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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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Human proteases that activate FLUAV-HA for cell entry also activate batFLUAV-HAL.(A) HEK-293T cells were transfected with plasmids encoding HA or HAL proteins and either trypsin treated or cotransfected with plasmids encoding type II transmembrane serine proteases. Transfection of empty vector served as negative control. Cleavage of HA/HAL proteins was analyzed by SDS-PAGE and Western blotting, employing antibodies against FLUAV-HA (α-FLUAV) and the FLAG epitope (α-FLAG). Detection of ß-actin served as loading control. Signals corresponding to uncleaved precursor proteins are marked by black circles, while products of proteolytic cleavage are indicated by white circles. The results were confirmed in a separate experiment. To assess proteolytic activation of HA/HAL proteins, vesicular stomatitis virus-based pseudotypes (VSVpp) were produced in cells transfected to express the indicated type II transmembrane serine proteases (B) or different amounts of TMPRSS2 (C). Pseudotypes were either directly used for transduction of EpoNi/22.1 cells (black bars) or previously treated with trypsin (white bars). At 24 h post inoculation, transduction efficiency was measured by quantification of the activity of VSVpp-encoded luciferase in cell lysates. For normalization, transduction by HA- or HAL-bearing pseudotypes that were produced in the absence of type II transmembrane serine protease expression (empty vector) and not treated with trypsin was set as 1. The result of a single representative experiment carried out with quadruplicate samples is presented. Similar results were obtained in three 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.g006: Human proteases that activate FLUAV-HA for cell entry also activate batFLUAV-HAL.(A) HEK-293T cells were transfected with plasmids encoding HA or HAL proteins and either trypsin treated or cotransfected with plasmids encoding type II transmembrane serine proteases. Transfection of empty vector served as negative control. Cleavage of HA/HAL proteins was analyzed by SDS-PAGE and Western blotting, employing antibodies against FLUAV-HA (α-FLUAV) and the FLAG epitope (α-FLAG). Detection of ß-actin served as loading control. Signals corresponding to uncleaved precursor proteins are marked by black circles, while products of proteolytic cleavage are indicated by white circles. The results were confirmed in a separate experiment. To assess proteolytic activation of HA/HAL proteins, vesicular stomatitis virus-based pseudotypes (VSVpp) were produced in cells transfected to express the indicated type II transmembrane serine proteases (B) or different amounts of TMPRSS2 (C). Pseudotypes were either directly used for transduction of EpoNi/22.1 cells (black bars) or previously treated with trypsin (white bars). At 24 h post inoculation, transduction efficiency was measured by quantification of the activity of VSVpp-encoded luciferase in cell lysates. For normalization, transduction by HA- or HAL-bearing pseudotypes that were produced in the absence of type II transmembrane serine protease expression (empty vector) and not treated with trypsin was set as 1. The result of a single representative experiment carried out with quadruplicate samples is presented. Similar results were obtained in three 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: FLUAV-HA is synthesized as an inactive precursor and requires activation by host cell proteases to be responsive to low pH, the trigger for HA-driven membrane fusion [12–14]. Members of the TTSP family activate FLUAV-HA in cell culture [33, 54–58] and TMPRSS2 was previously shown to be essential for FLUAV-HA activation and viral spread in mice [59]. Therefore, we asked whether TTSPs able to activate FLUAV-HA can also activate batFLUAV-HAL. For this, we first investigated batFLUAV-HAL cleavage by TMPRSS2, DESC-1 and MSPL, and compared it to cleavage by trypsin. Cleavage of the 1918-HA served as positive control. We found that 1918-HA was efficiently processed by all proteases tested, as expected. Moreover, we could show that coexpression of TMPRSS2, DESC-1 and MSPL, and trypsin treatment resulted in cleavage of the HAL precursor (HAL0) determined by the appearance of bands corresponding to the HAL2 subunit (Fig 6A). While HAL18 was comparably cleaved by all tested TTSPs, HAL17 cleavage by TMPRSS2 was more pronounced than proteolysis by DESC-1 and MSPL (Fig 6A). Moreover, HAL18 was generally more sensitive to cleavage by TTSPs than HAL17 (Fig 6A). In order to assess whether batFLUAV-HAL cleavage by TTSPs also leads to HAL activation for host cell entry, we produced pseudotypes harboring batFLUAV-HAL (HAL17 or HAL18) in the presence of TMPRSS2, DESC-1 and MSPL. As a control, pseudotypes bearing 1918-HA and -NA were included in this experiment. The pseudotypes were treated with trypsin to activate HA/HAL or were mock-treated before addition to EpoNi/22.1 cells. Pseudotypes bearing 1918-HA and -NA and produced in the presence of TMPRSS2, DESC-1 and MSPL or treated with trypsin robustly transduced target cells (Fig 6B). In contrast, infectivity of FLUAV-HA pseudotypes produced in the absence of TTSPs or not treated with trypsin was in the background range (Fig 6B). Similarly, batFLUAV-HAL-bearing pseudotypes were activated by trypsin or TTSPs, including TMPRSS2 (Fig 6B). However, differences in the activation of HAL17 and HAL18 were observed and correlated with the efficiency of HAL protein cleavage, as determined above (Fig 6A). Thus, expression of TMPRSS2 but not DESC-1 and MSPL conferred robust infectivity to HAL17-bearing pseudotypes while all proteases were able to efficiently activate HAL18. Moreover, transfection of escalating amounts of TMPRSS2-encoding plasmids increased infectivity of HAL17-bearing pseudotypes in a concentration-dependent manner. In contrast, transfection of even the lowest amount of TMPRSS2 plasmid was sufficient to confer maximal infectivity of HAL18-bearing pseudotypes, confirming that the efficiency of TMPRSS2-mediated activation of HAL is subtype specific (Fig 6C). In sum, proteolytic activation of batFLUAV-HAL is critical for HAL-driven cell entry and proteases able to activate HA can also activate HAL.


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)

Human proteases that activate FLUAV-HA for cell entry also activate batFLUAV-HAL.(A) HEK-293T cells were transfected with plasmids encoding HA or HAL proteins and either trypsin treated or cotransfected with plasmids encoding type II transmembrane serine proteases. Transfection of empty vector served as negative control. Cleavage of HA/HAL proteins was analyzed by SDS-PAGE and Western blotting, employing antibodies against FLUAV-HA (α-FLUAV) and the FLAG epitope (α-FLAG). Detection of ß-actin served as loading control. Signals corresponding to uncleaved precursor proteins are marked by black circles, while products of proteolytic cleavage are indicated by white circles. The results were confirmed in a separate experiment. To assess proteolytic activation of HA/HAL proteins, vesicular stomatitis virus-based pseudotypes (VSVpp) were produced in cells transfected to express the indicated type II transmembrane serine proteases (B) or different amounts of TMPRSS2 (C). Pseudotypes were either directly used for transduction of EpoNi/22.1 cells (black bars) or previously treated with trypsin (white bars). At 24 h post inoculation, transduction efficiency was measured by quantification of the activity of VSVpp-encoded luciferase in cell lysates. For normalization, transduction by HA- or HAL-bearing pseudotypes that were produced in the absence of type II transmembrane serine protease expression (empty vector) and not treated with trypsin was set as 1. The result of a single representative experiment carried out with quadruplicate samples is presented. Similar results were obtained in three 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.g006: Human proteases that activate FLUAV-HA for cell entry also activate batFLUAV-HAL.(A) HEK-293T cells were transfected with plasmids encoding HA or HAL proteins and either trypsin treated or cotransfected with plasmids encoding type II transmembrane serine proteases. Transfection of empty vector served as negative control. Cleavage of HA/HAL proteins was analyzed by SDS-PAGE and Western blotting, employing antibodies against FLUAV-HA (α-FLUAV) and the FLAG epitope (α-FLAG). Detection of ß-actin served as loading control. Signals corresponding to uncleaved precursor proteins are marked by black circles, while products of proteolytic cleavage are indicated by white circles. The results were confirmed in a separate experiment. To assess proteolytic activation of HA/HAL proteins, vesicular stomatitis virus-based pseudotypes (VSVpp) were produced in cells transfected to express the indicated type II transmembrane serine proteases (B) or different amounts of TMPRSS2 (C). Pseudotypes were either directly used for transduction of EpoNi/22.1 cells (black bars) or previously treated with trypsin (white bars). At 24 h post inoculation, transduction efficiency was measured by quantification of the activity of VSVpp-encoded luciferase in cell lysates. For normalization, transduction by HA- or HAL-bearing pseudotypes that were produced in the absence of type II transmembrane serine protease expression (empty vector) and not treated with trypsin was set as 1. The result of a single representative experiment carried out with quadruplicate samples is presented. Similar results were obtained in three 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: FLUAV-HA is synthesized as an inactive precursor and requires activation by host cell proteases to be responsive to low pH, the trigger for HA-driven membrane fusion [12–14]. Members of the TTSP family activate FLUAV-HA in cell culture [33, 54–58] and TMPRSS2 was previously shown to be essential for FLUAV-HA activation and viral spread in mice [59]. Therefore, we asked whether TTSPs able to activate FLUAV-HA can also activate batFLUAV-HAL. For this, we first investigated batFLUAV-HAL cleavage by TMPRSS2, DESC-1 and MSPL, and compared it to cleavage by trypsin. Cleavage of the 1918-HA served as positive control. We found that 1918-HA was efficiently processed by all proteases tested, as expected. Moreover, we could show that coexpression of TMPRSS2, DESC-1 and MSPL, and trypsin treatment resulted in cleavage of the HAL precursor (HAL0) determined by the appearance of bands corresponding to the HAL2 subunit (Fig 6A). While HAL18 was comparably cleaved by all tested TTSPs, HAL17 cleavage by TMPRSS2 was more pronounced than proteolysis by DESC-1 and MSPL (Fig 6A). Moreover, HAL18 was generally more sensitive to cleavage by TTSPs than HAL17 (Fig 6A). In order to assess whether batFLUAV-HAL cleavage by TTSPs also leads to HAL activation for host cell entry, we produced pseudotypes harboring batFLUAV-HAL (HAL17 or HAL18) in the presence of TMPRSS2, DESC-1 and MSPL. As a control, pseudotypes bearing 1918-HA and -NA were included in this experiment. The pseudotypes were treated with trypsin to activate HA/HAL or were mock-treated before addition to EpoNi/22.1 cells. Pseudotypes bearing 1918-HA and -NA and produced in the presence of TMPRSS2, DESC-1 and MSPL or treated with trypsin robustly transduced target cells (Fig 6B). In contrast, infectivity of FLUAV-HA pseudotypes produced in the absence of TTSPs or not treated with trypsin was in the background range (Fig 6B). Similarly, batFLUAV-HAL-bearing pseudotypes were activated by trypsin or TTSPs, including TMPRSS2 (Fig 6B). However, differences in the activation of HAL17 and HAL18 were observed and correlated with the efficiency of HAL protein cleavage, as determined above (Fig 6A). Thus, expression of TMPRSS2 but not DESC-1 and MSPL conferred robust infectivity to HAL17-bearing pseudotypes while all proteases were able to efficiently activate HAL18. Moreover, transfection of escalating amounts of TMPRSS2-encoding plasmids increased infectivity of HAL17-bearing pseudotypes in a concentration-dependent manner. In contrast, transfection of even the lowest amount of TMPRSS2 plasmid was sufficient to confer maximal infectivity of HAL18-bearing pseudotypes, confirming that the efficiency of TMPRSS2-mediated activation of HAL is subtype specific (Fig 6C). In sum, proteolytic activation of batFLUAV-HAL is critical for HAL-driven cell entry and proteases able to activate HA can also activate HAL.

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