Figure 1. Expression of the SARS-CoV 9b protein in the cytoplasm and translocation into the nucleus in SARS-CoV infected Vero cells.
A. Localization pattern of the 9b protein at various time points in SARS-CoV infected cells. Chamber-culture slides (BD Falcon) were seeded with Vero E6 cells to form confluent monolayers. Chambers were inoculated with SARS-CoV (Tor-3 strain) at an MOI of 5 TCID50 per cell. Infected cells were fixed in 2% paraformaldehyde in PBS, pH = 7, for 15 minutes at room temperature, at 12, 18, 24, 36 and 48 hours post infection. Slides were imaged using a Zeiss LSM700 Laser Scanning Microscope with Zen2009 version 5.5 software. Three-dimensional volume projections were rendered in “Volume Viewer” using “ImageJ” version 1.43 g. Figure shows merged 2-channel confocal images of SARS-CoV infected Vero E6 cells stained with anti-9b antibodies (green: Alexa Fluor 488) and DAPI (blue), imaged at 12, 18, 36 and 48 h post-infection under a 63 x oil immersion lens. Nuclear inclusions of 9b are numerous at 36 and 48 h post-infection (an example is arrowed). B. Two and three-dimensional confocal imaging of the time course of infection with SARS-CoV labelled with antibody for 9b (green) and DAPI counter-stain for nuclei (blue). The first column shows zoomed close-ups of typical examples of an uninfected cell, and infected cells at 12, 24, 36, and 48 h post-infection respectively. Column 2 shows projections of three-dimensional reconstructions, using maximum intensity in the xy orientation (looking from the top view), made with the images taken of the cells shown in column 1. Column 3 is a projection of the three-dimensional reconstruction in the zx orientation (side view). Column 4 shows sections through the three-dimensional reconstructions of the nucleus, viewed at a 45 degree angle to the culture surface. The bright spots in the nucleus are the Alexa-labelled 9b antibody positive regions within the nucleus, clearly indicating that these are within the nuclear membrane.