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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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Entry driven by batFLUAV-HAL relies on an acidic pH.EpoNi/22.1 cells incubated for 3 h in the absence (black bars) or presence (white bars) of ammonium chloride (50 mM) were subsequently inoculated with trypsin-treated vesicular stomatitis virus-based pseudotypes (VSVpp) harboring the indicated viral glycoproteins. At 1 h post inoculation, the inoculum was removed and the cells were further incubated for 18–20 h in the presence or absence of ammonium chloride before transduction efficiency was measured by quantification of the activity of VSVpp-encoded luciferase. For each of the different pseudotypes, transduction efficiency (given as percentage on a linear scale) was normalized against the respective control (water). The result of a single representative experiment carried out with quadruplicate samples is shown. Similar results were obtained in two independent experiments carried out with separate pseudotype preparations. Error bars indicate standard deviations. A two-tailed, unpaired student’s t-test was used to test statistical significance (* = p < 0.05).
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pone.0152134.g005: Entry driven by batFLUAV-HAL relies on an acidic pH.EpoNi/22.1 cells incubated for 3 h in the absence (black bars) or presence (white bars) of ammonium chloride (50 mM) were subsequently inoculated with trypsin-treated vesicular stomatitis virus-based pseudotypes (VSVpp) harboring the indicated viral glycoproteins. At 1 h post inoculation, the inoculum was removed and the cells were further incubated for 18–20 h in the presence or absence of ammonium chloride before transduction efficiency was measured by quantification of the activity of VSVpp-encoded luciferase. For each of the different pseudotypes, transduction efficiency (given as percentage on a linear scale) was normalized against the respective control (water). The result of a single representative experiment carried out with quadruplicate samples is shown. Similar results were obtained in two independent experiments carried out with separate pseudotype preparations. Error bars indicate standard deviations. A two-tailed, unpaired student’s t-test was used to test statistical significance (* = p < 0.05).

Mentions: Endosomal low pH triggers FLUAV-HA for membrane fusion. Therefore, we investigated whether increasing the endosomal pH in EpoNi/22.1 cells by ammonium chloride treatment impacts HAL-driven entry. As expected, ammonium chloride treatment led to a decrease in transduction efficiency mediated by pseudotypes bearing the HA-proteins of FLUAV of the H1N1 and H2N2 subtype and VSV-G (Fig 5). In contrast, pseudotype entry orchestrated by NiV-F and -G was unaffected, again in keeping with published data [52, 53]. Finally, HAL-driven entry was markedly reduced by ammonium chloride, demonstrating that the membrane fusion activity of batFLUAV-HAL is triggered by acidification (Fig 5).


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)

Entry driven by batFLUAV-HAL relies on an acidic pH.EpoNi/22.1 cells incubated for 3 h in the absence (black bars) or presence (white bars) of ammonium chloride (50 mM) were subsequently inoculated with trypsin-treated vesicular stomatitis virus-based pseudotypes (VSVpp) harboring the indicated viral glycoproteins. At 1 h post inoculation, the inoculum was removed and the cells were further incubated for 18–20 h in the presence or absence of ammonium chloride before transduction efficiency was measured by quantification of the activity of VSVpp-encoded luciferase. For each of the different pseudotypes, transduction efficiency (given as percentage on a linear scale) was normalized against the respective control (water). The result of a single representative experiment carried out with quadruplicate samples is shown. Similar results were obtained in two independent experiments carried out with separate pseudotype preparations. Error bars indicate standard deviations. A two-tailed, unpaired student’s t-test was used to test statistical significance (* = p < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0152134.g005: Entry driven by batFLUAV-HAL relies on an acidic pH.EpoNi/22.1 cells incubated for 3 h in the absence (black bars) or presence (white bars) of ammonium chloride (50 mM) were subsequently inoculated with trypsin-treated vesicular stomatitis virus-based pseudotypes (VSVpp) harboring the indicated viral glycoproteins. At 1 h post inoculation, the inoculum was removed and the cells were further incubated for 18–20 h in the presence or absence of ammonium chloride before transduction efficiency was measured by quantification of the activity of VSVpp-encoded luciferase. For each of the different pseudotypes, transduction efficiency (given as percentage on a linear scale) was normalized against the respective control (water). The result of a single representative experiment carried out with quadruplicate samples is shown. Similar results were obtained in two independent experiments carried out with separate pseudotype preparations. Error bars indicate standard deviations. A two-tailed, unpaired student’s t-test was used to test statistical significance (* = p < 0.05).
Mentions: Endosomal low pH triggers FLUAV-HA for membrane fusion. Therefore, we investigated whether increasing the endosomal pH in EpoNi/22.1 cells by ammonium chloride treatment impacts HAL-driven entry. As expected, ammonium chloride treatment led to a decrease in transduction efficiency mediated by pseudotypes bearing the HA-proteins of FLUAV of the H1N1 and H2N2 subtype and VSV-G (Fig 5). In contrast, pseudotype entry orchestrated by NiV-F and -G was unaffected, again in keeping with published data [52, 53]. Finally, HAL-driven entry was markedly reduced by ammonium chloride, demonstrating that the membrane fusion activity of batFLUAV-HAL is triggered by acidification (Fig 5).

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