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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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Surface glycoproteins of batFLUAV enable pseudotype entry into different bat but not human, simian and canine cell lines.Vesicular stomatitis virus-based pseudotypes (VSVpp) harboring no or the indicated viral glycoproteins were either left untreated (white bars) or treated with trypsin (black bars), before they were inoculated onto mammalian cell lines of human (HEK-293T, Huh7), simian (Vero) and canine (MDCK) origin (A) or bat cell lines (RoNi/7, EidNi/41, HypNi/1.1, EpoNi/22.1, CpKd) representing five different bat species (B). At 18–20 h post inoculation, the transduction efficiency was measured by quantification of the activity of the VSVpp-encoded luciferase (given as counts per second, cps, on a logarithmic scale). The result of a single representative experiment carried out with quadruplicate samples is shown. Similar results were obtained in four 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.g002: Surface glycoproteins of batFLUAV enable pseudotype entry into different bat but not human, simian and canine cell lines.Vesicular stomatitis virus-based pseudotypes (VSVpp) harboring no or the indicated viral glycoproteins were either left untreated (white bars) or treated with trypsin (black bars), before they were inoculated onto mammalian cell lines of human (HEK-293T, Huh7), simian (Vero) and canine (MDCK) origin (A) or bat cell lines (RoNi/7, EidNi/41, HypNi/1.1, EpoNi/22.1, CpKd) representing five different bat species (B). At 18–20 h post inoculation, the transduction efficiency was measured by quantification of the activity of the VSVpp-encoded luciferase (given as counts per second, cps, on a logarithmic scale). The result of a single representative experiment carried out with quadruplicate samples is shown. Similar results were obtained in four 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: We found that none of the human, simian and canine cell lines was susceptible to entry driven by batFLUAV surface proteins (Fig 2A). In contrast, pseudotypes bearing VSV-G, NiV-F/G or WSN-HA/NA could readily enter these cells, whereas pseudotypes that harbored 1918- or H2N2-HA/NA required activation by exogenous trypsin for efficient transduction (Fig 2A). These results are in agreement with expectations, since activation of WSN-HA is known to be independent of trypsin [42–44], although viral infectivity can be enhanced by trypsin treatment.


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)

Surface glycoproteins of batFLUAV enable pseudotype entry into different bat but not human, simian and canine cell lines.Vesicular stomatitis virus-based pseudotypes (VSVpp) harboring no or the indicated viral glycoproteins were either left untreated (white bars) or treated with trypsin (black bars), before they were inoculated onto mammalian cell lines of human (HEK-293T, Huh7), simian (Vero) and canine (MDCK) origin (A) or bat cell lines (RoNi/7, EidNi/41, HypNi/1.1, EpoNi/22.1, CpKd) representing five different bat species (B). At 18–20 h post inoculation, the transduction efficiency was measured by quantification of the activity of the VSVpp-encoded luciferase (given as counts per second, cps, on a logarithmic scale). The result of a single representative experiment carried out with quadruplicate samples is shown. Similar results were obtained in four 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.g002: Surface glycoproteins of batFLUAV enable pseudotype entry into different bat but not human, simian and canine cell lines.Vesicular stomatitis virus-based pseudotypes (VSVpp) harboring no or the indicated viral glycoproteins were either left untreated (white bars) or treated with trypsin (black bars), before they were inoculated onto mammalian cell lines of human (HEK-293T, Huh7), simian (Vero) and canine (MDCK) origin (A) or bat cell lines (RoNi/7, EidNi/41, HypNi/1.1, EpoNi/22.1, CpKd) representing five different bat species (B). At 18–20 h post inoculation, the transduction efficiency was measured by quantification of the activity of the VSVpp-encoded luciferase (given as counts per second, cps, on a logarithmic scale). The result of a single representative experiment carried out with quadruplicate samples is shown. Similar results were obtained in four 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: We found that none of the human, simian and canine cell lines was susceptible to entry driven by batFLUAV surface proteins (Fig 2A). In contrast, pseudotypes bearing VSV-G, NiV-F/G or WSN-HA/NA could readily enter these cells, whereas pseudotypes that harbored 1918- or H2N2-HA/NA required activation by exogenous trypsin for efficient transduction (Fig 2A). These results are in agreement with expectations, since activation of WSN-HA is known to be independent of trypsin [42–44], although viral infectivity can be enhanced by trypsin treatment.

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