Analysis of parainfluenza virus-5 hemagglutinin-neuraminidase protein mutants that are blocked in internalization and degradation

Jessica G. Robach; Robert A. Lamb

doi:10.1016/j.virol.2010.06.049

Analysis of parainfluenza virus-5 hemagglutinin-neuraminidase protein mutants that are blocked in internalization and degradation

Jessica G. Robach, Robert A. Lamb^*

^*Corresponding author for this work

Molecular Biosciences

Research output: Contribution to journal › Article › peer-review

7 Scopus citations

Abstract

The PIV-5 hemagglutinin-neuraminidase (HN) protein is a multifunctional protein with sialic acid binding, neuraminidase and fusion promotion activity. HN is internalized by clathrin-mediated endocytosis and degraded. HN lacks internalization signals in its cytoplasmic tail but a single glutamic acid present at residue 37 at the putative transmembrane/ectodomain boundary is critical. We rescued rPIV-5 with mutations E37D or E37K, which have been shown to impair or abolish HN internalization, respectively. These viruses exhibited growth properties similar to wild-type (wt) virus but are impaired for fitness in tissue culture. Biochemical analysis of HN activities showed differences between HN E37D and HN E37K in fusion promotion and incorporation of HN and F into virions. Furthermore, oligomeric analyses indicate that HN E37 mutants perturb the tetrameric organization of HN, probably by destabilizing the dimer-of-dimers interface.

Original language	English (US)
Pages (from-to)	189-201
Number of pages	13
Journal	Virology
Volume	406
Issue number	2
DOIs	https://doi.org/10.1016/j.virol.2010.06.049
State	Published - Oct 2010

Funding

We are grateful to David Waning and Anthony Schmitt who originally made the HN E37D and HN E37K constructs, respectively. We are grateful to George Leser for many discussions about HN internalization experiments. The Northwestern University Keck Biophysics Facility was used for the circular dichroism measurements and the facility is supported by a Cancer Center Support Grant ( NCI CA060553 ). This research was supported in part by a Public Health Services grant AI-23173 from the National Institute of Allergy and Infectious Diseases . J.G.R. was supported by predoctoral training grant T32 AI060523 from the National Institute of Allergy and Infectious Diseases . R.A.L. is an investigator of the Howard Hughes Medical Institute.

Keywords

Degradation
Hemagglutinin-neuraminidase protein
Paramyxoviruses
Protein oligomerization
Reverse genetics
Viral fitness

ASJC Scopus subject areas

Virology

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10.1016/j.virol.2010.06.049

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title = "Analysis of parainfluenza virus-5 hemagglutinin-neuraminidase protein mutants that are blocked in internalization and degradation",

abstract = "The PIV-5 hemagglutinin-neuraminidase (HN) protein is a multifunctional protein with sialic acid binding, neuraminidase and fusion promotion activity. HN is internalized by clathrin-mediated endocytosis and degraded. HN lacks internalization signals in its cytoplasmic tail but a single glutamic acid present at residue 37 at the putative transmembrane/ectodomain boundary is critical. We rescued rPIV-5 with mutations E37D or E37K, which have been shown to impair or abolish HN internalization, respectively. These viruses exhibited growth properties similar to wild-type (wt) virus but are impaired for fitness in tissue culture. Biochemical analysis of HN activities showed differences between HN E37D and HN E37K in fusion promotion and incorporation of HN and F into virions. Furthermore, oligomeric analyses indicate that HN E37 mutants perturb the tetrameric organization of HN, probably by destabilizing the dimer-of-dimers interface.",

keywords = "Degradation, Hemagglutinin-neuraminidase protein, Paramyxoviruses, Protein oligomerization, Reverse genetics, Viral fitness",

author = "Robach, \{Jessica G.\} and Lamb, \{Robert A.\}",

note = "Funding Information: We are grateful to David Waning and Anthony Schmitt who originally made the HN E37D and HN E37K constructs, respectively. We are grateful to George Leser for many discussions about HN internalization experiments. The Northwestern University Keck Biophysics Facility was used for the circular dichroism measurements and the facility is supported by a Cancer Center Support Grant ( NCI CA060553 ). This research was supported in part by a Public Health Services grant AI-23173 from the National Institute of Allergy and Infectious Diseases . J.G.R. was supported by predoctoral training grant T32 AI060523 from the National Institute of Allergy and Infectious Diseases . R.A.L. is an investigator of the Howard Hughes Medical Institute.",

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N2 - The PIV-5 hemagglutinin-neuraminidase (HN) protein is a multifunctional protein with sialic acid binding, neuraminidase and fusion promotion activity. HN is internalized by clathrin-mediated endocytosis and degraded. HN lacks internalization signals in its cytoplasmic tail but a single glutamic acid present at residue 37 at the putative transmembrane/ectodomain boundary is critical. We rescued rPIV-5 with mutations E37D or E37K, which have been shown to impair or abolish HN internalization, respectively. These viruses exhibited growth properties similar to wild-type (wt) virus but are impaired for fitness in tissue culture. Biochemical analysis of HN activities showed differences between HN E37D and HN E37K in fusion promotion and incorporation of HN and F into virions. Furthermore, oligomeric analyses indicate that HN E37 mutants perturb the tetrameric organization of HN, probably by destabilizing the dimer-of-dimers interface.

AB - The PIV-5 hemagglutinin-neuraminidase (HN) protein is a multifunctional protein with sialic acid binding, neuraminidase and fusion promotion activity. HN is internalized by clathrin-mediated endocytosis and degraded. HN lacks internalization signals in its cytoplasmic tail but a single glutamic acid present at residue 37 at the putative transmembrane/ectodomain boundary is critical. We rescued rPIV-5 with mutations E37D or E37K, which have been shown to impair or abolish HN internalization, respectively. These viruses exhibited growth properties similar to wild-type (wt) virus but are impaired for fitness in tissue culture. Biochemical analysis of HN activities showed differences between HN E37D and HN E37K in fusion promotion and incorporation of HN and F into virions. Furthermore, oligomeric analyses indicate that HN E37 mutants perturb the tetrameric organization of HN, probably by destabilizing the dimer-of-dimers interface.

KW - Degradation

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KW - Protein oligomerization

KW - Reverse genetics

KW - Viral fitness

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Analysis of parainfluenza virus-5 hemagglutinin-neuraminidase protein mutants that are blocked in internalization and degradation

Abstract

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Keywords

ASJC Scopus subject areas

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