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. 2009 May 1;5(5):e1000403. doi: 10.1371/journal.ppat.1000403

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Yi et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Rate zonal centrifugation of cell lysates derived from VEE replicon cell lines expressing GFP, Flag-H77-NS2, or Flag-H77-NS2(SG) following transfection with the HJ3, HJ3-5, and HJ3-5(SG) RNA. A total of 40 fractions were collected from each gradient: the titer of infectious virus in fractions from HJ3-5 transfected cells was reduced in scale 40-fold to allow it to be plotted on the same graph as the trans-complemented infectious particles present in lysates of Flag-H77-NS2 cells transfected with the mutant HJ3-5(SG) RNA. Cell lysates were prepared three days following transfection by multiple rounds of freeze-thawing. The top panel shows the sedimentation profile of infectious HJ3-5 and trans-complemented HJ3-5(SG)-derived particles. The bottom panel shows immunoblot detection of core protein in fractions 1–20 of the gradients. The fractions containing the peak infectivity are indicated with dashed lines. Slow-sedimenting core antigen was present in fractions 6–13, while fast-sedimenting particles were found in fractions 14–19. (B) Core antigen measured by a quantitative ELISA assay in cell lysates or supernatant fluid from FT3-7 cells transfected with HJ3-5 RNA, with or without the S168G mutation. (C) Equilibrium density gradient ultracentrifugation of particles present in VEE replicon cell lysates expressing GFP or Flag-H77-NS2, three days post-transfection of the HJ3-5 or HJ3-5(SG) RNA. Infectious virus titer of each fraction is shown in the top panel, with the scale reduced 80-fold for HJ3-5 to allow it to be plotted in the same graph as the trans-complemented HJ3-5(SG) –derived virus. The relative distribution of core antigen present in each gradient fraction, as determined by immunoblotting, is shown in the lower panel.