Limits...
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.

Show MeSH

Related in: MedlinePlus

CD81 depletion does not affect viral protein expression and transport.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 t-test was performed for all of the numerical data, and the p value of the data is shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3795033&req=5

ppat-1003701-g004: CD81 depletion does not affect viral protein expression and transport.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 t-test was performed for all of the numerical data, and the p value of the data is shown.

Mentions: In the subsequent experiments, we aimed to determine which post-entry step(s) of the viral infection process are CD81 dependent. To this end, we examined the effect of CD81 on viral protein expression, viral protein trafficking, and the assembly and egress of progeny viruses. Our initial results in Figure 1E suggested that CD81 knockdown did not directly affect viral NP expression. This was further substantiated by infecting cells with various viral doses across different time points using the acid-bypass treatment. As shown in Figure 4A, the fraction of NP+ cells and NP expression level increased with the viral dose and infection time, while there was no significant difference between control and CD81-knockdown cells. To further validate the finding, we measured the expression of another cytosolic viral protein, M1. Similar to the results on NP, CD81-knockdown cells infected by influenza viruses using the acid-bypass treatment did not exhibit a difference in the fraction of M1+ cells or the level of M1 protein expression, when compared to the control cells (Figure 4B). These data suggest that CD81 does not play a role in viral gene expression for cytosolic viral proteins.


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)

CD81 depletion does not affect viral protein expression and transport.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 t-test was performed for all of the numerical data, and the p value of the data is shown.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003701-g004: CD81 depletion does not affect viral protein expression and transport.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 t-test was performed for all of the numerical data, and the p value of the data is shown.
Mentions: In the subsequent experiments, we aimed to determine which post-entry step(s) of the viral infection process are CD81 dependent. To this end, we examined the effect of CD81 on viral protein expression, viral protein trafficking, and the assembly and egress of progeny viruses. Our initial results in Figure 1E suggested that CD81 knockdown did not directly affect viral NP expression. This was further substantiated by infecting cells with various viral doses across different time points using the acid-bypass treatment. As shown in Figure 4A, the fraction of NP+ cells and NP expression level increased with the viral dose and infection time, while there was no significant difference between control and CD81-knockdown cells. To further validate the finding, we measured the expression of another cytosolic viral protein, M1. Similar to the results on NP, CD81-knockdown cells infected by influenza viruses using the acid-bypass treatment did not exhibit a difference in the fraction of M1+ cells or the level of M1 protein expression, when compared to the control cells (Figure 4B). These data suggest that CD81 does not play a role in viral gene expression for cytosolic viral proteins.

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