Intracellular logistics : sorting nexins in retrograde transport of endogenous and exogenous cargo

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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