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Dual function of CD81 in influenza virus uncoating and budding.

He J, Sun E, Bujny MV, Kim D, Davidson MW, Zhuang X - PLoS Pathog. (2013)

Bottom Line: In this work, we examined the effect of CD81 depletion on the major steps of the influenza infection.CD81 knockdown led to a budding defect and resulted in elongated budding virions with a higher propensity to remain attached to the plasma membrane.Taken together, these results demonstrate important roles of CD81 in both entry and budding stages of the influenza infection cycle.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, United States of America ; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, United States of America.

ABSTRACT
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.

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A major fraction of viruses are trafficked to and fuse in CD81-positive endosomes.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 in situ 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.
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ppat-1003701-g003: A major fraction of viruses are trafficked to and fuse in CD81-positive endosomes.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 in situ 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.

Mentions: Next, we probed the role of CD81 in virus fusion. Influenza virus is trafficked from early endosomes to maturing endosomes, in which the low pH environment triggers conformational changes in HA that mediate viral fusion with the endosomal membrane [2], [9], [41]. In addition to being distributed on the plasma membrane, CD81 also showed substantial colocalization with early and maturing endosomes, which are Rab5 positive (Rab5+) (Figure 3A) [41]. About 30–35% Rab5+ endosomes contained CD81 (Figure 3A), suggesting that CD81 is enriched in a sub-population of these endosomes. To probe whether influenza virus particles are delivered into CD81+ endosomes, we allowed Alexa Fluor 647-labeled X-31 to internalize for 15 minutes and immunostained the cells for CD81. As shown in Figure 3B, a substantial fraction (∼54%) of virus colocalized with CD81+ endosomes.


Dual function of CD81 in influenza virus uncoating and budding.

He J, Sun E, Bujny MV, Kim D, Davidson MW, Zhuang X - PLoS Pathog. (2013)

A major fraction of viruses are trafficked to and fuse in CD81-positive endosomes.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 in situ 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3795033&req=5

ppat-1003701-g003: A major fraction of viruses are trafficked to and fuse in CD81-positive endosomes.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 in situ 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.
Mentions: Next, we probed the role of CD81 in virus fusion. Influenza virus is trafficked from early endosomes to maturing endosomes, in which the low pH environment triggers conformational changes in HA that mediate viral fusion with the endosomal membrane [2], [9], [41]. In addition to being distributed on the plasma membrane, CD81 also showed substantial colocalization with early and maturing endosomes, which are Rab5 positive (Rab5+) (Figure 3A) [41]. About 30–35% Rab5+ endosomes contained CD81 (Figure 3A), suggesting that CD81 is enriched in a sub-population of these endosomes. To probe whether influenza virus particles are delivered into CD81+ endosomes, we allowed Alexa Fluor 647-labeled X-31 to internalize for 15 minutes and immunostained the cells for CD81. As shown in Figure 3B, a substantial fraction (∼54%) of virus colocalized with CD81+ endosomes.

Bottom Line: In this work, we examined the effect of CD81 depletion on the major steps of the influenza infection.CD81 knockdown led to a budding defect and resulted in elongated budding virions with a higher propensity to remain attached to the plasma membrane.Taken together, these results demonstrate important roles of CD81 in both entry and budding stages of the influenza infection cycle.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, United States of America ; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus