scholarly journals Disulfide bond formation during the folding of influenza virus hemagglutinin.

1992 ◽  
Vol 118 (2) ◽  
pp. 227-244 ◽  
Author(s):  
M S Segal ◽  
J M Bye ◽  
J F Sambrook ◽  
M J Gething
Keyword(s):  
Influenza Virus ◽  
Disulfide Bonds ◽  
Intracellular Transport ◽  
Cellular Protein ◽  
Cysteine Residues ◽  
Mutant Proteins ◽  
Influenza Virus Hemagglutinin ◽  
Stalk Domain ◽  
Do So

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.

scholarly journals Natural and directed antigenic drift of the H1 influenza virus hemagglutinin stalk domain

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Christopher S. Anderson ◽  
Sandra Ortega ◽  
Francisco A. Chaves ◽  
Amelia M. Clark ◽  
Hongmei Yang ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Viral Fitness ◽  
Antigenic Drift ◽  
Virus Infections ◽  
Head Region ◽  
Immune Pressure ◽  
Stalk Domain ◽  
Ha Protein

Abstract The induction of antibodies specific for the influenza HA protein stalk domain is being pursued as a universal strategy against influenza virus infections. However, little work has been done looking at natural or induced antigenic variability in this domain and the effects on viral fitness. We analyzed human H1 HA head and stalk domain sequences and found substantial variability in both, although variability was highest in the head region. Furthermore, using human immune sera from pandemic A/California/04/2009 immune subjects and mAbs specific for the stalk domain, viruses were selected in vitro containing mutations in both domains that partially contributed to immune evasion. Recombinant viruses encoding amino acid changes in the HA stalk domain replicated well in vitro, and viruses incorporating two of the stalk mutations retained pathogenicity in vivo. These findings demonstrate that the HA protein stalk domain can undergo limited drift under immune pressure and the viruses can retain fitness and virulence in vivo, findings which are important to consider in the context of vaccination targeting this domain.

scholarly journals Modification of Cysteine Residues In Vitro and In Vivo Affects the Immunogenicity and Antigenicity of Major Histocompatibility Complex Class I–restricted Viral Determinants

1999 ◽  
Vol 189 (11) ◽  
pp. 1757-1764 ◽  
Author(s):  
Weisan Chen ◽  
Jonathan W. Yewdell ◽  
Rodney L. Levine ◽  
Jack R. Bennink
Keyword(s):  
Influenza Virus ◽  
Posttranslational Modifications ◽  
Synthetic Peptides ◽  
Influenza Virus Infection ◽  
Lymphocytic Choriomeningitis ◽  
Reducing Agents ◽  
Cysteine Residues ◽  
T Cell Lines

In studying the subdominant status of two cysteine-containing influenza virus nuclear protein (NP) determinants (NP39–47 and NP218–226) restricted by H-2Kd, we found that the antigenicity of synthetic peptides was enhanced 10–100-fold by treatment with reducing agents, despite the fact that the affinity for Kd was not enhanced. Reducing agents also markedly enhanced the immunogenicity of cysteine-containing peptides, as measured by propagation of long-term T cell lines in vitro. Similar enhancing effects were obtained by substituting cysteine with alanine or serine in the synthetic peptides, demonstrating that sulfhydryl modification of cysteine is responsible for the impaired antigenicity and immunogenicity of NP39–47 and NP218–226. We found similar effects for two widely studied, cysteine-containing peptides from lymphocytic choriomeningitis virus. The major modifications of cysteine-containing synthetic peptides are cysteinylation and dimerization occurring through cysteine residues. We demonstrate that both of these modifications occur in cells synthesizing a cytosolic NP218–226 minigene product and, further, that T cells specific for cysteinylated NP218–226 are induced by influenza virus infection in mice, demonstrating that this modification occurs in vivo. These findings demonstrate that posttranslational modifications affect the immunogenicity and antigenicity of cysteine-containing viral peptides and that this must be considered in studying the status of such peptides in immunodominance hierarchies.

scholarly journals Publisher Correction: Natural and directed antigenic drift of the H1 influenza virus hemagglutinin stalk domain

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Christopher S. Anderson ◽  
Sandra Ortega ◽  
Francisco A. Chaves ◽  
Amelia M. Clark ◽  
Hongmei Yang ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Antigenic Drift ◽  
Influenza Virus Hemagglutinin ◽  
Stalk Domain

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

1988 ◽  
Vol 11 ◽  
pp. 19
Author(s):  
Christiane Will ◽  
Kazumichi Kuroda ◽  
Wolfgang Garten ◽  
Hans-Dieter Klenk
Keyword(s):  
Influenza Virus ◽  
Intracellular Transport ◽  
Influenza Virus Hemagglutinin

scholarly journals Conformational modulation of influenza virus hemagglutinin: characterization and in vivo efficacy of monomeric form

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Jong Hyeon Seok ◽  
Jeongwon Kim ◽  
Dan Bi Lee ◽  
Ki Joon Cho ◽  
Ji-Hye Lee ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Monomeric Form ◽  
In Vivo Efficacy ◽  
Influenza Virus Hemagglutinin

scholarly journals A Novel Cellular Protein, VPEF, Facilitates Vaccinia Virus Penetration into HeLa Cells through Fluid Phase Endocytosis

2008 ◽  
Vol 82 (16) ◽  
pp. 7988-7999 ◽  
Author(s):  
Cheng-Yen Huang ◽  
Tsai-Yi Lu ◽  
Chi-Horng Bair ◽  
Yuan-Shau Chang ◽  
Jeng-Kuan Jwo ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Hela Cells ◽  
Intracellular Transport ◽  
Fluid Phase ◽  
Cellular Protein ◽  
Cell Entry ◽  
Surface Lipid ◽  
Fluid Phase Endocytosis

ABSTRACT Vaccinia virus is a large DNA virus that infects many cell cultures in vitro and animal species in vivo. Although it has been used widely as a vaccine, its cell entry pathway remains unclear. In this study, we showed that vaccinia virus intracellular mature virions bound to the filopodia of HeLa cells and moved toward the cell body and entered the cell through an endocytic route that required a dynamin-mediated pathway but not a clathrin- or caveola-mediated pathway. Moreover, virus penetration required a novel cellular protein, vaccinia virus penetration factor (VPEF). VPEF was detected on cell surface lipid rafts and on vesicle-like structures in the cytoplasm. Both vaccinia virus and dextran transiently colocalized with VPEF, and, importantly, knockdown of VPEF expression blocked vaccinia virus penetration as well as intracellular transport of dextran, suggesting that VPEF mediates vaccinia virus entry through a fluid uptake endocytosis process in HeLa cells. Intracellular VPEF-containing vesicles did not colocalize with Rab5a or caveolin but partially colocalized with Rab11, supporting the idea that VPEF plays a role in vesicle trafficking and recycling in HeLa cells. In summary, this study characterized the mechanism by which vaccinia virus enters HeLa cells and identified a cellular factor, VPEF, that is exploited by vaccinia virus for cell entry through fluid phase endocytosis.

scholarly journals A Bridging [4Fe-4S] Cluster and Nucleotide Binding Are Essential for Function of the Cfd1-Nbp35 Complex as a Scaffold in Iron-Sulfur Protein Maturation

2012 ◽  
Vol 287 (15) ◽  
pp. 12365-12378 ◽  
Author(s):  
Daili J. A. Netz ◽  
Antonio J. Pierik ◽  
Martin Stümpfig ◽  
Eckhard Bill ◽  
Anil K. Sharma ◽  
...  
Keyword(s):  
Protein Function ◽  
Nucleotide Binding ◽  
Protein Assembly ◽  
Cysteine Residues ◽  
Binding Motif ◽  
S Protein ◽  
Mutant Proteins ◽  
Iron Sulfur ◽  
Sulfur Protein

The essential P-loop NTPases Cfd1 and Nbp35 of the cytosolic iron-sulfur (Fe-S) protein assembly machinery perform a scaffold function for Fe-S cluster synthesis. Both proteins contain a nucleotide binding motif of unknown function and a C-terminal motif with four conserved cysteine residues. The latter motif defines the Mrp/Nbp35 subclass of P-loop NTPases and is suspected to be involved in transient Fe-S cluster binding. To elucidate the function of these two motifs, we first created cysteine mutant proteins of Cfd1 and Nbp35 and investigated the consequences of these mutations by genetic, cell biological, biochemical, and spectroscopic approaches. The two central cysteine residues (CPXC) of the C-terminal motif were found to be crucial for cell viability, protein function, coordination of a labile [4Fe-4S] cluster, and Cfd1-Nbp35 hetero-tetramer formation. Surprisingly, the two proximal cysteine residues were dispensable for all these functions, despite their strict evolutionary conservation. Several lines of evidence suggest that the C-terminal CPXC motifs of Cfd1-Nbp35 coordinate a bridging [4Fe-4S] cluster. Upon mutation of the nucleotide binding motifs Fe-S clusters could no longer be assembled on these proteins unless wild-type copies of Cfd1 and Nbp35 were present in trans. This result indicated that Fe-S cluster loading on these scaffold proteins is a nucleotide-dependent step. We propose that the bridging coordination of the C-terminal Fe-S cluster may be ideal for its facile assembly, labile binding, and efficient transfer to target Fe-S apoproteins, a step facilitated by the cytosolic iron-sulfur (Fe-S) protein assembly proteins Nar1 and Cia1 in vivo.

scholarly journals Protein Synthesis Shut-Off Induced by Influenza Virus Infection Is Independent of PKR Activity

2000 ◽  
Vol 74 (18) ◽  
pp. 8781-8784 ◽  
Author(s):  
Thomas Zürcher ◽  
Rosa María Marión ◽  
Juan Ortín
Keyword(s):  
Protein Synthesis ◽  
Influenza Virus ◽  
Influenza Virus Infection ◽  
Nucleocytoplasmic Transport ◽  
Cellular Protein ◽  
Metabolic Labeling ◽  
Ns1 Protein ◽  
Wild Type ◽  
Mrna Polyadenylation

ABSTRACT The role of PKR activity in influenza virus-induced cell shut-off was studied by infection of PKR+ or PKR− cell cultures and metabolic labeling in vivo. No differences in the synthesis of viral proteins or the decay of cellular protein synthesis were observed. To investigate the relevance of the inhibition of cellular pre-mRNA polyadenylation and nucleocytoplasmic transport in virus-induced shut-off, we carried out similar experiments with mutant viruses lacking C-terminal sequences of NS1 protein. No differences in the shut-off induced by mutant versus wild-type viruses were observed, indicating that these nuclear events are not relevant for shut-off. The analysis of cytoplasmic mRNA stability indicated that the accumulation of viral mRNA during the infection correlated with the progressive decay of cellular mRNA, in both the wild type and an NS1 deletion mutant.

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

1988 ◽  
Vol 107 (6) ◽  
pp. 2059-2073 ◽  
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.

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

1989 ◽  
Vol 108 (3) ◽  
pp. 821-832 ◽  
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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