Figure 4
Comparison of XMRV and MXMRV. A) Organization of chimeric expression plasmids is shown. pMXMRV encodes M-MuLV glyco-gag fused to XMRV Gag protein. To abolish glyco-gag expression from MXMRV virus (MX), the pMXMRV mutants, pMX-3 + 4 and pMX CTG/CA were generated by substituting nucleotides in or adjacent to the glyco-gag intiation codon (underlined) analogous to glyco-gag negative mutations generated by other investigators (indicated). B) Viruses harvested from 293 T cells transfected with the different XMRV expression plasmids. In addition, released viruses were infected into 293 T cells or DU-145 cells and producer cultures were obtained. In this panel the expression of glyco-gag, poly-Gag and Capsid (CA) proteins in the cell lysates and released viruses from the infected 293 T cells were detected by SDS-PAGE and Western blotting with anti-p30CA antibody. C) Quantification of the relative % Gag release for pMXMRV compared to pVP62 in the transiently transfected 293 T cells is shown. D) Quantification of XMRV and MXMRV released from the infected DU145 cells. E) Buoyant density in 20-55% sucrose gradients of XMRV (diamonds) and MXMRV (squares) released from infected 293 T cells. The densities of each sucrose gradient fraction were measured, and the amount of viral CA protein in each fraction was quantified by SDS-PAGE and Western blotting. Distribution of the percent of total CA in each gradient fraction was calculated, and the results are displayed relative to the densities of each fraction. The peak densities for XMRV and MXMRV are indicated.