Detection of fast-sedimenting, core-containing HJ3-5(SG) particles with physical properties similar to infectious virus.

<p>(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 <i>trans</i>-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.