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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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IFITM-mediated restriction of virus-endosome fusion in different cell types.(A) IFITM3-mediated inhibition of viral fusion with different cell types. BlaM-Vpr carrying pseudoviruses (IAVpp, VSVpp and LASVpp, MOI = 1) were bound to IFITM3- or vector-transduced A549, MDCK, CV1, HeLaH1 or CHO cells in the cold. Fusion was allowed to proceed for 90 min at 37°C and was measured by the BlaM assay, as described in Materials and Methods. ND, not determined. Data are means and SEM from 2 independent triplicate experiments. (B) IFITM3 expression patterns in A549, MDCK and CHO cells transduced with an empty vector (left) or IFITM3 (right). Cells were fixed, permeabilized and immunostained for IFITM3 (red), as described in Materials and Methods. The nuclear stain, Hoechst-3342, is shown in blue. (C) IFITM3 restricts fusion of influenza virus-like particles containing β-lactamase reporter protein fused to the influenza matrix protein-1 (BlaM1). Experiments were carried out as described above. Data are means and SEM from 2 independent triplicate experiments. (D) Exposure to low pH overcomes the IFITM3-mediated block of IAVpp fusion. To force pseudovirus fusion at the plasma membrane, A549 cells transduced with IFITM1, IFITM3 or an empty vector were pretreated with 50 nM BafA1 for 30 min at 37°C or left untreated. IAVpp/BlaM-Vpr pseudoviruses (MOI = 1) were bound to cells of in the cold and exposed to either a pre-warmed pH 5.0 MES-citrate buffer or neutral buffer for 10 min at 37°C and further incubated in growth medium (with or without BafA1) for 90 min at 37°C. Data are means and SEM from 2 independent triplicate experiments. ***, P<0.001 by two-tailed t-test.
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ppat-1004048-g001: IFITM-mediated restriction of virus-endosome fusion in different cell types.(A) IFITM3-mediated inhibition of viral fusion with different cell types. BlaM-Vpr carrying pseudoviruses (IAVpp, VSVpp and LASVpp, MOI = 1) were bound to IFITM3- or vector-transduced A549, MDCK, CV1, HeLaH1 or CHO cells in the cold. Fusion was allowed to proceed for 90 min at 37°C and was measured by the BlaM assay, as described in Materials and Methods. ND, not determined. Data are means and SEM from 2 independent triplicate experiments. (B) IFITM3 expression patterns in A549, MDCK and CHO cells transduced with an empty vector (left) or IFITM3 (right). Cells were fixed, permeabilized and immunostained for IFITM3 (red), as described in Materials and Methods. The nuclear stain, Hoechst-3342, is shown in blue. (C) IFITM3 restricts fusion of influenza virus-like particles containing β-lactamase reporter protein fused to the influenza matrix protein-1 (BlaM1). Experiments were carried out as described above. Data are means and SEM from 2 independent triplicate experiments. (D) Exposure to low pH overcomes the IFITM3-mediated block of IAVpp fusion. To force pseudovirus fusion at the plasma membrane, A549 cells transduced with IFITM1, IFITM3 or an empty vector were pretreated with 50 nM BafA1 for 30 min at 37°C or left untreated. IAVpp/BlaM-Vpr pseudoviruses (MOI = 1) were bound to cells of in the cold and exposed to either a pre-warmed pH 5.0 MES-citrate buffer or neutral buffer for 10 min at 37°C and further incubated in growth medium (with or without BafA1) for 90 min at 37°C. Data are means and SEM from 2 independent triplicate experiments. ***, P<0.001 by two-tailed t-test.

Mentions: We chose to focus on IFITM3 to study the mechanism of IAV restriction because this protein potently inhibits infection in vitro and in vivo[8]–[10]. Since published data suggest that IFITM3 likely inhibits the viral fusion step, a direct virus-cell fusion assay was employed to evaluate the extent of restriction in different cell lines [23]. HIV-1 particles carrying the β-lactamase-Vpr (BlaM-Vpr) chimera and pseudotyped with the influenza HA and NA proteins from the H1N1 A/WSN/33 strain (referred to as IAVpp) were allowed to fuse with cells transduced with an empty vector or with an IFITM3-expressing vector. The resulting cytosolic BlaM activity was measured as previously described [24]. Out of several cell lines tested, A549 and MDCK cells over-expressing IFITM3 were least permissive to IAVpp fusion (Fig. 1A). In agreement with the previous reports [2], [13], we found that IFITM3 over-expression partially inhibited VSV G glycoprotein-mediated fusion of pseudoviruses (VSVpp) carrying the BlaM-Vpr chimera (Fig. 1A). Similar to inhibition of IAVpp fusion, the IFITM3-mediated restriction of VSVpp was most potent in A549 and MDCK cells. As expected, fusion of particles pseudotyped with the Lassa fever virus glycoprotein (LASVpp), which directs virus entry through an IFITM3-resistant pathway [2], [6], was not considerably affected by IFITM3 over-expression.


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)

IFITM-mediated restriction of virus-endosome fusion in different cell types.(A) IFITM3-mediated inhibition of viral fusion with different cell types. BlaM-Vpr carrying pseudoviruses (IAVpp, VSVpp and LASVpp, MOI = 1) were bound to IFITM3- or vector-transduced A549, MDCK, CV1, HeLaH1 or CHO cells in the cold. Fusion was allowed to proceed for 90 min at 37°C and was measured by the BlaM assay, as described in Materials and Methods. ND, not determined. Data are means and SEM from 2 independent triplicate experiments. (B) IFITM3 expression patterns in A549, MDCK and CHO cells transduced with an empty vector (left) or IFITM3 (right). Cells were fixed, permeabilized and immunostained for IFITM3 (red), as described in Materials and Methods. The nuclear stain, Hoechst-3342, is shown in blue. (C) IFITM3 restricts fusion of influenza virus-like particles containing β-lactamase reporter protein fused to the influenza matrix protein-1 (BlaM1). Experiments were carried out as described above. Data are means and SEM from 2 independent triplicate experiments. (D) Exposure to low pH overcomes the IFITM3-mediated block of IAVpp fusion. To force pseudovirus fusion at the plasma membrane, A549 cells transduced with IFITM1, IFITM3 or an empty vector were pretreated with 50 nM BafA1 for 30 min at 37°C or left untreated. IAVpp/BlaM-Vpr pseudoviruses (MOI = 1) were bound to cells of in the cold and exposed to either a pre-warmed pH 5.0 MES-citrate buffer or neutral buffer for 10 min at 37°C and further incubated in growth medium (with or without BafA1) for 90 min at 37°C. Data are means and SEM from 2 independent triplicate experiments. ***, P<0.001 by two-tailed t-test.
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Related In: Results  -  Collection

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ppat-1004048-g001: IFITM-mediated restriction of virus-endosome fusion in different cell types.(A) IFITM3-mediated inhibition of viral fusion with different cell types. BlaM-Vpr carrying pseudoviruses (IAVpp, VSVpp and LASVpp, MOI = 1) were bound to IFITM3- or vector-transduced A549, MDCK, CV1, HeLaH1 or CHO cells in the cold. Fusion was allowed to proceed for 90 min at 37°C and was measured by the BlaM assay, as described in Materials and Methods. ND, not determined. Data are means and SEM from 2 independent triplicate experiments. (B) IFITM3 expression patterns in A549, MDCK and CHO cells transduced with an empty vector (left) or IFITM3 (right). Cells were fixed, permeabilized and immunostained for IFITM3 (red), as described in Materials and Methods. The nuclear stain, Hoechst-3342, is shown in blue. (C) IFITM3 restricts fusion of influenza virus-like particles containing β-lactamase reporter protein fused to the influenza matrix protein-1 (BlaM1). Experiments were carried out as described above. Data are means and SEM from 2 independent triplicate experiments. (D) Exposure to low pH overcomes the IFITM3-mediated block of IAVpp fusion. To force pseudovirus fusion at the plasma membrane, A549 cells transduced with IFITM1, IFITM3 or an empty vector were pretreated with 50 nM BafA1 for 30 min at 37°C or left untreated. IAVpp/BlaM-Vpr pseudoviruses (MOI = 1) were bound to cells of in the cold and exposed to either a pre-warmed pH 5.0 MES-citrate buffer or neutral buffer for 10 min at 37°C and further incubated in growth medium (with or without BafA1) for 90 min at 37°C. Data are means and SEM from 2 independent triplicate experiments. ***, P<0.001 by two-tailed t-test.
Mentions: We chose to focus on IFITM3 to study the mechanism of IAV restriction because this protein potently inhibits infection in vitro and in vivo[8]–[10]. Since published data suggest that IFITM3 likely inhibits the viral fusion step, a direct virus-cell fusion assay was employed to evaluate the extent of restriction in different cell lines [23]. HIV-1 particles carrying the β-lactamase-Vpr (BlaM-Vpr) chimera and pseudotyped with the influenza HA and NA proteins from the H1N1 A/WSN/33 strain (referred to as IAVpp) were allowed to fuse with cells transduced with an empty vector or with an IFITM3-expressing vector. The resulting cytosolic BlaM activity was measured as previously described [24]. Out of several cell lines tested, A549 and MDCK cells over-expressing IFITM3 were least permissive to IAVpp fusion (Fig. 1A). In agreement with the previous reports [2], [13], we found that IFITM3 over-expression partially inhibited VSV G glycoprotein-mediated fusion of pseudoviruses (VSVpp) carrying the BlaM-Vpr chimera (Fig. 1A). Similar to inhibition of IAVpp fusion, the IFITM3-mediated restriction of VSVpp was most potent in A549 and MDCK cells. As expected, fusion of particles pseudotyped with the Lassa fever virus glycoprotein (LASVpp), which directs virus entry through an IFITM3-resistant pathway [2], [6], was not considerably affected by IFITM3 over-expression.

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