The influenza hemagglutinin precursor as an acid-sensitive probe of the biosynthetic pathway.

Abstract

The hemagglutinin of influenza virus (HA), an acid‐activated membrane fusion protein, is synthesized in the endoplasmic reticulum and transported through the Golgi complex to the cell surface of infected cells as an uncleaved, fusion‐incompetent precursor, HA0. The mature, proteolytically activated HA is known to undergo a rapid, irreversible, acid‐induced conformational change which mediates membrane fusion and virus penetration. On the basis of antigenic modifications and the acquisition of trypsin susceptibility, we demonstrate here that HA0, while unable to cause fusion, is acid sensitive. It undergoes irreversible conformational changes quite similar to those of HA at mildly acidic pH (pH less than 6.0). The ectodomain of HA0 does not, however, acquire hydrophobic properties and the changes occur in a less concerted manner (the pH dependence is much broader and the rate of conversion slower). These differences are likely to account for the inability of acid‐treated HA0 to trigger membrane fusion. It was shown, moreover, that HA0 acquired its acid‐sensitive properties immediately following trimerization in the endoplasmic reticulum. Since HA0 did not convert to the acid form at any point during its intracellular transport, we concluded that the trans‐Golgi compartment, known to be more acidic than the cytosol and involved in constitutive membrane transport, is not likely to have a pH less than 6.0.

Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry

“…3C). Many, although not all, pH-dependent viral surface glycoproteins, such as inf luenza HA, are inactivated by pretreatment with low pH before contact with cells because of the induction of premature, irreversible conformational rearrangements in the glycoprotein (23). The G glycoprotein of VSV is notable for undergoing reversible acid pH-induced conformational changes, and thus is not inactivated by pretreatment at acid pH (24).…”

Influenza Virus M2 Protein Ion Channel Activity Helps To Maintain Pandemic 2009 H1N1 Virus Hemagglutinin Fusion Competence during Transport to the Cell Surface

“…To detect pH-induced conformational changes in HA, HA-pseudoviruses made in serum-free medium in the absence of HAT were subjected to limited proteolysis by trypsin as described previously (32)(33)(34)(35)(36)(37). In brief, HA-pseudovirus supernatant samples were mixed with 10% n-dodecyl ␤-D-maltoside (DDM) to a final concentration of 1% DDM and incubated at 37°C for 1 h. The samples were then digested with TPCK-treated trypsin (Pierce) at a final concentration of 100 g/ml at room temperature for 20 h. The trypsin-digested samples were then resolved by nonreducing SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA) for Western blot analysis.…”

Intermonomer Interactions in Hemagglutinin Subunits HA1 and HA2 Affecting Hemagglutinin Stability and Influenza Virus Infectivity

“…Protease sensitivity and Western blotting. The whole viral particles and purified HA proteins have successfully been used to demonstrate the acidic pH-induced HA conformational changes and HA sensitivity to proteolysis (15,38,(42)(43)(44)(45)(46). Following the acidic pH-induced conformational change of HA, L-(tosylamido-2-phenyl ethyl) chloromethyl ketone (TPCK)-treated trypsin digests the HA1 subunit but not the HA2 subunit (42).…”