Influenza A Virus Hemagglutinin Trimerization Completes Monomer Folding and Antigenicity

Abstract

c Influenza A virus (IAV) remains an important human pathogen largely because of antigenic drift, the rapid emergence of antibody escape mutants that precludes durable vaccination. The most potent neutralizing antibodies interact with cognate epitopes in the globular "head" domain of hemagglutinin (HA), a homotrimeric glycoprotein. The H1 HA possesses five distinct regions defined by a large number of mouse monoclonal antibodies (MAbs), i.e., Ca1, Ca2, Cb, Sa, and Sb. Ca1-Ca2 sites require HA trimerization to attain full antigenicity, consistent with their locations on opposite sides of the trimer interface. Here, we show that full antigenicity of Cb and Sa sites also requires HA trimerization, as revealed by immunofluorescence microscopy of IAVinfected cells and biochemically by pulse-chase radiolabeling experiments. Surprisingly, epitope antigenicity acquired by HA trimerization persists following acid triggering of the globular domains dissociation and even after proteolytic release of monomeric heads from acid-treated HA. Thus, the requirement for HA trimerization by trimer-specific MAbs mapping to the Ca, Cb, and Sa sites is not dependent upon the bridging of adjacent monomers in the native HA trimer. Rather, complete antigenicity of HA (and, by inference, immunogenicity) requires a final folding step that accompanies its trimerization. Once this conformational change occurs, HA trimers themselves would not necessarily be required to induce a highly diverse neutralizing response to epitopes in the globular domain.T he influenza A virus (IAV) hemagglutinin (HA) glycoprotein attaches virions to target cells by binding terminal sialic acid residues on cell surface glycans (1, 2). As a prototypical homotrimeric type I integral membrane protein, HA is synthesized in the endoplasmic reticulum (ER) of infected cells and transported through the Golgi complex (GC) to the plasma membrane (PM), where it is incorporated into budding virions. A variable number (depending on the strain) of N-linked oligosaccharides are added cotranslationally as HA is extruded into the ER through the translocon and subsequently trimmed and modified extensively during HA transport to the cell surface. In addition, the cytoplasmic COOH terminus of HA is palmitoylated during its intracellular transport, likely in an early GC compartment (3).HA folding begins cotranslationally, as shown by the acquisition of intrachain disulfide bonds and by the binding of monoclonal antibodies (MAbs) specific for discontinuous epitopes within HA to nascent chains (4). Initial folding of HA monomers is likely completed shortly after chain termination (within 1 or 2 min), whereas HA trimerization occurs with a half-life of ϳ5 min (5, 6). The localization of the later process appears to be strain (and perhaps subtype) specific, with prototype H2 (7) and H3 (8) HAs trimerizing in the ER while the A/Puerto Rico/8/34 (PR8) H1 HA trimerizes in either the ER-Golgi intermediate compartment or the cis-GC (3, 6).HA is of great medical importance by virtue of its recog...

Complete mapping of viral escape from neutralizing antibodies

“…Neutralization assays confirmed that the P234K mutation escaped H17-L10, while the P234V mutation caused no change in antibody sensitivity (Fig 5B). Interestingly, in HA's structure, site 234 is on a neighboring protomer relative to all the other mutations strongly selected by H17-L10 (S2 Fig). Our finding that escape mutations from H17-L10 cross the HA trimer interface is consistent with the fact that this antibody only recognizes trimeric HA [41]. Escape mutations at such epitopes are discernible because mutational antigenic profiling uses actual viruses that display intact HA; such conformational epitopes might not be properly displayed in the modified forms of viral glycoproteins often used in other high-throughput methods such as phage and yeast display.…”

Influenza A virus hemagglutinin specific antibodies interfere with virion neuraminidase activity via two distinct mechanisms

“…All Abs used are mouse IgG mAbs and have been described previously (Brooke et al , 2013; Gerhard et al , 1981; Magadan et al , 2013). …”

Eliciting unnatural immune responses by activating cryptic epitopes in viral antigens

“…The selection criteria included: 1 ) conservation among H1, H2, H3, and H5 subtypes; 2 ) sites containing ≥6 residues as a minimum length required for a peptide to elicit an antibody response; and 3 ) sites where >50% residues had a conservation score of 0.9–1.0 calculated from multiple sequence alignment. Previously, Sahini et al (10) used the monomer conformation of H3 (PDB: 1HGJ), but here, we used the trimer structure as a more-relevant conformation for the present purpose, considering that influenza HA is a homotrimeric membrane glycoprotein, and some antigenic sites require HA trimerization (39). To determine whether the predicted sites are suitable for binding to their specific antibodies, structural characteristics, such as accessible surface area and polarity, were analyzed in HA trimers ( Fig.…”