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

TLDR

Hemagglutinin (HA) is a homotrimeric integral membrane glycoprotein that is cotranslationally inserted into the endoplasmic reticulum as HA0 and traffics to the cell surface via the Golgi complex. The study aimed to investigate the kinetics and cellular location of the assembly reaction that leads to HA0 trimerization. The authors detected quaternary structure formation by three independent criteria: recognition of a trimer‑specific epitope by monoclonal antibodies, acquisition of trypsin resistance, and co‑sedimentation of stable complexes with mature HA0 trimer in Triton X‑100 sucrose gradients. Oligomer formation is a post‑translational event with a half‑time of ~7.5 min, occurring in the ER and followed by rapid transport to the Golgi, where a stabilization event occurs upon exit or plasma‑membrane arrival; about 10 % of newly synthesized HA0 form aberrant trimers that are retained in the ER, and correct quaternary folding is essential for ER export, with additional subunit interaction changes during the secretory pathway.

Abstract

The hemagglutinin (HA) of influenza virus is a homotrimeric integral membrane glycoprotein. It is cotranslationally inserted into the endoplasmic reticulum as a precursor called HA0 and transported to the cell surface via the Golgi complex. We have, in this study, investigated the kinetics and cellular location of the assembly reaction that results in HA0 trimerization. Three independent criteria were used for determining the formation of quaternary structure: the appearance of an epitope recognized by trimer-specific monoclonal antibodies; the acquisition of trypsin resistance, a characteristic of trimers; and the formation of stable complexes which cosedimented with the mature HA0 trimer (9S20,w) in sucrose gradients containing Triton X-100. The results showed that oligomer formation is a posttranslational event, occurring with a half time of approximately 7.5 min after completion of synthesis. Assembly occurs in the endoplasmic reticulum, followed almost immediately by transport to the Golgi complex. A stabilization event in trimer structure occurs when HA0 leaves the Golgi complex or reaches the plasma membrane. Approximately 10% of the newly synthesized HA0 formed aberrant trimers which were not transported from the endoplasmic reticulum to the Golgi complex or the plasma membrane. Taken together the results suggested that formation of correctly folded quaternary structure constitutes a key event regulating the transport of the protein out of the endoplasmic reticulum. Further changes in subunit interactions occur as the trimers move along the secretory pathway.