A point mutation in the transmembrane domain of the hemagglutinin of influenza virus stabilizes a hemifusion intermediate that can transit to fusion

G. B. Melikyan, R. M. Markosyan, M. G. Roth, F. S. Cohen

Research output: Contribution to journalArticle

85 Citations (Scopus)

Abstract

A hemagglutinin (HA) of influenza virus having a single semiconserved Gly residue within the transmembrane domain mutated to Leu (G520L) was expressed on cells; these cells were bound to red blood cells. By decreasing pH at 23°C rather than 37°C, an intermediate with properties expected of hemifusion just as the membranes are about to transit to full fusion was captured. As evidence: 1) increasing temperature to 37°C at neutral pH allowed fusion to proceed; 2) after achieving the intermediate, the two membranes did not separate from each other after proteolytic cleavage of G520L because cells treated with proteinase K could not fuse upon temperature increase but could fuse upon the addition of chlorpromazine; and 3) at the point of the intermediate, adding exogenous lipids known to promote or inhibit the creation of hemifusion did not significantly alter the lipid dye spread that occurred upon increasing temperature, implying that at the intermediate, contacting membrane leaflets had already merged. A stable intermediate of hemifusion that could transit to fusion was also generated for wild-type HA, but pH had to be reduced at the significantly lower temperature of 4°C. The fusion pores generated by G520L did not enlarge, whereas those induced by wild-type HA did. The finding that a state of transitional hemifusion can be readily obtained via a point mutation without the need for unusually low temperature supports the hypothesis that hemifusion occurs before pore formation.

Original languageEnglish (US)
Pages (from-to)3765-3775
Number of pages11
JournalMolecular Biology of the Cell
Volume11
Issue number11
StatePublished - 2000

Fingerprint

Hemagglutinins
Orthomyxoviridae
Point Mutation
Temperature
Membranes
Lipids
Endopeptidase K
Influenza A virus
Chlorpromazine
Coloring Agents
Erythrocytes

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

A point mutation in the transmembrane domain of the hemagglutinin of influenza virus stabilizes a hemifusion intermediate that can transit to fusion. / Melikyan, G. B.; Markosyan, R. M.; Roth, M. G.; Cohen, F. S.

In: Molecular Biology of the Cell, Vol. 11, No. 11, 2000, p. 3765-3775.

Research output: Contribution to journalArticle

@article{76238db14970496c9610754fb6bc8b81,
title = "A point mutation in the transmembrane domain of the hemagglutinin of influenza virus stabilizes a hemifusion intermediate that can transit to fusion",
abstract = "A hemagglutinin (HA) of influenza virus having a single semiconserved Gly residue within the transmembrane domain mutated to Leu (G520L) was expressed on cells; these cells were bound to red blood cells. By decreasing pH at 23°C rather than 37°C, an intermediate with properties expected of hemifusion just as the membranes are about to transit to full fusion was captured. As evidence: 1) increasing temperature to 37°C at neutral pH allowed fusion to proceed; 2) after achieving the intermediate, the two membranes did not separate from each other after proteolytic cleavage of G520L because cells treated with proteinase K could not fuse upon temperature increase but could fuse upon the addition of chlorpromazine; and 3) at the point of the intermediate, adding exogenous lipids known to promote or inhibit the creation of hemifusion did not significantly alter the lipid dye spread that occurred upon increasing temperature, implying that at the intermediate, contacting membrane leaflets had already merged. A stable intermediate of hemifusion that could transit to fusion was also generated for wild-type HA, but pH had to be reduced at the significantly lower temperature of 4°C. The fusion pores generated by G520L did not enlarge, whereas those induced by wild-type HA did. The finding that a state of transitional hemifusion can be readily obtained via a point mutation without the need for unusually low temperature supports the hypothesis that hemifusion occurs before pore formation.",
author = "Melikyan, {G. B.} and Markosyan, {R. M.} and Roth, {M. G.} and Cohen, {F. S.}",
year = "2000",
language = "English (US)",
volume = "11",
pages = "3765--3775",
journal = "Molecular Biology of the Cell",
issn = "1059-1524",
publisher = "American Society for Cell Biology",
number = "11",

}

TY - JOUR

T1 - A point mutation in the transmembrane domain of the hemagglutinin of influenza virus stabilizes a hemifusion intermediate that can transit to fusion

AU - Melikyan, G. B.

AU - Markosyan, R. M.

AU - Roth, M. G.

AU - Cohen, F. S.

PY - 2000

Y1 - 2000

N2 - A hemagglutinin (HA) of influenza virus having a single semiconserved Gly residue within the transmembrane domain mutated to Leu (G520L) was expressed on cells; these cells were bound to red blood cells. By decreasing pH at 23°C rather than 37°C, an intermediate with properties expected of hemifusion just as the membranes are about to transit to full fusion was captured. As evidence: 1) increasing temperature to 37°C at neutral pH allowed fusion to proceed; 2) after achieving the intermediate, the two membranes did not separate from each other after proteolytic cleavage of G520L because cells treated with proteinase K could not fuse upon temperature increase but could fuse upon the addition of chlorpromazine; and 3) at the point of the intermediate, adding exogenous lipids known to promote or inhibit the creation of hemifusion did not significantly alter the lipid dye spread that occurred upon increasing temperature, implying that at the intermediate, contacting membrane leaflets had already merged. A stable intermediate of hemifusion that could transit to fusion was also generated for wild-type HA, but pH had to be reduced at the significantly lower temperature of 4°C. The fusion pores generated by G520L did not enlarge, whereas those induced by wild-type HA did. The finding that a state of transitional hemifusion can be readily obtained via a point mutation without the need for unusually low temperature supports the hypothesis that hemifusion occurs before pore formation.

AB - A hemagglutinin (HA) of influenza virus having a single semiconserved Gly residue within the transmembrane domain mutated to Leu (G520L) was expressed on cells; these cells were bound to red blood cells. By decreasing pH at 23°C rather than 37°C, an intermediate with properties expected of hemifusion just as the membranes are about to transit to full fusion was captured. As evidence: 1) increasing temperature to 37°C at neutral pH allowed fusion to proceed; 2) after achieving the intermediate, the two membranes did not separate from each other after proteolytic cleavage of G520L because cells treated with proteinase K could not fuse upon temperature increase but could fuse upon the addition of chlorpromazine; and 3) at the point of the intermediate, adding exogenous lipids known to promote or inhibit the creation of hemifusion did not significantly alter the lipid dye spread that occurred upon increasing temperature, implying that at the intermediate, contacting membrane leaflets had already merged. A stable intermediate of hemifusion that could transit to fusion was also generated for wild-type HA, but pH had to be reduced at the significantly lower temperature of 4°C. The fusion pores generated by G520L did not enlarge, whereas those induced by wild-type HA did. The finding that a state of transitional hemifusion can be readily obtained via a point mutation without the need for unusually low temperature supports the hypothesis that hemifusion occurs before pore formation.

UR - http://www.scopus.com/inward/record.url?scp=0033730718&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033730718&partnerID=8YFLogxK

M3 - Article

C2 - 11071905

AN - SCOPUS:0033730718

VL - 11

SP - 3765

EP - 3775

JO - Molecular Biology of the Cell

JF - Molecular Biology of the Cell

SN - 1059-1524

IS - 11

ER -