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The Hemagglutinin of Bat-Associated Influenza Viruses Is Activated by TMPRSS2 for pH-Dependent Entry into Bat but Not Human Cells.

Hoffmann M, Krüger N, Zmora P, Wrensch F, Herrler G, Pöhlmann S - PLoS ONE (2016)

Bottom Line: In contrast, sialic acids were dispensable for HAL-driven entry.Collectively, these results identify viral and cellular factors governing host cell entry driven by batFLUAV surface proteins.They suggest that the absence of a functional receptor precludes entry of batFLUAV into human cells while other prerequisites for entry, HAL activation and protonation, are met in target cells of human origin.

View Article: PubMed Central - PubMed

Affiliation: Infection Biology Unit, German Primate Center, Göttingen, Germany.

ABSTRACT
New World bats have recently been discovered to harbor influenza A virus (FLUAV)-related viruses, termed bat-associated influenza A-like viruses (batFLUAV). The internal proteins of batFLUAV are functional in mammalian cells. In contrast, no biological functionality could be demonstrated for the surface proteins, hemagglutinin (HA)-like (HAL) and neuraminidase (NA)-like (NAL), and these proteins need to be replaced by their human counterparts to allow spread of batFLUAV in human cells. Here, we employed rhabdoviral vectors to study the role of HAL and NAL in viral entry. Vectors pseudotyped with batFLUAV-HAL and -NAL were able to enter bat cells but not cells from other mammalian species. Host cell entry was mediated by HAL and was dependent on prior proteolytic activation of HAL and endosomal low pH. In contrast, sialic acids were dispensable for HAL-driven entry. Finally, the type II transmembrane serine protease TMPRSS2 was able to activate HAL for cell entry indicating that batFLUAV can utilize human proteases for HAL activation. Collectively, these results identify viral and cellular factors governing host cell entry driven by batFLUAV surface proteins. They suggest that the absence of a functional receptor precludes entry of batFLUAV into human cells while other prerequisites for entry, HAL activation and protonation, are met in target cells of human origin.

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HAL of batFLUAV are robustly expressed and incorporated into rhabdoviral particles.(A) BHK-21 cells were transfected with the indicated HAL proteins harboring a C-terminal FLAG antigenic tag and protein expression was analyzed by immunofluorescence microscopy of permeabilized cells (magnification, 10x). Cells transfected with an empty expression vector served as negative control. Nuclei were visualized by DAPI staining. Similar results were obtained in a separate experiment. (B) For analysis of HAL incorporation into rhabdoviral particles, vesicular stomatitis virus (VSV)-based pseudotypes were pelleted though a 20% sucrose cushion and analyzed by SDS-PAGE and Western blotting with antibodies against the FLAG tag (α-FLAG), VSV glycoprotein (α-VSV-G) and matrix protein (α-VSV-M). The numbers on the left side of the blots indicate the molecular weight in kilo Daltons (kDa). The results were confirmed in an independent experiment.
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pone.0152134.g001: HAL of batFLUAV are robustly expressed and incorporated into rhabdoviral particles.(A) BHK-21 cells were transfected with the indicated HAL proteins harboring a C-terminal FLAG antigenic tag and protein expression was analyzed by immunofluorescence microscopy of permeabilized cells (magnification, 10x). Cells transfected with an empty expression vector served as negative control. Nuclei were visualized by DAPI staining. Similar results were obtained in a separate experiment. (B) For analysis of HAL incorporation into rhabdoviral particles, vesicular stomatitis virus (VSV)-based pseudotypes were pelleted though a 20% sucrose cushion and analyzed by SDS-PAGE and Western blotting with antibodies against the FLAG tag (α-FLAG), VSV glycoprotein (α-VSV-G) and matrix protein (α-VSV-M). The numbers on the left side of the blots indicate the molecular weight in kilo Daltons (kDa). The results were confirmed in an independent experiment.

Mentions: To test whether HAL17 and HAL18 are comparably expressed and incorporated into rhabdoviral pseudotypes, both proteins were equipped with a C-terminal FLAG epitope (HAL17-FLAG, HAL18-FLAG), since no batFLUAV-HAL-specific antibody was available. Upon transfection of BHK-21 cells similar numbers of HAL-expressing cells were detected by fluorescence microscopy (Fig 1A) and the intensity of the fluorescence signals emitted by the cells was comparable, indicating robust expression of both batFLUAV-HAL proteins. In order to assess HAL incorporation into rhabdoviral pseudotypes, we pelleted pseudotype preparations through a sucrose cushion and subjected the samples to SDS-PAGE and immunoblotting. Using antibodies specific for the FLAG epitope, VSV-G and VSV matrix protein (VSV-M), we found that VSV-G, as expected, as well as both HAL17 and HAL18 proteins were incorporated into particles, with incorporation of HAL17 being more efficient than that of HAL18. (Fig 1B). Thus, both HAL17 and HAL18 were robustly expressed and incorporated into VSVpp, allowing their functional characterization.


The Hemagglutinin of Bat-Associated Influenza Viruses Is Activated by TMPRSS2 for pH-Dependent Entry into Bat but Not Human Cells.

Hoffmann M, Krüger N, Zmora P, Wrensch F, Herrler G, Pöhlmann S - PLoS ONE (2016)

HAL of batFLUAV are robustly expressed and incorporated into rhabdoviral particles.(A) BHK-21 cells were transfected with the indicated HAL proteins harboring a C-terminal FLAG antigenic tag and protein expression was analyzed by immunofluorescence microscopy of permeabilized cells (magnification, 10x). Cells transfected with an empty expression vector served as negative control. Nuclei were visualized by DAPI staining. Similar results were obtained in a separate experiment. (B) For analysis of HAL incorporation into rhabdoviral particles, vesicular stomatitis virus (VSV)-based pseudotypes were pelleted though a 20% sucrose cushion and analyzed by SDS-PAGE and Western blotting with antibodies against the FLAG tag (α-FLAG), VSV glycoprotein (α-VSV-G) and matrix protein (α-VSV-M). The numbers on the left side of the blots indicate the molecular weight in kilo Daltons (kDa). The results were confirmed in an independent experiment.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4814062&req=5

pone.0152134.g001: HAL of batFLUAV are robustly expressed and incorporated into rhabdoviral particles.(A) BHK-21 cells were transfected with the indicated HAL proteins harboring a C-terminal FLAG antigenic tag and protein expression was analyzed by immunofluorescence microscopy of permeabilized cells (magnification, 10x). Cells transfected with an empty expression vector served as negative control. Nuclei were visualized by DAPI staining. Similar results were obtained in a separate experiment. (B) For analysis of HAL incorporation into rhabdoviral particles, vesicular stomatitis virus (VSV)-based pseudotypes were pelleted though a 20% sucrose cushion and analyzed by SDS-PAGE and Western blotting with antibodies against the FLAG tag (α-FLAG), VSV glycoprotein (α-VSV-G) and matrix protein (α-VSV-M). The numbers on the left side of the blots indicate the molecular weight in kilo Daltons (kDa). The results were confirmed in an independent experiment.
Mentions: To test whether HAL17 and HAL18 are comparably expressed and incorporated into rhabdoviral pseudotypes, both proteins were equipped with a C-terminal FLAG epitope (HAL17-FLAG, HAL18-FLAG), since no batFLUAV-HAL-specific antibody was available. Upon transfection of BHK-21 cells similar numbers of HAL-expressing cells were detected by fluorescence microscopy (Fig 1A) and the intensity of the fluorescence signals emitted by the cells was comparable, indicating robust expression of both batFLUAV-HAL proteins. In order to assess HAL incorporation into rhabdoviral pseudotypes, we pelleted pseudotype preparations through a sucrose cushion and subjected the samples to SDS-PAGE and immunoblotting. Using antibodies specific for the FLAG epitope, VSV-G and VSV matrix protein (VSV-M), we found that VSV-G, as expected, as well as both HAL17 and HAL18 proteins were incorporated into particles, with incorporation of HAL17 being more efficient than that of HAL18. (Fig 1B). Thus, both HAL17 and HAL18 were robustly expressed and incorporated into VSVpp, allowing their functional characterization.

Bottom Line: In contrast, sialic acids were dispensable for HAL-driven entry.Collectively, these results identify viral and cellular factors governing host cell entry driven by batFLUAV surface proteins.They suggest that the absence of a functional receptor precludes entry of batFLUAV into human cells while other prerequisites for entry, HAL activation and protonation, are met in target cells of human origin.

View Article: PubMed Central - PubMed

Affiliation: Infection Biology Unit, German Primate Center, Göttingen, Germany.

ABSTRACT
New World bats have recently been discovered to harbor influenza A virus (FLUAV)-related viruses, termed bat-associated influenza A-like viruses (batFLUAV). The internal proteins of batFLUAV are functional in mammalian cells. In contrast, no biological functionality could be demonstrated for the surface proteins, hemagglutinin (HA)-like (HAL) and neuraminidase (NA)-like (NAL), and these proteins need to be replaced by their human counterparts to allow spread of batFLUAV in human cells. Here, we employed rhabdoviral vectors to study the role of HAL and NAL in viral entry. Vectors pseudotyped with batFLUAV-HAL and -NAL were able to enter bat cells but not cells from other mammalian species. Host cell entry was mediated by HAL and was dependent on prior proteolytic activation of HAL and endosomal low pH. In contrast, sialic acids were dispensable for HAL-driven entry. Finally, the type II transmembrane serine protease TMPRSS2 was able to activate HAL for cell entry indicating that batFLUAV can utilize human proteases for HAL activation. Collectively, these results identify viral and cellular factors governing host cell entry driven by batFLUAV surface proteins. They suggest that the absence of a functional receptor precludes entry of batFLUAV into human cells while other prerequisites for entry, HAL activation and protonation, are met in target cells of human origin.

Show MeSH
Related in: MedlinePlus