Influenza A virus hemagglutinin trimerization completes monomer folding and antigenicity

Abstract

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 IAV-infected 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.

Fig 1

Biochemical characterization of HA maturation, trafficking, and assembly with specific anti-HA MAbs. (A to C) IAV PR8-infected MDCK cells were labeled with [³⁵S]Met for 2 min at 37°C, chased for the indicated times at the same temperature, and lysed with an ice-cold extraction buffer supplemented (+NEM) or not (−NEM) with NEM. Detergent lysates were then precipitated with MAbs specific to HA monomers (Y8-10C2, A), HA trimers (H17-L2, B), or both HA species (H28-E23, C). Immunocollected proteins were boiled in SDS sample buffer supplemented (+DTT) or not (−DTT) with dithiothreitol and analyzed by SDS-PAGE and fluorography. IT1 and IT2, HA folding intermediate species; pHA, processed HA; rHA, reduced HA. (D) The amount of immunoprecipitated rHA was quantified by densitometry and expressed in arbitrary units (a.u.). (E to G) MDCK cells infected with IAV PR8 were radiolabeled and chased at 20°C instead of 37°C. Cell lysates made at the end of each chase time point were subjected to IP with the Y8-10C2 (E), H17-L2 (F), or H28-E23 (G) MAbs. (H) The amount of precipitated rHA was quantified as described for panel D. The values to the left of panels A to C and E to G are molecular sizes in kilodaltons.

Fig 2

Specificity of anti-HA MAbs to ER-located HA monomers or trimerized HA lying on the GC. (A to C) IAV PR8-infected MDCK cells were labeled with [³⁵S]Met and chased at 37°C as described in the legend to Fig. 1A to C. HA species were depleted from cell lysates with an irrelevant MAb to the vesicular stomatitis virus N protein (10G-4; no depletion), the HA monomer-specific Y8-10C2 MAb (mHA depleted), or the HA trimer-specific H17-L2 MAb (tHA depleted). After two rounds of depletion, cell extracts were incubated with the Y8-10C2 (A), H17-L2 (B), or H28-E23 (C) MAbs. Immunoprecipitated HA species were analyzed under nonreducing conditions by SDS-PAGE and fluorography. (D) Bar graph representing the percentage of pooled HA-associated radioactivity (from 0 to 20 min) relative to the total HA (100%) in nondepleted cell lysates. (E to V) MDCK cells were infected with IAV PR8 in the absence (no treatment) or presence of 10 μM monensin. Cells were fixed, permeabilized, and incubated with the Y8-10C2 (E to J), H17-L2 (K to P), or H28-E23 (Q to V) MAbs (green channel) and rabbit polyclonal Abs to NA (red channel). DNA was labeled with DAPI (blue channel). Stained cells were examined by confocal fluorescence microscopy. Bars, 10 μm. Arrowheads point to NA colocalization with HA monomers on the nuclear envelope (ER) of cells labeled with Y8-10C2 and H28-E23. The values to the left of panels A to C are molecular sizes in kilodaltons.

Fig 3

Reactivity of various anti-HA MAbs assayed by immunofluorescence microscopy. MDCK cells were infected with IAV PR8 in the absence (no treatment) or presence of 10 μM monensin as described in the legend to Fig. 2E to V. HA was labeled on fixed and permeabilized cells with specific MAbs to the HA Ca (H17-L10, A to F), Cb (H35-C10, G to L), Sa (H9-A22, M to R), and Sb (IC5-4F8, S to X) antigenic sites (green channel). NA was detected with rabbit polyclonal Abs (red channel). DNA was labeled with DAPI (blue channel). Stained cells were examined by confocal fluorescence microscopy. Bars, 10 μm. Arrowheads point to NA colocalization with HA monomers on the nuclear envelope (ER) of cells labeled with IC5-4F8.

Fig 4

Targeting of the HA Ca, Cb, and Sa antigenic sites by HA trimer-specific MAbs. (A, C, E, and G) PyMOL images of the crystal structure of the IAV PR8 HA trimer (46) (RSCB protein database entry 1RU7) showing amino acid substitutions (red, H3 numbering scheme) on mutants that escape MAbs specific for the HA Ca (A), Cb (C), Sa (E), and Sb (G) antigenic sites. Each of the subunits within the HA oligomer are displayed in light green, pink, and purple. (B, D, F, and H) Detergent extracts from [³⁵S]Met-labeled and chased cells were left untreated or depleted for HA monomers or trimers as described in the legend to Fig. 2A to C before incubation with specific MAbs to the HA Ca (H17-L10, B), Cb (H35-C10, D), Sa (H9-A22, F), and Sb (IC5-4F8, H) antigenic sites. Immunocollected HA species were visualized by SDS-PAGE under nonreducing conditions, followed by fluorography. The values to the left of panels B, D, F, and H are molecular sizes in kilodaltons.

Fig 5

HA trimer-specific MAbs to the Ca, Cb, and Sa antigenic sites bind to monomers derived from oligomerized HA. (A and B) Untreated (pH 7.5) and acid-triggered (pH 5) recHA₃ were incubated with increasing amounts of trypsin for 15 min at 37°C. Digestion was stopped with 1 mM PMSF. recHA1- (A) or recHA2 (B)-derived fragments were then visualized by immunoblotting (IB) with the anti-HA CM-1 or RA5-22 MAbs, respectively. (C and D) In addition to a cocktail of protein standards, native recHA₃, acid-treated recHA₃, and the monomeric, tryptic fragments derived from recHA1 (HA tops) (sample 7, A) were fractionated by ultracentrifugation on layered 5 to 25% sucrose gradients as described in Materials and Methods. Sucrose gradient fractions were then analyzed by reducing SDS-PAGE and immunoblotting with the CM-1 MAb (D). The amounts of sedimented standards and recHA proteins in each fraction were quantified by densitometry and expressed in arbitrary units (a.u.) (C). (E) The recHA₃ pH 7.5, recHA₃ pH 5, and monomeric tryptic recHA1 proteins were subjected to IP at a high (300 mM) or low (150 mM) salt concentration with the HA monomer-specific Y8-10C2 MAb; the HA trimer-reactive H17-L2, H17L-10, H35-C10, and H9-A22 MAbs; or MAbs H28-E23 and IC5-4F8, which recognize both HA species. (F) The amount of immunoprecipitated HA tops was quantified as described in the legend to Fig. 1D. The values to the left of panels A, B, D, and E are molecular sizes in kilodaltons.