Figure 1.
cVACs are formed during γ-herpesvirus infection and have liquid properties. (A) Time-lapse imaging of cVAC formation. COS-7 cells were infected with mCherry-ORF52 recombinant virus at a MOI of 3 and images were collected from 12 hpi (00:00:00). cVACs formed gradually in the cytoplasm over time. Bar: 5 μm. (B) CLEM images of a 100-nm ultrathin section of a cell infected by mEosEM-ORF52 recombinant virus. (a) Direct correlation between green fluorescence (representing ORF52) and TEM in a 293T cell. (b and c) Green fluorescence of cVACs in cytoplasm and TEM image of the whole 293T cell, respectively, after infection by recombinant virus. (i and ii) Enlarged images of the boxed areas in panel a, showing tegumentation and secondary envelopment of viral capsids in ORF52 puncta. Scale bars: 3 µm (main image); 1 µm (zoomed-in images). Black arrowhead: cVAC; white arrowheads: capsids; yellow arrowheads: gold nanoparticles used as fiducial markers for registration of light and electron microscopy images. (C) Co-localization of ORF33 (homologue of UL16 in HSV-1 and UL94 in HCMV) and ORF45 with cVACs. COS-7 cells were infected with mCherry-ORF52 virus (MOI = 3), and indirect immunofluorescence assay was performed at 24 hpi. ORF33 was detected using a mouse anti-ORF33 monoclonal antibody and ORF45 was detected using a rabbit anti-ORF45 polyclonal antibody. Secondary antibodies were conjugated with Alexa Fluor 488 (ORF33, green channel) or Alexa Fluor 647 (ORF45, blue channel). Bar: 5 μm. (D) Co-localization of ORF33 and ORF38 (homologue of UL11 in HSV-1 and UL99 in HCMV) with cVACs. The experiment was conducted as in C, except that ORF38 was detected using a rabbit anti-ORF38 polyclonal antibody. Bar: 5 μm. (E) FRAP of a cVAC in COS-7 cells at 37°C (box: bleach site; Nu, nucleus). These images are representative of at least three cells in which the cVAC puncta were photobleached. Bar: 5 μm. (F) Quantification of FRAP of cVAC puncta. Fluorescence intensities of cVACs were normalized to background and plotted over a 60-s time course. Shown are means ± SD. n = 3 cVAC puncta.