Dual Function of CD81 in Influenza Virus Uncoating and Budding
Figure 6
CD81 is enriched at specific sub-viral sites of budding virions and CD81 knockdown impairs virus scission.
A) CD81 knockdown does not change the number of budding virions attached to infected cells. siRNA-treated cells were infected with WSN virus with the acid-bypass treatment for 15 hours. Cells were directly fixed for transmission electron microscopy and the number of budding virus particles per cell cross-section is quantified for over 250 sections, and presented in the dot plot. A two-tailed student t-test was performed and the p value is provided. B) CD81 knockdown causes a substantial reduction in the number of released virus particles. siRNA-treated cells were infected with WSN virus with the acid-bypass treatment for 17 hours. The amount of viral M1 protein in the supernatant was probed with ELISA. The number of M1 positive and HA positive virus particles in the supernatant was counted using immunofluorescence imaging. The error bar is standard deviation from three independent measurements. C) CD81 localizes at the tip of growing X-31 viruses during the early budding stages. Cells were infected with X-31 for 12 hours and CD81 was immunogold labeled for electron microscopy. An enlarged image of the area in the white box is shown in the upper right corner. Scale bar: 100 nm. D) CD81 mainly localizes at the tip and budding neck of the X-31 viruses during late budding stages. Similar to (C) except the infection time was 16 hours. Scale bar: 200 nm. E) Distribution of gold particles in budding X-31 viruses at 16 hour post infection. To align the virus particles, the length of each virus is normalized to 1, with its middle point assigned with coordinate value of 0. For individual gold particles on the budding virus, their coordinate values were calculated based on their relative distance to the middle point. Coordinates with negative values correspond to positions close to the plasma membrane. A total of 105 budding viruses were analyzed. F) Budding WSN viruses exhibit a spherical morphology with fully enclosed membrane envelope in control siRNA-treated cells. A549 cells were infected with virus with the acid-bypass treatment for 13 hours. The region in the white box is magnified and shown in the upper right corner. Scale bar: 200 nm. G) Budding WSN viruses are more elongated in CD81 siRNA treated A549 cells. A substantial fraction of budding viruses have an open membrane neck connected to the plasma membrane (indicated by arrowheads). The region in the white box is magnified and shown in the upper right corner. Scale bar: 200 nm. H) Budding WSN viruses are elongated upon CD81 depletion, as shown by the distribution of budding virus length in control or CD81-knockdown cells.