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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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NAL of batFLUAV has no impact on the transduction efficiency of vectors bearing HAL.Vesicular stomatitis virus-based pseudotypes (VSVpp) harboring the indicated surface proteins of FLUAV or batFLUAV were treated with trypsin before inoculation of MDCK and EpoNi/22.1 cells. At 18–20 h post inoculation, the transduction efficiency was measured by quantification of the activity of the VSVpp-encoded luciferase. The combined data from three independent experiments (quadruplicate samples) with separate pseudotype preparations are shown. Transduction efficiencies were normalized against pseudotypes harboring only HA or HAL (set as 1) and are given as x-fold changes on a logarithmic scale. Error bars indicate standard error of the mean. A two-tailed, paired student’s t-test was used to test statistical significance (* = p < 0.05).
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pone.0152134.g003: NAL of batFLUAV has no impact on the transduction efficiency of vectors bearing HAL.Vesicular stomatitis virus-based pseudotypes (VSVpp) harboring the indicated surface proteins of FLUAV or batFLUAV were treated with trypsin before inoculation of MDCK and EpoNi/22.1 cells. At 18–20 h post inoculation, the transduction efficiency was measured by quantification of the activity of the VSVpp-encoded luciferase. The combined data from three independent experiments (quadruplicate samples) with separate pseudotype preparations are shown. Transduction efficiencies were normalized against pseudotypes harboring only HA or HAL (set as 1) and are given as x-fold changes on a logarithmic scale. Error bars indicate standard error of the mean. A two-tailed, paired student’s t-test was used to test statistical significance (* = p < 0.05).

Mentions: The NA proteins of human FLUAV facilitate release of progeny particles from infected cells by removing sialic acids from the cell surface. To study the impact of the batFLUAV-NAL on transduction efficiency, we produced pseudotypes bearing batFLUAV-HAL, -NAL or both proteins. For comparison, we generated pseudotypes harboring WSN-HA, WSN-NA or both proteins. These pseudotypes were then used for inoculation of MDCK (inoculated with pseudotypes bearing WSN proteins) and EpoNi/22.1 (inoculated with pseudotypes bearing WSN or batFLUAV proteins) cells. Pseudotypes harboring only WSN-NA were not able to transduce target cells (Fig 3) while pseudotypes bearing either WSN-HA alone or in combination with WSN-NA transduced both MDCK and EpoNi/22.1, as expected. Transduction efficiency was ~500–1,500-fold higher when WSN-NA was expressed in cells used for pseudotype production, in keeping with the findings that the presence of NA is required for efficient release of HA-bearing vectors and infectious FLUAV [32, 45, 46]. Pseudotypes harboring NAL were not infectious while pseudotypes bearing HAL robustly transduced EpoNi/22.1 cells, indicating that batFLUAV-HAL, like WSN-HA, is sufficient to mediate host cell entry. However, unlike WSN-NA, the expression of NAL in pseudotype producer cells did not increase transduction efficiency of HAL-harboring pseudotypes (Fig 3), suggesting that NAL is not required for release and/or infectivity of HAL containing particles, at least in the experimental system chosen.


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)

NAL of batFLUAV has no impact on the transduction efficiency of vectors bearing HAL.Vesicular stomatitis virus-based pseudotypes (VSVpp) harboring the indicated surface proteins of FLUAV or batFLUAV were treated with trypsin before inoculation of MDCK and EpoNi/22.1 cells. At 18–20 h post inoculation, the transduction efficiency was measured by quantification of the activity of the VSVpp-encoded luciferase. The combined data from three independent experiments (quadruplicate samples) with separate pseudotype preparations are shown. Transduction efficiencies were normalized against pseudotypes harboring only HA or HAL (set as 1) and are given as x-fold changes on a logarithmic scale. Error bars indicate standard error of the mean. A two-tailed, paired 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.g003: NAL of batFLUAV has no impact on the transduction efficiency of vectors bearing HAL.Vesicular stomatitis virus-based pseudotypes (VSVpp) harboring the indicated surface proteins of FLUAV or batFLUAV were treated with trypsin before inoculation of MDCK and EpoNi/22.1 cells. At 18–20 h post inoculation, the transduction efficiency was measured by quantification of the activity of the VSVpp-encoded luciferase. The combined data from three independent experiments (quadruplicate samples) with separate pseudotype preparations are shown. Transduction efficiencies were normalized against pseudotypes harboring only HA or HAL (set as 1) and are given as x-fold changes on a logarithmic scale. Error bars indicate standard error of the mean. A two-tailed, paired student’s t-test was used to test statistical significance (* = p < 0.05).
Mentions: The NA proteins of human FLUAV facilitate release of progeny particles from infected cells by removing sialic acids from the cell surface. To study the impact of the batFLUAV-NAL on transduction efficiency, we produced pseudotypes bearing batFLUAV-HAL, -NAL or both proteins. For comparison, we generated pseudotypes harboring WSN-HA, WSN-NA or both proteins. These pseudotypes were then used for inoculation of MDCK (inoculated with pseudotypes bearing WSN proteins) and EpoNi/22.1 (inoculated with pseudotypes bearing WSN or batFLUAV proteins) cells. Pseudotypes harboring only WSN-NA were not able to transduce target cells (Fig 3) while pseudotypes bearing either WSN-HA alone or in combination with WSN-NA transduced both MDCK and EpoNi/22.1, as expected. Transduction efficiency was ~500–1,500-fold higher when WSN-NA was expressed in cells used for pseudotype production, in keeping with the findings that the presence of NA is required for efficient release of HA-bearing vectors and infectious FLUAV [32, 45, 46]. Pseudotypes harboring NAL were not infectious while pseudotypes bearing HAL robustly transduced EpoNi/22.1 cells, indicating that batFLUAV-HAL, like WSN-HA, is sufficient to mediate host cell entry. However, unlike WSN-NA, the expression of NAL in pseudotype producer cells did not increase transduction efficiency of HAL-harboring pseudotypes (Fig 3), suggesting that NAL is not required for release and/or infectivity of HAL containing particles, at least in the experimental system chosen.

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