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...

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Purpose Transitioning the medical care of children with perinatally-acquired HIV from pediatric care to internal medicine practices has become increasingly important as newer therapies prolong survival. The study aims to describe challenges to caring for these adolescents and the potential barriers to transitioning them to internal medicine-based care. Methods Qualitative study in which data were gathered from open-ended interviews conducted from November 2005-April 2006 with 18 adolescents with HIV, 15 of their principal guardians, and 9 pediatric health care providers from the Yale Pediatric AIDS Care Program, New Haven, Connecticut. Results Issues of stigma played a prominent role in both the challenges to care and barriers to transitioning care. Challenges to care were: (1) poor adherence to medication regimens; (2) adolescent sexuality; and (3) disorganized social environments. Potential barriers to transitioning care were: (1) families’ negative perceptions of and experiences with stigma of HIV disease--which undermined the desire to meet new providers; (2) perceived and actual lack of autonomy-- pediatric providers feared that staff in adult clinics would demand a level of independence that adolescents did not have; and (3) difficulty letting-go of relationships-- adolescents, guardians, and providers described a familial relationship and expressed anxiety about terminating their relationships. Conclusion Understanding these challenges and barriers can inform both pediatric and adult HIV care providers and enable them to create successful transition programs, with the goal of improving retention and follow-up to care.

Abstract. Transport of the vesicular stomatitis virusencoded glycoprotein (G protein) between the endoplasmic reticulum (ER) and the cis Golgi compartment has been reconstituted in a cell-free system. Transfer is measured by the processing of the high mannose (mans_gGlcNAc2) ER form of G protein to the mansGlcNAc2 form by the cis Golgi enzyme a-mannosidase I. G protein is rapidly and efficiently transported to the Golgi complex by a process resembling that observed in vivo. G protein is trimmed from the high mannose form to the mansGlcNAc2 form without the appearance of the intermediate man6_TGlcNAcz oligosaccharide species, as is observed in vivo. G protein is found in a sealed membrane-bound compartment before and after incubation. Processing in vitro is sensitive to detergent, and the Golgi ~t-mannosidase I inhibitor 1-deoxymannorjirimycin. Transport between the ER and Golgi complex in vitro requires the addition of a high speed supematant (cytosol) of cell homogenates, and requires energy in the form of ATP. Efficient reconstitution of export of protein from the ER requires the preparation of homogenates from mitotic cell populations in which the nuclear envelope, ER, and Golgi compartments have been physiologically disassembled before cell homogenization. These results suggest that the high efficiency of transport observed here may require reassembly of functional organelles in vitro.

The influenza hemagglutinin precursor (HA0) and many other glycoproteins fold and oligomerize in the endoplasmic reticulum (ER). Only correctly folded oligomers are transported to the cell surface. To analyse the rules which determine this type of ER sorting, we have extended our analysis of hemagglutinin transport to two soluble, anchor‐free recombinant HA0s derived from X31/A/Aichi/68 and A/Japan/305/57 influenza A. The results showed that individual monomers rapidly acquired a folded structure similar to that of monomeric membrane‐anchored HA0. They were efficiently transported and secreted, but oligomerization was not required for secretion. Trimers or higher order complexes were either not formed (X31 HA0), or appeared during passage through the late compartments of the secretory pathway, with no effect on the rate of transport (Japan HA0). However, when initial folding was disturbed by inhibition of N‐linked glycosylation, anchor‐free X31 HA0 was misfolded and retained in the ER as disulfide‐linked complexes associated with binding protein, BiP (GRP78). The complexes were similar to those seen for the nonglycosylated membrane‐bound HA0, but instead of forming immediately after synthesis they appeared with a half‐time of 6 min. Taken together, the data demonstrate that the structural criteria that makes the anchor‐free HA0 transport competent are less stringent than those for the membrane form; they must fold correctly but do not need to oligomerize.