Dual Function of CD81 in Influenza Virus Uncoating and Budding

As an obligatory pathogen, influenza virus co-opts host cell machinery to harbor infection and to produce progeny viruses. In order to characterize the virus-host cell interactions, several genome-wide siRNA screens and proteomic analyses have been performed recently to identify host factors involved in influenza virus infection. CD81 has emerged as one of the top candidates in two siRNA screens and one proteomic study. The exact role played by CD81 in influenza infection, however, has not been elucidated thus far. In this work, we examined the effect of CD81 depletion on the major steps of the influenza infection. We found that CD81 primarily affected virus infection at two stages: viral uncoating during entry and virus budding. CD81 marked a specific endosomal population and about half of the fused influenza virus particles underwent fusion within the CD81-positive endosomes. Depletion of CD81 resulted in a substantial defect in viral fusion and infection. During virus assembly, CD81 was recruited to virus budding site on the plasma membrane, and in particular, to specific sub-viral locations. For spherical and slightly elongated influenza virus, CD81 was localized at both the growing tip and the budding neck of the progeny viruses. CD81 knockdown led to a budding defect and resulted in elongated budding virions with a higher propensity to remain attached to the plasma membrane. Progeny virus production was markedly reduced in CD81-knockdown cells even when the uncoating defect was compensated. In filamentous virus, CD81 was distributed at multiple sites along the viral filament. Taken together, these results demonstrate important roles of CD81 in both entry and budding stages of the influenza infection cycle

The mammalian phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) regulates endosome-to-TGN retrograde transport

The yeast gene fab1 and its mammalian orthologue Pip5k3 encode the phosphatidylinositol 3-phosphate [PtdIns(3)P] 5-kinases Fab1p and PIKfyve, respectively, enzymes that generates phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P(2)]. A shared feature of fab1Delta yeast cells and mammalian cells overexpressing a kinase-dead PIKfyve mutant is the formation of a swollen vacuolar phenotype: a phenotype that is suggestive of a conserved function for these enzymes and their product, PtdIns(3,5)P(2), in the regulation of endomembrane homeostasis. In the current study, fixed and live cell imaging has established that, when overexpressed at low levels in HeLa cells, PIKfyve is predominantly associated with dynamic tubular and vesicular elements of the early endosomal compartment. Moreover, through the use of small interfering RNA, it has been shown that suppression of PIKfyve induces the formation of swollen endosomal structures that maintain their early and late endosomal identity. Although internalisation, recycling and degradative sorting of receptors for epidermal growth factor and transferrin was unperturbed in PIKfyve suppressed cells, a clear defect in endosome to trans-Golgi-network (TGN) retrograde traffic was observed. These data argue that PIKfyve is predominantly associated with the early endosome, from where it regulates retrograde membrane trafficking to the TGN. It follows that the swollen endosomal phenotype observed in PIKfyve-suppressed cells results primarily from a reduction in retrograde membrane fission rather than a defect in multivesicular body biogenesis

A major fraction of viruses are trafficked to and fuse in CD81-positive endosomes.

<p>A) CD81 substantially colocalizes with Rab5. A549 cells were electroporated with CD81-mEmerald and RFP-Rab5. At 24 hours, the cells were fixed and imaged. An enlarged image of the boxed region is shown on the right. Scale bar: 10 µm. B) Influenza virus particles traffick into CD81+ endosomes. A549 cells were cold bound with Alexa Fluor 647-labeled X-31 virus (red) on ice for 30 minutes and then chased for 15 minutes at 37°C. The samples were fixed and immunostained against CD81 (green). An enlarged image of the boxed region is shown on the right. All of the images are confocal XY cross sections. Scale bar: 10 µm. C) An influenza virus particle enters and fuses within a CD81-positive endosome after entry. Live-cell confocal imaging of DiD-labeled X-31 added <i>in situ</i> to CD81-mEmearld expressing A549 cells maintained at 37°C. The images were collected with a 0.5 s interval. C-1) Several snapshots taken at different time points with the virus indicated by the white circles. C-2) The fluorescence signal of the indicated DiD-labeled virus as a function of time. Note that there is a sudden increase of DiD signal at 515 s, which indicates a viral fusion event. D) Influenza virus can also fuse in a CD81-negative endosome. D-1) Several snapshots taken at different time points with the virus indicated by the white circles. D-2) The fluorescence signal of the indicated DiD labeled virus as a function of time. The virus particle fused at 422 s. E) Among 61 virus particles tracked from binding to fusion, 52±8% enter and fuse within CD81+ endosomes whereas the remaining 48±8% fuse in CD81- endosomes. The results are taken for four independent experiments, and the ±error indicates the standard deviation derived from these experiments. F) Virus fusion is impaired upon CD81 depletion. DiD-labeled X-31 was allowed to bind with A549 cells on ice for 30 minutes, and then chased for the indicated times at 37°C. Cells were trypsinized and fixed immediately, and analyzed by flow cytometry. The increase in the DiD intensity versus the initial DiD intensity is plotted. The error bars are standard deviation derived from duplicate experiments.</p

CD81 is not required for virus binding, internalization or delivery into early endosomes.

<p>A) CD81-knockdown does not affect virus binding, as measured by flow cytometry. The magenta, blue and orange curves correspond to the intensity profiles measured for cells without adding viruses, control cells after influenza virus binding, and CD81-knockdown cells after influenza virus binding, respectively. B) The number of virus particles internalized is not affected by CD81 knockdown. The number of internalized virus particles was shown in a dot plot, with the middle line representing the mean value, and top/bottom line representing standard deviation. At least 40 randomly chosen cells were analyzed for each condition. C) The percent of virus particles colocalizing with early endosome is not affected by CD81 knockdown. Early endosomes were immunostained with anti-EEA1 antibody. Data was plotted similarly as in (B). At least 40 randomly chosen cells were analyzed for each condition. D) CD81 depletion does not affect RSV or pseudo-typed MLV infection. siRNA-treated A549 cells were infected with different doses of RSV and pseudo-typed MLV virus for 24 hours. For RSV virus infection, RSV fusion protein expression was quantified by flow cytometry, while for pseudo-typed MLV virus, the GFP signal was analyzed. A two-tailed student <i>t-test</i> was performed for all of the numerical data, and the p value of the data is shown.</p

CD81 is recruited to the virus budding sites.

<p>A) CD81 is recruited to the virus budding zone in X-31 infected cells. A549 cells were infected with X-31 for 16 hours. Cells were stained with anti-CD81 antibody (green) and anti-PB1 antibody (red). Images are confocal XY cross-sections. Scale bar: 10 µm. B) CD81 is incorporated into budding filamentous virions of Udorn-infected cells. A549 cells were infected with Udorn virus for 16 hours, and stained with anti-CD81 antibody and anti-PB1 antibody. Scale bar: 10 µm. C) Remaining CD81 in CD81-knockdown cells is incorporated into budding filamentous viruses of Udorn infected cells. Similar to (B) except that CD81-knockdown cells were used. The CD81 expression level in Udorn-infected cells was calculated based on confocal images of more than 100 cells, and was found to be decreased by ∼88% upon CD81 depletion as compared to control cells. The amount of CD81 per viral filament was reduced by 63% compared to that in untreated cells. Scale bar: 10 µm.</p

CD81 is involved in both early and late stages of influenza virus infection.

<p>A) A549 cells were treated with non-targeting control siRNA, or CD81 siRNA1 for 48 hours and immunostained with anti-CD81 antibody. Images are maximum projections of confocal z-stacks. B) A549 cells were treated with control or CD81 siRNA for 48 hours. Cells were harvested for western blotting with indicated antibodies. Tubulin was used as a loading control. C) Influenza virus infection is impaired by CD81 depletion. A549 cells were treated with siRNAs or mock treated for 48 hours and subsequently infected with X-31, WSN, or Udorn at a MOI of <0.1 for 36 hours. The viral titer in the supernatant was determined by plaque assays. The shaded bar indicates the infectivity measured in cells mock treated with a transfection solution that contains no siRNA; the hollow bars indicated the infectivity measured in cells treated with control, non-targeting siRNA; the black solid bars indicate the infectivity measured in cells treated with CD81 siRNA. D) The number of infected cells expressing viral NP is reduced by about 50% upon CD81- knockdown. siRNA-treated A549 cells were infected with WSN, X-31, or Udorn viruses with a MOI of <0.1 for 8 hours without acid bypass and the fraction of cells expressing NP was measured through flow cytometry. E) Viral NP expression is unaffected upon CD81 knockdown when influenza infection is induced by the acid-bypass treatment to eliminate the entry defect. siRNA-treated A549 cells were allowed to bind with WSN or X-31 virus on ice for 1 hour, treated with warm low pH PBS buffer (pH 4.5) for 2 minutes. After 8 hours, cells were collected and stained against NP for flow cytometry analysis. F) Virus titer in the supernatant is reduced by ∼50% or more in CD81-knockdown cells infected by influenza viruses through the acid-bypass treatment. Briefly virus was allowed to bind with control or CD81-knockdown cells on ice for 1 hour. Unbound virus particles were washed out and low pH buffer was added in for 2 minutes to trigger virus fusion at the plasma membrane. At 17 hours post infection, supernatant was collected and the viral titer was assayed by plaque assay. For <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003701#ppat-1003701-g001" target="_blank">figure 1C–1F</a>, the error bars are standard deviation derived from three independent experiments. A two-tailed student <i>t-test</i> was performed for all of the numerical data, and the p value of the data is shown. A p value smaller than 0.05 indicates there is a statistically significant difference.</p

CD81 depletion does not affect viral protein expression and transport.

<p>A) CD81 knockdown does not affect the expression of viral NP protein in cells infected by influenza viruses with the acid-bypass treatment. Experiments were performed similarly as in 1E) except that the expression levels are evaluated at different time point post infection and with different dose of viruses. The percent of NP-expression cells and the NP expression level in NP+ cells are plotted. B) CD81 knockdown does not affect the expression of viral M1 protein in cells infected by influenza viruses with the acid-bypass treatment. Experiments were performed similarly as in (A) except cells were immunostained for M1. The percent of M1+ cells and the M1 expression level in the M1+ cells are plotted. C) CD81 knockdown does not affect the expression of viral M2 protein in cells infected by influenza viruses with the acid-bypass treatment. Experiments were performed similarly as in (A) except cells were immunostained for M2. The percent of M2+ cells and the M2 expression level in the M2+ cells are plotted. D) CD81 knockdown does not affect the amount of M2 protein trafficked to the cell surface in cells infected by influenza viruses with the acid-bypass treatment. Experiments were performed similarly as in (C) except cells were stained for M2 without permeabilization. The percent of M2+ cells and the surface M2 expression level in M2+ cells are plotted. E) CD81 knockdown does not affect the amount of NA protein trafficked to the cell surface in cells infected by influenza viruses with the acid-bypass treatment. The NA expression level was estimated from confocal images in control or CD81 siRNA treated cells infected by X-31virus. A two-tailed student <i>t-test</i> was performed for all of the numerical data, and the p value of the data is shown.</p

CD81 is enriched at specific sub-viral sites of budding virions and CD81 knockdown impairs virus scission.

<p>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 <i>t-test</i> 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.</p