Structure of the Newcastle disease virus F protein in the post-fusion conformation

Abstract

The paramyxovirus F protein is a class I viral membrane fusion protein which undergoes a significant refolding transition during virus entry. Previous studies of the Newcastle disease virus, human parainfluenza virus 3 and parainfluenza virus 5 F proteins revealed differences in the pre- and post-fusion structures. The NDV Queensland (Q) F structure lacked structural elements observed in the other two structures, which are key to the refolding and fusogenic activity of F. Here we present the NDV Australia-Victoria (AV) F protein post-fusion structure and provide EM evidence for its folding to a pre-fusion form. The NDV AV F structure contains heptad repeat elements missing in the previous NDV Q F structure, forming a post-fusion six-helix bundle (6HB) similar to the post-fusion hPIV3 F structure. Electrostatic and temperature factor analysis of the F structures points to regions of these proteins that may be functionally important in their membrane fusion activity.

Figure 1. Protein construct design for NDV F constructs

(A, B). Construct design of the NDV fusion proteins, solF and F-GCNt, which are expressed in the post-fusion and pre-fusion conformations, respectively. The solF construct is truncated before the F transmembrane and cytoplasmic domains, contains a mutated cleavage activation site of a single R residue, indicated with an arrow labeled C.S., and contains an appended protease cleavage site and 6His tag for purification indicated in dark grey and labeled in the figure. The F1 and F2 fragments generated after proteolytic activation of F are indicated, along with the key structural elements for fusion HRA, HRB and the fusion peptide. The NDV F-GCNt construct contains a C-terminal GCN-trimerization domain in frame with the native HRB heptad repeat sequence, upstream of the 6His tag. The inserted GCNt trimerization domain is added to stabilize the protein in the pre-fusion state. (C, D). Negative stained electron microscopy of the NDV pre-fusion and post-fusion F proteins. (C). The phenotype of the NDV post-fusion F construct is that of an elongated triangle or golf tee as seen previously for the PIV5 and hPIV3 post-fusion F proteins. (D). The phenotype of the NDV pre-fusion F protein is that of a spherical head with a thin tail observed only in select orientations similar to the lollipop phenotype observed for the PIV5 pre-fusion construct studied previously.

Figure 2. Crystal structure of the NDV AV F protein

(A). Structures of the two antiparallel NDV F monomers from two different trimers present in the asymmetric unit. The two trimers lie along crystallographic 3-fold axes, which are indicated in the Figure. (B) Structure of the biological NDV F trimer observed when the crystallographic symmetry mates of one of the monomers are generated. Chains are colored in blue, pink and magenta. The head and 6-helix bundle domain stalk regions are indicated to the right. (C) Example sigmaA weighted electron density maps calculated using the final refined structure. The electron density map is contoured at 0.9σ and shows a segment of the 6HB with a model for the central HRA helix (residues E156, N162 and Q174 are indicated) flanked by two HRB helices from neighboring crystallographically-related symmetry mates. (D) An example of poor electron density observed in the 430 loop is shown. The electron density map represented is identical to that in panel (C). Electron density for residues G431 and D430 is partial and may be due to the flexibility of G431 and the low resolution (3.5 Å) of the diffraction data obtained.

Figure 3. Comparison of the NDV AV and partial NDV Q F ectodomain structures

(A). Structure of the previously determined NDV Q F protein. (B). Structure of the NDV AV F ectodomain. Note the absence of the N-terminal region of the HRA three-helix bundle and the entire HRB helix in the NDV Q F structure, leading to a shortening of the stalk region. The NDV AV F structure shows overall very close correspondence with the NDV Q structure in the head and neck regions, while containing the post-fusion 6HB.

Figure 4. Comparison of the NDV F and hPIV3 post-fusion structures

(A, B) Global superposition of the NDV AV F (blue) and hPIV3 F post-fusion structures (green). Best correspondence is observed between the combination of DIII and HRB segments, leading to displacement of HRA, DII, and portions of DI. (C). Close-up of the overlay of DI and DII domains. Positional displacements, relative to the superposed DIII domains, can be viewed as twisting motions (indicated by arrows) at the junctions of DI/DIII and DI/DII domains.

Figure 5. Electrostatic profiles of the NDV, hPIV3 and PIV5 protein structures

(A,B) NDV F. (C,D). hPIV3 F. (E,F). PIV5 F. “Side” and “end-on” views of the proteins are shown in paired panels A&B, C&D and E&F. Arrows point to conserved regions of positive charge on the apices of the pre-and post-fusion F timers. Note that the PIV5 F trimer in panel (F) is oriented with the pre-fusion stalk pointed toward the reader out of the plane of view, in order to maintain a consistent orientation of the DI-DIII head domains. Electrostatic charge is contoured at -1 to +1 kT/e.

Figure 6. Electrostatic charge on the surfaces of the post-fusion influenza HA2, Ebola GP2 and HIV gp41

(A) The bacterially expressed influenza HA2 fragment structure. (2) The Ebola virus GP2 protein. (C) The HIV gp41 six-helix bundle. All structures are contoured at -1 to +1 kT/e.

Figure 7. Temperature factor analysis of the pre-and post-fusion F structures

Normalized temperature factors were calculated from the NDV AV, hPIV3 and PIV5 F structures as described in Materials and Methods. The normalized values are plotted bar graph form in panels A, C and E and also displayed as color ramps on monomers of the three structures in panels B, D and F. Color ramps are from blue (low values) to yellow (high values) of the normalized temperature factors. Note that normalization of the temperature factors is relative to the overall mean and maximum values observed for each structure, yielding values that drop below 0 for regions that are better ordered than average and values above 0 for progressively more disordered regions. (A,B) NDV F. (C,D). hPIV3 F. (E,F). PIV5 F. Regions indicated by numbers 1 to 3 are discussed in the text.