Secretion of fowl plague virus haemagglutinin from insect cells requires elimination of both hydrophobic domains

Abstract

In the present study we have investigated the role of the hydrophobic domains of the fowl plague virus (FPV) haemagglutinin (HA) on its intracellular transport and maturation in insect cells. To this end processing of full-length HA (A +) has been compared to that of two truncated forms lacking either the cytoplasmic domain and the transmembrane domain (A-) or lacking the entire HA2 subunit, i.e. the transmembrane domain and the fusion peptide (HA~). All glycosylation sites present on A-and HA~-were glycosylated, indicating that both truncated forms were completely translocated in the endoplasmic reticulum. Unlike A +, A-and HA~ did not form trimers as indicated by cross-linking, gradient centrifugation and studies employing conformation-specific antibodies. Whereas HA~ was efficiently secreted, A-was retained in the cells in an apparently membrane-bound form. The data show that the carboxy-terminal transmembrane region is essential for the formation and stability of the trimers of the FPV HA. These observations also indicate that, under certain conditions, the fusion peptide of the FPV HA can serve as a membrane anchor.

Isolation and immunogenic properties of a monomeric form of the HA1 subunit of the influenza virus haemagglutinin from infected cells

“…2a) or the carbohydrate detection kit (Fig. 1, lane 5) between cHA 1 and sHA r This sHAa isolated from the allantoic fluid can be considered as the naturally occurring analogue to the secreted form of FPV HA which has been found in the culture medium of cells that were transfected with a truncated HA gene that lacked the HA 2 coding sequence (Kretzschmar et al, 1992) in accordance with previous findings (Gething & Sambrook, 1982). (a) (b) could enhance the vaccine.…”

A Secreted Form of the Major Histocompatibility Complex Class II-associated Invariant Chain Inhibiting T Cell Activation

“…Ii immune complexes from [ 35 S]methionine/cysteine-labeled cells were resolved by SDS-PAGE, and proteins were transferred to polyvinylidene difluoride membrane. IiP25 immobilized on the membrane was subjected to automated Edman degradation, and amino acid-containing fractions were analyzed for their 35 S content, indicative of the presence of methionine and/or cysteine. Radioactivity was eluted after 1, 6, 14, and 23 cycles (Fig.…”

“…As a source of IiP25, a recombinant form of IiP25 was generated by fusing the coding sequence for IiP25 (amino acid residues 99 -216) to the signal sequence of the hemagglutinin molecule of fowl plaque virus (see under "Experimental Procedures") (35). When Chinese hamster ovary cells were stably transfected with this construct, IiP25 could readily be detected in the culture medium of metabolically labeled and chased cells, indicating transport through the secretory pathway (data not shown).…”

“…The amount of 35 S eluted in each fraction was determined for each sequenator cycle by liquid scintillation methods.…”

“…Cells, Viruses and Antibodies-The human melanoma cell line Mel JuSo (34) Plasmid Constructions-cDNA encoding IiP25 was constructed by cloning the signal sequence of hemagglutinin from fowl plaque virus (strain A/fowl plaque virus/Rostock/34(H7N1 (35)) in front of methionine 99 of Ii. Briefly, cDNA encoding amino acid 1-18 of fowl plaque virus-hemagglutinin was amplified by polymerase chain reaction using 5Ј-gcgaattcgatacaaaatgaac-3Ј and 5Ј-aacgcatgccatttgtgggaatgac-3Ј as primers and recombinant AcMHPV virus encoding fowl plaque virushemagglutinin (kindly provided by Dr. H.-D. Klenk) as a template.…”