Abstract. The folding of influenza hemagglutinin (HAO) in the ER was analyzed in tissue culture cells by following the formation of intrachain disulfides after short (1 min) radioactive pulses . While some disulfide bonds were already formed on the nascent chains, the subunits acquired their final disulfide composition and antigenic epitopes posttranslationally. Two posttranslational folding intermediates were identified . In CHO cells constitutively expressing HAO, mature HAO subunits were formed with a half time of 3 min and their folding reached completion at 22 min . The rate of folding was highly dependent on N mammalian cells, protein folding occurs in three distinct environments : the cytosol, the mitochondria, and the ER. The prevailing conditions in each of these com partments are quite different, and therefore the folding processes display unique properties. For several secretory proteins, it has been shown that folding begins cotranslationally from the NH2-terminus and proceeds towards the COOH-terminus as the polypeptides enter the ER (Bergman and Kuehl, 1979;Jaenicke, 1987). The final outcome is significantly affected by covalent cotranslational modifications such as N-linked glycosylation or the removal of NH2-terminal signal sequences, and by protein disulfide isomerase-catalyzed formation ofdisulfide bonds (Freedman et al., 1984;Rose and Doms, 1988;Randall and Hardy, 1989) . In many cases, the involvement of other folding enzymes, such as proline isomerase, and chaperonins such as binding protein (BiP/GRP78)' is likely to be important (Freedman, 1989 ;Pelham, 1989;Rothman, 1989) . The ionic conditions and redox potential in the ER lumen, which are quite distinct from those in the cytosol, may also play an important role. The cell biological aspects of protein folding, however, are poorly understood.In this paper, we have analyzed the folding of influenza hemagglutinin . The hemagglutinin is a type I transmembrane glycoprotein (84 kD, 549 amino acids) with multiple folding domains (Wilson et al ., 1981 ;Wiley and Skehel, 1987). It is exceptionally well characterized in terms of cell type and expression system, and thus regulated by factors other than the sequence of the protein alone. Exposure of cells to stress conditions increased the level of glucose regulated proteins, including BiP, and decreased the folding rate. The efficiency of folding and subsequent trimerization was not dependent on the rate of translation, nor on temperature between 37 and 15°C; however, the rates of folding and trimerization decreased with decreasing temperature . Whereas the rate of folding was independent of expression level, trimerization was accelerated at higher levels of expression. structure, function, and intracellular transport . In infected or transfected cells, the ectodomain (513 amino acids) is cotranslationally translocated into the ER. Signal peptide cleavage and N-linked glycosylation on five to seven sites occur cotranslationally. When expressed at a high level, the protein forms homotrimers with a ha...

We have characterized the association between the binding protein, BiP (also known as GRP 78), and misfolded forms of the influenza virus hemagglutinin precursor, HA0. BiP is a heat-shock-related protein that binds to unassembled immunoglobulin heavy chain and to a variety of misfolded proteins in the lumen of the ER. A small fraction (5-10%) of newly synthesized HA0 in CV-1 cells was found to be misfolded and retained in the ER. When glycosylation was blocked with tunicamycin, all of the HA0 produced was similarly misfolded. The misfolded HA0 was retained as relatively small (9-25-S) complexes associated with BiP. In these complexes the top domains of HA0 were correctly folded judging by their reactivity with monoclonal antibodies, but the polypeptides were cross-linked via anomalous interchain disulfides. The association with BiP was non-covalent and easily broken by warming to 37 degrees C or by adding ATP to the lysate. Pulse-chase experiments showed that HA0's self-association into complexes occurred immediately after synthesis and was followed rapidly by BiP association. The misfolded, BiP- associated HA0 was not transported to the plasma membrane but persisted as complexes in the ER for a long period of time before degradation (t1/2 = 6 h). The results suggested that BiP may be part of a quality control system in the ER and that one of its functions is to detect and retain misfolded proteins.

The majority of human rhinoviruses use intercellular adhesion molecule 1 (ICAM-1) as a cell surface receptor. Two soluble forms of ICAM-1, one corresponding to the entire extracellular portion [tICAM(453)] and one corresponding to the two N-terminal immunoglobulin-like domains [tICAM(185)], have been produced, and their effects on virus-receptor binding, virus infectivity, and virus integrity have been examined. Results from competitive binding experiments indicate that the virus binding site is largely contained within the two N-terminal domains of ICAM-1. Virus infectivity studies indicate that tICAM(185) prevents infection by direct competition for receptor binding sites on virus, while tICAM(453) prevents infection at concentrations 10-fold lower than that needed to inhibit binding and apparently acts at the entry or uncoating steps. Neutralization by both forms of soluble ICAM-1 requires continual presence of ICAM-1 during the infection and is largely reversible. Both forms of soluble ICAM-1 can alter rhinovirus to yield subviral noninfectious particles lacking the viral subunit VP4 and the RNA genome, thus mimicking virus uncoating in vivo, although this irreversible modification of rhinovirus is not the major mechanism of virus neutralization.

Viral receptors serve both to target viruses to specific cell types and to actively promote the entry of bound virus into cells. Human rhinoviruses (HRVs) can form complexes in vitro with a truncated soluble form of the HRV cell surface receptor, ICAM-1. These complexes appear to be stoichiometric, with approximately 60 ICAM molecules bound per virion or 1 ICAM-1 molecule per icosahedral face of the capsid. The complex can have two fates, either dissociating to yield free virus and free ICAM-1 or uncoating to break down to an 80S empty capsid which has released VP4, viral RNA, and ICAM-1. This uncoating in vitro mimics the uncoating of virus during infection of cells. The stability of the virus-receptor complex is dependent on temperature and the rhinovirus serotype. HRV serotype 14 (HRV14)-ICAM-1 complexes rapidly uncoat, HRV16 forms a stable virus-ICAM complex which does not uncoat detectably at 34 degrees C, and HRV3 has an intermediate phenotype. Rhinovirus can also uncoat after exposure to mildly acidic pH. The sensitivities of individual rhinovirus serotypes to ICAM-1-mediated virus uncoating do not correlate with uncoating promoted by incubation at low pH, suggesting that these two means of virus destabilization occur by different mechanisms. Soluble ICAM-1 and low pH do not act synergistically to promote uncoating. The rate of uncoating does appear to be inversely related to virus affinity for its receptor.

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