Assembly of influenza hemagglutinin trimers and its role in intracellular transport.

Copeland, Constance S.; Doms, Robert W.; Bolzau, E; Webster, Robert G.; Helenius, Ari

doi:10.1083/jcb.103.4.1179

Cited by 397 publications

(374 citation statements)

References 63 publications

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“…In vivo, F2 loses its epitope around the time of HA trimerization, and HA is not likely to trimerize in this diluted system in the absence of membranes 18,19 . Indeed, HA trimers were not detectable after folding in the lysate: HA did not precipitate with the trimer-specific antibody N2, and did not reach trypsin resistance as it does in vivo 20 (data not shown). Taken together, the results show that in a cell lysate in the absence of an intact ER, HA monomers folded into their proper, native conformation with high efficiency.…”

Section: Ha Folded In Vitro Acquires Correct Epitopesmentioning

confidence: 94%

“…To probe for different conformations, two mouse monoclonal IgG antibodies, F1 and F2, made against nondenatured HA2 were used 6 . The mouse monoclonal antibodies HC3, HC19 and HC100, mapped against epitopes A, B and E, respectively, all in the top domain of HA, have been described 7,20,35,36 . For coimmunoprecipitation with known chaperones, cells were either lysed in 2% (w/v) CHAPS in HBS (50 mM HEPES, pH 7.6, 200 mM NaCl) or in 0.5% (v/v) Triton X-100 lysis buffer (as described above).…”

Section: Immunoprecipitation and Sds-pagementioning

confidence: 99%

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A critical step in the folding of influenza virus HA determined with a novel folding assay

Maggioni

Liscaljet

Braakman

2005

Nat Struct Mol Biol

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Most principles of protein folding emerged from refolding studies in vitro on small, soluble proteins, because large ones tend to misfold and aggregate. We developed a folding assay allowing the study of large proteins in detergent such that the extent of cellular assistance required for proper folding can be determined. We identified a critical step in the in vivo folding pathway of influenza virus hemagglutinin (HA). Only the formation of the first few disulfides in the top domain of HA required the intact endoplasmic reticulum. After that, HA proceeded to fold efficiently in a very dilute solution, despite its size and complexity. This study paves the way for detailed structural analyses during the folding of complex proteins.Protein folding is the process by which linear polypeptide chains acquire their biologically active three-dimensional conformation. Although all information needed to reach the native conformation is encoded by the amino acid sequence 1 , protein folding inside the cell generally is assisted by molecular chaperones and folding enzymes 2-4 . They mainly protect nascent chains and newly synthesized proteins from misfolding and aggregation. Chaperones and folding enzymes reside in almost every compartment of eukaryotic cells.The endoplasmic reticulum (ER) is highly specialized for the folding of membrane and secretory proteins. One of the bestcharacterized model proteins used to study protein folding in the ER is the influenza virus HA. This protein is not only crucial for influenza virus infection but also an important target for the immune system. HA is a multidomain glycoprotein of 84 kDa with six disulfide bonds and seven N-linked glycans. It folds in the ER, trimerizes and is transported to the plasma membrane, where it is incorporated into new virions. The folding of HA has been studied extensively both in complete cells [5][6][7][8][9][10][11] and in microsomes 12,13 , using radioactive labeling of newly synthesized HA or truncated ribosome-bound nascent chains 14 . Although these studies yielded fairly detailed information on the HA folding process, further molecular insight would require biophysical studies on purified protein, which would disregard the role of the cell.To start bridging the gap between folding studies in vitro and in intact cells, we developed an assay that allows the study of post-translational HA folding in a cell-free system without membranes. We replaced the chase period in the well-established pulse-chase folding assay 6 with a folding analysis in solution: the protein is translated and translocated in intact cells, after which post-translational folding is studied in a detergent cell lysate. Using this 'in vitro chase' assay, we found that HA can fold in a detergent cell lysate without ER assistance, provided that the first folding step occurs in an intact ER. RESULTS HA can fold in a detergent cell lysateWhen studied with the pulse-chase assay in intact cells, newly synthesized HA ran as a single band in a reducing SDS-PAGE gel (Fig. 1a, R) and as three ban...

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Section: Ha Folded In Vitro Acquires Correct Epitopesmentioning

confidence: 94%

Section: Immunoprecipitation and Sds-pagementioning

confidence: 99%

A critical step in the folding of influenza virus HA determined with a novel folding assay

Maggioni

Liscaljet

Braakman

2005

Nat Struct Mol Biol

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“…However, these observations should not be necessarily interpreted to suggest that GRP78-BiP is absent from the process of folding of mutant HC because either deletion mutant may continue to contain the other available binding sites. Newly synthesized molecules in the ER must properly fold and assemble before transport to the Golgi apparatus (Gething et al, 1986;Copeland et al, 1986). Although there is mounting evidence that molecular chaperones such as GRP78-BiP are involved in the folding of polypeptides, it must be stressed that additional factors may be essential in promoting folding of proteins in the secretory pathway.…”

Section: Discussionmentioning

confidence: 99%

Analysis in vivo of GRP78-BiP/substrate interactions and their role in induction of the GRP78-BiP gene.

Watowich

Lamb

1992

MBoC

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The endoplasmic reticulum (ER)-localized chaperone protein, GRP78-BiP, is involved in the folding and oligomerization of secreted and membrane proteins, including the simian virus 5 hemagglutinin-neuraminidase (HN) glycoprotein. To understand this interaction better, we have constructed a series of HN mutants in which specific portions of the extracytoplasmic domain have been deleted. Analysis of these mutant polypeptides expressed in CV-1 cells have indicated that GRP78-BiP binds to selective sequences in HN and that there exists more than a single site of interaction. Mutant polypeptides have been characterized that are competent and incompetent for association with GRP78-BiP. These mutants have been used to show that the induction of GRP78-BiP synthesis due to the presence of nonnative protein molecules in the ER is dependent on GRP78-BiP complex formation with its substrates. These studies have implications for the function of the GRP78-BiP protein and the mechanism by which the gene is regulated. INTRODUCTIONThe 78-kDa glucose-regulated/Ig heavy-chain binding protein (GRP78-BiP) is a member of the highly con-

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“…Unfolded or partially folded HAs reveal additional trypsin cleavage sites and produce a more complicated series of fragments when treated with the protease. Thus, the ability of a chimeric HA to be cleaved by trypsin into only HA1 and HA2 is a sensitive measure of the correct folding of the HA external domain (Copeland et al, 1986;Gething et al, 1986;Lazarovits et al, 1990). To determine whether chimeric HAs folded properly, the proteins were expressed in CV-1 cells infected with SV40 expression vectors, and the proteins were analyzed by pulse-chase protocols.…”

Section: Chimeras With Foreign Tm Domains Are Properlymentioning

confidence: 99%

Amino Acid Sequence Requirements of the Transmembrane and Cytoplasmic Domains of Influenza Virus Hemagglutinin for Viable Membrane Fusion

Melikyan

Lin

Roth

et al. 1999

MBoC

121

135

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The amino acid sequence requirements of the transmembrane (TM) domain and cytoplasmic tail (CT) of the hemagglutinin (HA) of influenza virus in membrane fusion have been investigated. Fusion properties of wild-type HA were compared with those of chimeras consisting of the ectodomain of HA and the TM domain and/or CT of polyimmunoglobulin receptor, a nonviral integral membrane protein. The presence of a CT was not required for fusion. But when a TM domain and CT were present, fusion activity was greater when they were derived from the same protein than derived from different proteins. In fact, the chimera with a TM domain of HA and truncated CT of polyimmunoglobulin receptor did not support full fusion, indicating that the two regions are not functionally independent. Despite the fact that there is wide latitude in the sequence of the TM domain that supports fusion, a point mutation of a semiconserved residue within the TM domain of HA inhibited fusion. The ability of a foreign TM domain to support fusion contradicts the hypothesis that a pore is composed solely of fusion proteins and supports the theory that the TM domain creates fusion pores after a stage of hemifusion has been achieved. INTRODUCTIONMembrane fusion is common to many cellular processes, such as exocytosis and intracellular trafficking. But only in the case of viruses have the proteins responsible for fusion been unambiguously identified. A number of common features have been found among viral fusion proteins. All are oligomerized. The monomer of each oligomer always contains a critical stretch of nonpolar amino acids, known as the fusion peptide (Hernandez et al., 1996;Durell et al., 1997). As crystallographically determined, fusion proteins from several different virus families have the same backbone structure of extended coiled-coil ␣-helices (Bullough et al., 1994;Chan et al., 1997). Because viral fusion proteins of unrelated viruses have common structural patterns, it is logical to assume that their mechanisms of fusion are similar as well. Furthermore, a complex of SNARES, proteins thought to be responsible for the constitutive fusion of intracellular trafficking and the regulated fusion that occurs in exocytosis (Sü dhof, 1995), has been crystallographically determined (Poirier et al., 1998;Sutton et al., 1998). It too displays a long coiled-coil region. Therefore it may well be that the mechanisms of viral fusion pertain more generally to cellular fusion. Influenza virus has long been the prototypic virus for study of fusion mechanisms. It ‡ Corresponding author. E-mail address: fcohen@rush.edu. Abbreviations used: CF, carboxyfluorescein; CPZ, chlorpromazine; CT, cytoplasmic tail; DMEM, Dulbecco's modified Eagle's medium; GPI, glycosylphosphatidylinositol; HA, hemagglutinin; PBS-A-S, PBS supplemented with 0.1% sodium azide and 5% calf serum; p.i., postinfection; pIgR, polyimmunoglobulin receptor; R18, octadecylrhodamine B; RBC, red blood cell; RD, tetramethylrhodamine-tagged dextran; TM, transmembrane; VSV, vesicular stomatitis virus; WT,...

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Assembly of influenza hemagglutinin trimers and its role in intracellular transport.

Cited by 397 publications

References 63 publications

A critical step in the folding of influenza virus HA determined with a novel folding assay

A critical step in the folding of influenza virus HA determined with a novel folding assay

Analysis in vivo of GRP78-BiP/substrate interactions and their role in induction of the GRP78-BiP gene.

Amino Acid Sequence Requirements of the Transmembrane and Cytoplasmic Domains of Influenza Virus Hemagglutinin for Viable Membrane Fusion

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