Mutants of the influenza virus hemagglutinin with a block in intracellular transport exhibit impaired trimer assembly

To study the importance of individual sulfhydryl residues during the folding and assembly in vivo of influenza virus hemagglutinin (HA), we have constructed and expressed a series of mutant HA proteins in which cysteines involved in three disulfide bonds have been substituted by serine residues. Investigations of the structure and intracellular transport of the mutant proteins indicate that (a) cysteine residues in the ectodomain are essential both for efficient folding of HA and for stabilization of the folded molecule; (b) cysteine residues in the globular portion of the ectodomain are likely to form native disulfide bonds rapidly and directly, without involvement of intermediate, nonnative linkages; and (c) cysteine residues in the stalk portion of the ectodomain also appear not to form intermediate disulfide bonds, even though they have the opportunity to do so, being separated from their correct partners by hundreds of amino acids including two or more other sulfhydryl residues. We propose a role for the cellular protein BiP in shielding the cysteine residues of the stalk domain during the folding process, thus preventing them from forming intermediate, nonnative disulfide bonds.

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Addition of carbohydrate side chains at novel sites on influenza virus hemagglutinin can modulate the folding, transport, and activity of the molecule.

The Journal of Cell Biology ◽

10.1083/jcb.107.6.2059 ◽

1988 ◽

Vol 107 (6) ◽

pp. 2059-2073 ◽

Cited By ~ 56

Author(s):

P Gallagher ◽

J Henneberry ◽

I Wilson ◽

J Sambrook ◽

M J Gething

Keyword(s):

Influenza Virus ◽

Intracellular Transport ◽

Secretory Pathway ◽

High Efficiency ◽

Biological Activities ◽

Side Chains ◽

Wild Type ◽

Temperature Dependent ◽

Influenza Virus Hemagglutinin ◽

General Tool

We have constructed and expressed a series of mutant influenza virus hemagglutinins, each containing a new consensus site for glycosylation in addition to the seven sites found on the wild-type protein. Oligosaccharide side chains were added with high efficiency at four of the five novel sites, located on areas of the protein's surface that are not normally shielded by carbohydrate. Investigations of the structure, intracellular transport, and biological activities of the mutant hemagglutinin molecules indicated that (a) supernumerary carbohydrate side chains can be used to shield or disrupt functional epitopes on the surface of hemagglutinin, and (b) the presence of an additional oligosaccharide may cause temperature-dependent defects in the transport of the glycoprotein. We discuss the addition of supernumerary oligosaccharides as a general tool for shielding chosen areas of the surface of proteins that enter or traverse the secretory pathway.

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Differential extractability of influenza virus hemagglutinin during intracellular transport in polarized epithelial cells and nonpolar fibroblasts.

The Journal of Cell Biology ◽

10.1083/jcb.108.3.821 ◽

1989 ◽

Vol 108 (3) ◽

pp. 821-832 ◽

Cited By ~ 146

Author(s):

J E Skibbens ◽

M G Roth ◽

K S Matlin

Keyword(s):

Influenza Virus ◽

Plasma Membrane ◽

Epithelial Cells ◽

Disulfide Bond ◽

Intracellular Transport ◽

Mdck Cells ◽

Apical Plasma Membrane ◽

Transport Pathway ◽

Influenza Virus Hemagglutinin ◽

Triton X

Biochemical changes in the influenza virus hemagglutinin during intracellular transport to the apical plasma membrane of epithelial cells were investigated in Madin-Darby canine kidney (MDCK) cells and in LLC-PK1 cells stably transfected with a hemagglutinin gene. After pulse-labeling a substantial fraction of hemagglutinin was observed to become insoluble in isotonic solutions of Triton X-100. Insolubility of hemagglutinin was detected late in the transport pathway after addition of complex sugars in the Golgi complex but before insertion of the protein in the plasma membrane. Insolubility was not dependent on oligosaccharide modification since deoxymannojirimycin (dMM), which inhibits mannose trimming, failed to prevent its onset. Insolubility was not due to assembly of virus particles at the plasma membrane because insoluble hemagglutinin was also observed in transfected cells. Hemagglutinin insolubility was also seen in MDCK cells cultured in suspension and in chick embryo fibroblasts, indicating that insolubility and plasma membrane polarity are not simply correlated. In addition to insolubility, an apparent transport-dependent reduction of the disulfide bond linking HA1 and HA2 in hemagglutinin was detected. Because of the timing of both insolubility and the loss of the disulfide bond, these modifications may be important in the delivery of the hemagglutinin to the cell surface.

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Monoclonal antibodies localize events in the folding, assembly, and intracellular transport of the influenza virus hemagglutinin glycoprotein

Cell ◽

10.1016/0092-8674(88)90426-6 ◽

1988 ◽

Vol 52 (6) ◽

pp. 843-852 ◽

Cited By ~ 40

Author(s):

Jonathan W. Yewdell ◽

Amy Yellen ◽

Thomas Bächi

Keyword(s):

Influenza Virus ◽

Monoclonal Antibodies ◽

Intracellular Transport ◽

Influenza Virus Hemagglutinin

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Mutations in the cytoplasmic domain of the influenza virus hemagglutinin affect different stages of intracellular transport.

The Journal of Cell Biology ◽

10.1083/jcb.100.3.704 ◽

1985 ◽

Vol 100 (3) ◽

pp. 704-714 ◽

Cited By ~ 71

Author(s):

C Doyle ◽

M G Roth ◽

J Sambrook ◽

M J Gething

Keyword(s):

Endoplasmic Reticulum ◽

Influenza Virus ◽

Golgi Apparatus ◽

Cell Surface ◽

Dna Sequences ◽

Cellular Localization ◽

Intracellular Transport ◽

Cytoplasmic Domain ◽

Biologically Active ◽

Influenza Virus Hemagglutinin

Mutations have been introduced into the cloned DNA sequences coding for influenza virus hemagglutinin (HA), and the resulting mutant genes have been expressed in simian cells by the use of SV40-HA recombinant viral vectors. In this study we analyzed the effect of specific alterations in the cytoplasmic domain of the HA molecule on its rate of biosynthesis and transport, cellular localization, and biological activity. Several of the mutants displayed abnormalities in the pathway of transport from the endoplasmic reticulum to the cell surface. One mutant HA remained within the endoplasmic reticulum; others were delayed in reaching the Golgi apparatus after core glycosylation had been completed in the endoplasmic reticulum, but then progressed at a normal rate from the Golgi apparatus to the cell surface; another was delayed in transport from the Golgi apparatus to the plasma membrane. However, two mutants were indistinguishable from wild-type HA in their rate of movement from the endoplasmic reticulum through the Golgi apparatus to the cell surface. We conclude that changes in the cytoplasmic domain can powerfully influence the rate of intracellular transport and the efficiency with which HA reaches the cell surface. Nevertheless, absolute conservation of this region of the molecule is not required for maturation and efficient expression of a biologically active HA on the surface of infected cells.

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