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Fig. S1. Subcellular localization of c-myc-syntaxin 3 (STX3) in HCMV-infected cells. BJ1 c-myc-STX3 cells HCMV infected for 5 days at 0.5 moi were fixed, permeabilized, stained with anti-c-myc (green) and anti-HCMV glycoprotein UL33 (red) antibodies and imaged by confocal microscopy (panels show two single optical sections ∼0.4 μm thick of the same field). Dapi staining of nuclei in blue. Higher-magnification views of the boxed areas are shown in the insets. Scale bars, 20 μm.

Fig. S2. Immunogold localization of syntaxin 3 (STX3) on the viral envelope of isolated HCMV viral particles.

A and B. Secreted viruses from HCMV-infected parental and c-myc-STX3-expressing BJ1 cells were harvested at 5 dpi, permeabilized with saponin or directly labelled with antibodies against STX3 (A) or c-myc (B) and 10 nm gold-coupled protein-A.

C. General structure of HCMV virions stained with 2% uranyl acetate; when viral membranes were partially disrupted, uranyl acetate revealed the nucleocapsids. Scale bars, 50 nm.

Fig. S3. Syntaxin 3 (STX3) expression in STX3 shRNA MeWo cells. MeWo cells untransduced, or transduced with non-target control and STX3 shRNAs were grown on glass coverslip for immunofluorescence analysis or lysed.

A. Cells were fixed, permeabilized and stained with anti-STX3 (red) antibodies. Dapi staining of nuclei in blue. Scale bars, 20 μm.

B. Cell lysates were analysed by Western blotting and per cent STX3 levels, normalized to actin loading control, are displayed bellow each lane (n = 2).

Fig. S4. Analysis of viral proteins with HCMV-positive human serum. BJ1 cells uninfected or infected with HCMV at 3 moi were lysed at 4 dpi. Cell lysates were analysed by Western blotting with HCMV antibody-positive sera.

Fig. S5. EM analysis of HCMV-infected syntaxin 3 (STX3) shRNA-expressing cell monolayers embedded in situ. BJ1 cells transduced with non-target control and STX3 shRNAs growing on Thermanox® coverslips were infected with RecCMV at 0.5 moi. At 5 dpi, cells were fixed and embedded in situ.

A. Diagram depicts sectioning parallel to the Thermanox® surface. Low-magnification EM micrographs of a cell from 2 to 5 μm selected serial sections. Scale bars, 5 μm.

B and C. High-magnification EM micrographs of HCMV-infected non-target control (B) and STX3 (C) shRNA-expressing cells. MTOC, microtubule-organizing centre; V, virion; DB, dense bodies. Scale bars, 200 nm.

Fig. S6. Subcellular localization of TGN46, transferrin receptor and cation-dependent mannose 6-phosphate receptor in STX3 shRNA MeWo cells. MeWo cells transduced with non-target control and STX3 shRNAs were grown on glass coverslip, fixed, permeabilized and stained with anti-trans-Golgi network marker TGN46 (red), anti-transferrin receptor, TfR (red), anti-cation-dependent mannose 6-phosphate receptor, M6PR (red) and anti-STX3 (green), antibodies. Dapi staining of nuclei in blue. Scale bars, 20 μm.

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