How a paramyxovirus fusion/entry complex adapts to escape a neutralizing antibody

Abstract

Paramyxoviruses including measles, Nipah, and parainfluenza viruses are public health threats with pandemic potential. Human parainfluenza virus type 3 (HPIV3) is a leading cause of illness in pediatric, older, and immunocompromised populations. There are no approved vaccines or therapeutics for HPIV3. Neutralizing monoclonal antibodies (mAbs) that target viral fusion are a potential strategy for mitigating paramyxovirus infection, however their utility may be curtailed by viral evolution that leads to resistance. Paramyxoviruses enter cells by fusing with the cell membrane in a process mediated by a complex consisting of a receptor binding protein (HN) and a fusion protein (F). Existing atomic resolution structures fail to reveal physiologically relevant interactions during viral entry. We present cryo-ET structures of pre-fusion HN-F complexes in situ on surfaces of virions that evolved resistance to an anti-HPIV3 F neutralizing mAb. Single mutations in F abolish mAb binding and neutralization. In these complexes, the HN protein that normally restrains F triggering has shifted to uncap the F apex. These complexes are more readily triggered to fuse. These structures shed light on the adaptability of the pre-fusion HN-F complex and mechanisms of paramyxoviral resistance to mAbs, and help define potential barriers to resistance for the design of mAbs.

Fig. 1. EV1 and EV2 resistance to inhibition by PIA174.

Model of the full-length HN/F complex with mutations for (A) EV1 and (B) EV2 indicated by red atomic spheres. C Vero cells were infected with mCherry-bearing parental, EV1, or EV2 HPIV3 and overlaid with serial dilutions of PIA174 FAb. Total fluorescence was measured 2 days post infection and y-axis shows the percent inhibition of total read fluorescence for each virus. D Inhibition of viral entry for EV1 and EV2 was quantitated by plaque counting normalized to no treatment. E Cryo-EM model of prefusion F (PDBID: 6MJZ) with both PIA174 and 3 × 1 structures showing location of L234 (green) and A194 (red) residues in relation to 3x and PIA174 mAbs. F, G A194T and L234F recognition by PIA174. Cells transfected with A194T, L234F, or parental Fs stabilized by Q162C-L168C, I213C-G230C, A463V, I474Y mutations were treated with 1ug/mL PIA174 Fab or 3 × 1 mAb and fluorescent secondary anti-human antibody. Fluorescence was quantitated. Data are means ± SE from three separate biological replicates for (C, D) and (F, G).

Fig. 2. Key PIA174 escape mutations alter the activatability of F.

Percent of F in pre-fusion state at different time points at 55 °C without (A) or with (B) PIA174 Ab fragment. C Rate of conformational change of uncleaved F with A194T or L234F measured by rate of lipopeptide capture at 37 °C with red blood cells. The HN bears a mutation (D216R) that makes it sialidase-deficient to maximize the HN-receptor contact and HN’s fusion promotion in order to compare the properties of the F proteins. D, E Triggering of F by heat (left) assessed at a range of temperatures with parental, A194T, or L234F Fs. F activation is quantitated by % of red blood cells (RBCs) released, bound, or fused with F-expressing cells. Activation of F by HN (right) was assessed at a range of temperatures for 60 min. with parental, A194T, L234F F. Data are means ± SE from at least three separate biological replicates for (A–E).

Fig. 3. HN escape mutations enhance fusion promotion of F.

A Cell-to-cell fusion measured by beta-galactosidase complementation with cells expressing HN or HA and parental F, A194T F, or L234F. Fusion values are normalized to max value in each experiment. B Cell-to-cell fusion measured by beta-galactosidase complementation with cells expressing parental F and parental HN, I243V, H552Q, or I243V/H552Q. Data are means ± SE from three separate biological replicates for (A, B).

Fig. 4. HN/F fusion complex subtomogram averages for EV1 and EV2 variants.

A Side views of the field strain viral HN-F fusion complex with prefusion F [Protein Data Bank (PDB) ID: 6MJZ] and HN (PDB ID: 4MZA) models fit into the density. B Side views of the EV1 HN- F reconstruction with prefusion F and HN models fit into the density. Insets show the interaction between the HN and F models for field strain and EV1 viral fusion complexes. C Overlay of the final subtomogram averages for field strain (blue) and EV1 (orange) fusion complexes. D 90 degrees rotated view (with respect to A) of the field strain viral fusion complex with prefusion F and HN models fit into the densities. E Side views of the EV2 HN-F reconstruction with prefusion F and HN models fit into the density. Insets show the interaction between the HN and F models for field strain and EV2 viral fusion complexes. F Overlay of the subtomogram averages for field strain (blue) and EV2 (green) fusion complexes. G Overlay of EV2 HN/F complex (green) with L234F HN/F complex (blue). H The buried surface area (residues of interaction with HN) on F for the field strain (blue), EV1 (orange), and EV2 (green). I Buried surface area (residues of interaction with HN) on F, overlaid with the model of PIA174 Fab to show overlap between Fab and HN interacting residues. Scale bars (A–F): 5 nm.

Fig. 5. Fitness cost of escape mutations.

A Human Airway Epithelial (HAE) cells were infected with EV1, EV2, or Parental HPIV3 and apical washes were titered daily up to 7 days post infection. Allele frequencies (points) and titers measured in plaque forming units (PFU; gray bars) of (B) EV1 at 0, 7 days post infection and (C) EV2 at 0, 2, 4, 5, and 7 days post infection. D Location of H552Q on the field strain HN model, which remained at 100% allele frequency in all wells of EV1, and compensatory mutations in EV2 wells that increased in allele frequency during the 7 days of infection for HN. Data are means ± SE from three separate biological replicates for (A).