Crystal Structure of the Hendra Virus Attachment G Glycoprotein Bound to a Potent Cross-Reactive Neutralizing Human Monoclonal Antibody

<div><p>The henipaviruses, represented by Hendra (HeV) and Nipah (NiV) viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in Australia, Southeast Asia, India and Bangladesh. The high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. Henipavirus entry is initiated by the attachment of the G envelope glycoprotein to host cell membrane receptors. Previously, henipavirus-neutralizing human monoclonal antibodies (hmAb) have been isolated using the HeV-G glycoprotein and a human naïve antibody library. One cross-reactive and receptor-blocking hmAb (m102.4) was recently demonstrated to be an effective post-exposure therapy in two animal models of NiV and HeV infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. Here, we report the crystal structure of the complex of HeV-G with m102.3, an m102.4 derivative, and describe NiV and HeV escape mutants. This structure provides detailed insight into the mechanism of HeV and NiV neutralization by m102.4, and serves as a blueprint for further optimization of m102.4 as a therapeutic agent and for the development of entry inhibitors and vaccines.</p></div

Germline mutations are affected by transcription.

<p><i>Panel A</i>, HGMD dataset; <i>y-axis</i>, as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003816#pgen-1003816-g001" target="_blank">Figure 1C</a>; <i>x-axis</i>, ratio of mutated NGNN sequences in protein coding genes containing the P2-guanine base on the non-transcribed (<i>NT</i>) <i>vs</i>. transcribed (<i>T</i>) strand; <i>solid circles</i>, HGMD dataset (<i>r</i>² 0.32, P(α)_0.05 0.991, P<0.001); <i>open circles</i>, 1000 Genomes Project dataset. <i>Panel B</i>, inherited splicing mutations dataset; <i>top</i>, cartoon of exon-intron boundaries showing the conserved GT and AG bases at the donor (<i>ds</i>) and acceptor (<i>as</i>) splice sites; <i>bottom</i>, graph of splicing mutations; <i>y-axis</i>, number of SBSs; <i>x-axis</i>, position of SBSs relative to +/−20 nt of splice junctions; <i>Panel C</i>, model for sequence-dependent SBSs in cancer and human inherited disease. In the first step, an electron is lost from within a tetranucleotide sequence, leaving a hole. In the second step, the hole migrates to and from various competing sites, including nearby bases and chromatin-associated amino acids (not shown), eventually being trapped by a guanine base. The resulting guanine radical cation either causes DNA-protein crosslinking or undergoes subsequent chemical modifications. If the modified base is not corrected by DNA repair, it may give rise to a mutation (X-Y base-pair) as a result of error-prone DNA polymerase synthesis during DNA replication (dashed arrow).</p

Neutralization efficacy of wild-type and neutralization-escape mutants by m102.4 mAb, m102.4 Fab and m102.3 Fab.

<p>Wild-type (WT) Nipah (NiV) and Hendra (HeV) viruses and their respective m102.4 neutralization escape mutants NiV-V507I and HeV-D582N were used to evaluate the neutralization efficacy of m102.4 mAb, m102.4 Fab and m102.3 Fab. Starting concentration of m102.4 was 100 µg/mL. The mAb and Fab concentrations at which 50% of the virus was neutralized are plotted. Error bars represent standard deviations. * t-test; p<0.001 compared to WT.</p

Vertical ionization potentials (VIPs) of guanine-centered DGN sequences.

<p>VIPs for the centrally (italicized) guanine computed at the M06-2X/6-31G(<i>d</i>) level of theory;</p>a<p>VIP of free unalkylated guanine;</p>b<p>from reference <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003816#pgen.1003816-Zaytseva1" target="_blank">[57]</a>.</p

Comparison of the m102.3-binding regions of HeV-G and NiV-G.

<p>The HeV-G (green) and NiV-G (purple) structures are superimposed and viewed from top. The m102.3 contacting residues of HeV-G and the corresponding residues of NiV-G are shown and labeled. Most residues are conserved between HeV-G and NiV-G except for three: T/S241, T/V507 and Y/F458.</p

Structure of the m102.3/HeV-G complex, and comparison to the ephrin-B2/HeV-G structure.

<p>A. Left: Overall Structure of the m102.3/HeV-G complex viewed from the side. CDR-H3 (magenta) of m102.3 inserts into the central cavity of HeV-G (green). Disulfide bonds are shown as yellow sticks. The five glycosylation sites of HeV-G are shown as grey spheres. Right: A close up view of the HeVG/m102.3 complex interface. Residues involved in the interaction are shown as stick figures and labeled. The solvent accessible surface of HeV-G central cavity region, viewed from top, is presented on the bottom. CDR-H3 residues (magenta) and R30 (cyan, on the light chain of Fab) and their contacting residues on HeV-G (green) are shown and labeled. B. Overall structure of the ephrin-B2 (orange)/HeV-G (blue) complex. HeV-G in the complex is in the same orientation as in panel A.</p

Comparison of the binding interfaces in the HeV-G/m102.3 and the HeV-G/ephrin-B2 complexes.

<p>A: The solvent accessible surface of the HeV-G molecule in the HeV-G/m102.3 complex viewed from the top. HeV-G is colored in green, except for the m102.3-contacting region that is colored in red (for the 1∶1 complex interface) and in blue (for the region contacted by another copy of the light chain in the hetero-tetrameric 2∶2 complex interface). B: The solvent accessible surface view of the HeV-G molecule in the HeV-G/ephrin-B2 complex viewed from the top. HeV-G is colored in grey, except for the ephrin-B2 contacting region, which is colored in red. C: The tip of the m102.3 CDR-H3 region (in magenta) bound in the HeV-G surface cavity (in green). D: The tip of the ephrin-B2 G-H loop (in yellow) bound in the HeV-G surface cavity (in grey).</p

SBSs and VIPs.

<p><i>Panel A</i>, whisker plot of the fractions of SBSs at G•C bp for the EWS and GWS datasets computed using AgilentV2 and Duke35 mappability counts, respectively; <i>red line</i>, mean; <i>black line</i>, median; <i>green lines</i>, average GC-contents in the mappable AgilentV2 (EWS) and Duke35 (GWS) sets. <i>Panel B</i>, NGRA sequences are enriched in SBSs in melanoma. <i>y-axis</i>, for each 4-member sequence combination with matching P1–P3 bases, the fraction of mutations at P4-A was divided by the average fraction of mutations at P4-(C/T/G); <i>x-axis</i>, P3 base composition; <i>R</i>, purine; <i>Y</i>, pyrimidine; mean ± SD; P-value from two-tailed <i>t</i>-test. <i>Panel C</i>, the <i>ln</i> of normalized fractions of mutated DGNN (D = A/G/T) sequences, <i>F_i</i>, for the seven cancer datasets with −logP ≥3 for DGRN>DGYN (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003816#pgen-1003816-t001" target="_blank">Table 1</a>) were combined and plotted as a function of the average absolute free energy of base stacking, ΔG(ν), for each of the 48 DGNN sequences. <i>Panel D</i>, three-dimensional model of the (5′-GGG-3′)•(5′-CCC-3′) trinucleotide showing the LUBMO (lowest unoccupied beta molecular orbital) of the ionized sequence. <i>Panel E</i>, plot of the normalized fractions (<i>log f_i</i>×10³) of mutated DGN sequences (Duke35 counts) for the Lung_nsc cancer dataset <i>vs.</i> VIPs; <i>outer circle</i>, 5′D base; <i>inner circle</i>, 3′N base; <i>blue</i>, adenine; <i>green</i>, guanine; <i>red</i>, thymine; <i>yellow</i>, cytosine. <i>Panel F</i>, agglomerative hierarchical clustering of 14 cancer genome datasets obtained from linear correlations with <i>ln</i> VIP values, as obtained from T_hg19 counts; <i>colored boxes</i>, elements found to be clustered at the 90% confidence interval.</p