Figure 1.
Analysis of purified influenza virus preparations.
(A) Electron micrograph of negatively stained, sucrose gradient purified influenza A/WSN/33 virus at 50,000× magnification. (B) SDS-PAGE separation of proteins in a purified influenza virus preparation. 15 ug of untreated (lane 1) or deglycosylated (lane 2) proteins were separated on an 8–16% polyacrylamide gel and stained with Coomassie blue. The positions of the viral proteins, identified by their predicted molecular weights, are indicated.
Table 1.
Virion-associated influenza virus proteins identified by mass spectrometry.
Table 2.
Cellular proteins identified in purified influenza virions by both gel fractionation and MudPIT LC-MS/MS analyses.
Table 3.
Cellular proteins in purified influenza virions identified only by MudPIT LC-MS/MS analysis.
Table 4.
Cellular proteins identified in purified influenza virions only by gel fractionation LC-MS/MS analysis.
Figure 2.
Confirmation of host protein incorporation into influenza virions derived from different cell lines.
Influenza A/WSN/33 virus was purified from the supernatant of infected A549 and Vero cells. 2 ug of purified virus derived from A549 and Vero cells (lane 1 and 2, respectively) and 10 ug of cellular extracts from uninfected A549 and Vero cells (lanes 3 and 4, respectively) were subjected to western blot analysis with antibodies against the following proteins: (A) Influenza hemagglutinin (HA0 and HA1 are visible), (B) Beta actin, (C) Annexin A5, (D) Cyclophilin A. Numbers to the left are molecular weight markers.
Figure 3.
The effect of protease treatment on influenza virion associated host proteins.
Purified influenza A/WSN/33 virus was either mock treated or subjected to overnight digestion with subtilisin followed by concentration through a sucrose cushion. 10 ug of mock infected cell lysate (lane 1) or influenza infected cell lysate (lane 2) and 2 ug of untreated influenza virions (lane 3) or protease treated influenza virions (lane 4) were then analyzed by western blot with antibodies against the indicated proteins. Numbers to the right are molecular weight markers.
Figure 4.
Gradient fractionation demonstrates co-purification of influenza virus and CD9.
Influenza A/WSN/33 virus was purified over (A) sucrose and (B) Optiprep gradients. Fractions were taken from the top and analyzed by western blot for the presence of NP, CD9 and MHC-I, as indicated. Numbers to the right are molecular weight markers.
Figure 5.
Immunogold labeling of host proteins in purified influenza virions.
Influenza virions purified from the supernatant of infected Vero cells were immunogold labeled with antibodies against (A) Hemagglutinin, (B) CD9, (C) CD81 and (D) normal mouse IgG. Labeled virus was negatively stained with sodium silicotungstate and visualized by electron microscopy (50,000× magnification). The number of gold particles per virion is shown below (n = the number of virions counted).