFU of Adtet-trans/cell alone (DR/trans) or with 100 and 20 PFU of AdtetUS3 and Adtet-trans (DR/US3)/cell, respectively, before fractionation and Western blotting. (B) Neo6 cells were infected with 120 PFU of Adtet-trans/cell alone (trans) or with 100 and 20 PFU of AdtetUS3 and Adtet-trans (US3)/cell, respectively, for 18 h. Infected cells were radiolabeled for 1 h, and the label was chased for 8 h. The cells were fractionated with Percoll gradients, and the fractions were collected from the top and numbered 1 to 16. The membrane fractions were solubilized in NP-40-DOC lysis buffer, and DR proteins were immunoprecipitated with MAb DA6.147. The samples were subjected to electrophoresis and phosphorimager analysis.
