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IFITM3 restricts influenza A virus entry by blocking the formation of fusion pores following virus-endosome hemifusion.

Desai TM, Marin M, Chin CR, Savidis G, Brass AL, Melikyan GB - PLoS Pathog. (2014)

Bottom Line: Consistent with this mechanism, excess cholesterol in late endosomes of IFITM-expressing cells has been reported to inhibit IAV entry.IFITM3 over-expression did not inhibit lipid mixing, but abrogated the release of viral content into the cytoplasm.We propose that IFITM3 interferes with pore formation either directly, through partitioning into the cytoplasmic leaflet of a hemifusion intermediate, or indirectly, by modulating the lipid/protein composition of this leaflet.

View Article: PubMed Central - PubMed

Affiliation: Division of Pediatric Infectious Diseases, Emory University Children's Center, Atlanta, Georgia, United States of America.

ABSTRACT
Interferon-induced transmembrane proteins (IFITMs) inhibit infection of diverse enveloped viruses, including the influenza A virus (IAV) which is thought to enter from late endosomes. Recent evidence suggests that IFITMs block virus hemifusion (lipid mixing in the absence of viral content release) by altering the properties of cell membranes. Consistent with this mechanism, excess cholesterol in late endosomes of IFITM-expressing cells has been reported to inhibit IAV entry. Here, we examined IAV restriction by IFITM3 protein using direct virus-cell fusion assay and single virus imaging in live cells. IFITM3 over-expression did not inhibit lipid mixing, but abrogated the release of viral content into the cytoplasm. Although late endosomes of IFITM3-expressing cells accumulated cholesterol, other interventions leading to aberrantly high levels of this lipid did not inhibit virus fusion. These results imply that excess cholesterol in late endosomes is not the mechanism by which IFITM3 inhibits the transition from hemifusion to full fusion. The IFITM3's ability to block fusion pore formation at a post-hemifusion stage shows that this protein stabilizes the cytoplasmic leaflet of endosomal membranes without adversely affecting the lumenal leaflet. We propose that IFITM3 interferes with pore formation either directly, through partitioning into the cytoplasmic leaflet of a hemifusion intermediate, or indirectly, by modulating the lipid/protein composition of this leaflet. Alternatively, IFITM3 may redirect IAV fusion to a non-productive pathway, perhaps by promoting fusion with intralumenal vesicles within multivesicular bodies/late endosomes.

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Analyses of the extent and kinetics of single IAV lipid mixing events.(A) The fraction of AF488-labeled particles undergoing lipid mixing in A549 transduced with an empty vector or IFITM3 and in MDCK cells. Control experiments in A549 cells were carried out in the presence of NH4Cl. Error bars are SEM from 11 independent experiments. ***, P<0.001. (B) The distribution of waiting times for onset of IAV lipid mixing in A549 and MDCK cells transduced with IFITM3 or an empty vector. The time intervals from shifting to 37°C to the onset of vDiD dequenching were determined, as described in Materials and Methods, and plotted as normalized fraction of events as a function of time. Pairwise comparison of all curves yields P>0.2. (C) Ensemble averages of initial vDiD dequenching profiles. The dequenching traces were aligned at the onset of hemifusion and averaged for each time point. Error bars are SEM. (D) The extent of vDiD dequenching was calculated based on I2/I1 ratio, as illustrated in Fig. 2B.
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ppat-1004048-g003: Analyses of the extent and kinetics of single IAV lipid mixing events.(A) The fraction of AF488-labeled particles undergoing lipid mixing in A549 transduced with an empty vector or IFITM3 and in MDCK cells. Control experiments in A549 cells were carried out in the presence of NH4Cl. Error bars are SEM from 11 independent experiments. ***, P<0.001. (B) The distribution of waiting times for onset of IAV lipid mixing in A549 and MDCK cells transduced with IFITM3 or an empty vector. The time intervals from shifting to 37°C to the onset of vDiD dequenching were determined, as described in Materials and Methods, and plotted as normalized fraction of events as a function of time. Pairwise comparison of all curves yields P>0.2. (C) Ensemble averages of initial vDiD dequenching profiles. The dequenching traces were aligned at the onset of hemifusion and averaged for each time point. Error bars are SEM. (D) The extent of vDiD dequenching was calculated based on I2/I1 ratio, as illustrated in Fig. 2B.

Mentions: Labeled viruses were allowed to enter A549-Vector cells, and the resulting lipid mixing activity was examined by single particle tracking. A fraction of virions exhibited a marked increase in the vDiD signal (Fig. 2A, B). Redistribution of vDiD was mediated by low pH-dependent conformational changes in the IAV HA glycoprotein, as evidenced by potent inhibition of lipid mixing by anti-HA antibodies (Fig. 2C) and by NH4Cl (Fig. 3A). Without simultaneous monitoring of the viral content release into the cytoplasm, vDiD dequenching does not discriminate between hemifusion (operationally defined as lipid mixing without content transfer [30]) and full fusion. To avoid over-interpreting dequenching events, we will refer to these events as lipid mixing or hemifusion. A similar vDiD dequenching pattern was observed in MDCK cells transduced with an empty vector (data not shown). Analysis of lipid mixing showed that 2.2±0.4% and 5.6±0.6% of cell-bound particles released vDiD in A549 and MDCK cells, respectively (Fig. 3A). By comparison, a much greater fraction of virions (38.3±0.6%) hemifused with CHO cells (data not shown), in agreement with the previously reported data [28].


IFITM3 restricts influenza A virus entry by blocking the formation of fusion pores following virus-endosome hemifusion.

Desai TM, Marin M, Chin CR, Savidis G, Brass AL, Melikyan GB - PLoS Pathog. (2014)

Analyses of the extent and kinetics of single IAV lipid mixing events.(A) The fraction of AF488-labeled particles undergoing lipid mixing in A549 transduced with an empty vector or IFITM3 and in MDCK cells. Control experiments in A549 cells were carried out in the presence of NH4Cl. Error bars are SEM from 11 independent experiments. ***, P<0.001. (B) The distribution of waiting times for onset of IAV lipid mixing in A549 and MDCK cells transduced with IFITM3 or an empty vector. The time intervals from shifting to 37°C to the onset of vDiD dequenching were determined, as described in Materials and Methods, and plotted as normalized fraction of events as a function of time. Pairwise comparison of all curves yields P>0.2. (C) Ensemble averages of initial vDiD dequenching profiles. The dequenching traces were aligned at the onset of hemifusion and averaged for each time point. Error bars are SEM. (D) The extent of vDiD dequenching was calculated based on I2/I1 ratio, as illustrated in Fig. 2B.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3974867&req=5

ppat-1004048-g003: Analyses of the extent and kinetics of single IAV lipid mixing events.(A) The fraction of AF488-labeled particles undergoing lipid mixing in A549 transduced with an empty vector or IFITM3 and in MDCK cells. Control experiments in A549 cells were carried out in the presence of NH4Cl. Error bars are SEM from 11 independent experiments. ***, P<0.001. (B) The distribution of waiting times for onset of IAV lipid mixing in A549 and MDCK cells transduced with IFITM3 or an empty vector. The time intervals from shifting to 37°C to the onset of vDiD dequenching were determined, as described in Materials and Methods, and plotted as normalized fraction of events as a function of time. Pairwise comparison of all curves yields P>0.2. (C) Ensemble averages of initial vDiD dequenching profiles. The dequenching traces were aligned at the onset of hemifusion and averaged for each time point. Error bars are SEM. (D) The extent of vDiD dequenching was calculated based on I2/I1 ratio, as illustrated in Fig. 2B.
Mentions: Labeled viruses were allowed to enter A549-Vector cells, and the resulting lipid mixing activity was examined by single particle tracking. A fraction of virions exhibited a marked increase in the vDiD signal (Fig. 2A, B). Redistribution of vDiD was mediated by low pH-dependent conformational changes in the IAV HA glycoprotein, as evidenced by potent inhibition of lipid mixing by anti-HA antibodies (Fig. 2C) and by NH4Cl (Fig. 3A). Without simultaneous monitoring of the viral content release into the cytoplasm, vDiD dequenching does not discriminate between hemifusion (operationally defined as lipid mixing without content transfer [30]) and full fusion. To avoid over-interpreting dequenching events, we will refer to these events as lipid mixing or hemifusion. A similar vDiD dequenching pattern was observed in MDCK cells transduced with an empty vector (data not shown). Analysis of lipid mixing showed that 2.2±0.4% and 5.6±0.6% of cell-bound particles released vDiD in A549 and MDCK cells, respectively (Fig. 3A). By comparison, a much greater fraction of virions (38.3±0.6%) hemifused with CHO cells (data not shown), in agreement with the previously reported data [28].

Bottom Line: Consistent with this mechanism, excess cholesterol in late endosomes of IFITM-expressing cells has been reported to inhibit IAV entry.IFITM3 over-expression did not inhibit lipid mixing, but abrogated the release of viral content into the cytoplasm.We propose that IFITM3 interferes with pore formation either directly, through partitioning into the cytoplasmic leaflet of a hemifusion intermediate, or indirectly, by modulating the lipid/protein composition of this leaflet.

View Article: PubMed Central - PubMed

Affiliation: Division of Pediatric Infectious Diseases, Emory University Children's Center, Atlanta, Georgia, United States of America.

ABSTRACT
Interferon-induced transmembrane proteins (IFITMs) inhibit infection of diverse enveloped viruses, including the influenza A virus (IAV) which is thought to enter from late endosomes. Recent evidence suggests that IFITMs block virus hemifusion (lipid mixing in the absence of viral content release) by altering the properties of cell membranes. Consistent with this mechanism, excess cholesterol in late endosomes of IFITM-expressing cells has been reported to inhibit IAV entry. Here, we examined IAV restriction by IFITM3 protein using direct virus-cell fusion assay and single virus imaging in live cells. IFITM3 over-expression did not inhibit lipid mixing, but abrogated the release of viral content into the cytoplasm. Although late endosomes of IFITM3-expressing cells accumulated cholesterol, other interventions leading to aberrantly high levels of this lipid did not inhibit virus fusion. These results imply that excess cholesterol in late endosomes is not the mechanism by which IFITM3 inhibits the transition from hemifusion to full fusion. The IFITM3's ability to block fusion pore formation at a post-hemifusion stage shows that this protein stabilizes the cytoplasmic leaflet of endosomal membranes without adversely affecting the lumenal leaflet. We propose that IFITM3 interferes with pore formation either directly, through partitioning into the cytoplasmic leaflet of a hemifusion intermediate, or indirectly, by modulating the lipid/protein composition of this leaflet. Alternatively, IFITM3 may redirect IAV fusion to a non-productive pathway, perhaps by promoting fusion with intralumenal vesicles within multivesicular bodies/late endosomes.

Show MeSH
Related in: MedlinePlus