Residues 315 and 369 in HN Protein Contribute to the Thermostability of Newcastle Disease Virus

Abstract

Thermostable Newcastle disease virus (NDV) vaccines have been widely used in areas where a "cold-chain" is not reliable. However, the molecular mechanism of NDV thermostability remains poorly understood. In this work, we constructed chimeric viruses by exchanging viral fusion (F) and/or hemagglutinin-neuraminidase (HN) genes between the heat-resistant strain HR09 and thermolabile strain La Sota utilizing a reverse genetic system. The results showed that only chimeras with HN derived from the thermostable virus exhibited a thermostable phenotype at 56°C. The hemagglutinin (HA) and neuraminidase (NA) activities of chimeras with HN derived from the HR09 strain were more thermostable than those containing HN from the La Sota strain. Then, we used molecular dynamics simulation at different temperatures (310 K and 330 K) to measure the HN protein of the La Sota strain. The conformation of an amino acid region (residues 315-375) was observed to fluctuate. Sequence alignment of the HN protein revealed that residues 315, 329, and 369 in the La Sota strain and thermostable strains differed. Whether the three amino acid substitutions affected viral thermostability was investigated. Three mutant viruses based on the thermolabile strain were generated by substituting one, two or three amino acids at positions 315, 369, and 329 in the HN protein. In comparison with the parental virus, the mutant viruses containing mutations S315P and I369V possessed higher thermostablity and HA titers, NA and fusion activities. Taken together, these data indicate that the HN gene of NDV is a major determinant of thermostability, and residues 315 and 369 have important effects on viral thermostability.

Keywords: HN gene; Newcastle disease; chimeric viruses; mutations; thermostability.

FIGURE 1

Schematic representation of the construction of chimeric NDVs. Black and white bars indicate the genes of the HR09 and La Sota strain, respectively. The corresponding gene fragments were fused by using the HiFi DNA assembly cloning method.

FIGURE 2

Growth kinetics of NDVs in chicken embryo fibroblasts (CEFs). CEFs were infected (multiplicity of infection = 0.01) with parental and different chimeric viruses. At the indicated time points, infected cells and culture supernatants were harvested for virus titration in CEFs. Mean and standard deviations are shown from three independent experiments.

FIGURE 3

The thermostability and neuraminidase (NA) activity tests for chimeric NDVs. Heat-inactivation kinetics of infectivity of parental and chimeric NDVs were determined at 56°C by performing an EID₅₀ (A), HA activity (B), and NA activity (C) assay. Values are the average of three independent tests.

FIGURE 4

Molecular dynamics simulation results for the HN protein of the strain La Sota over 18 ns at different temperatures. (A) The root mean square deviation (RMSD) curves of HN protein during the simulations at different temperatures where black is 310 K and red is 330 K. (B) The root mean square fluctuation (RMSF) calculation is based on the C_α atoms of HN protein at 310 K and 330 K, respectively. Significant differences are indicated by the green box.

FIGURE 5

Schematic representation showing the construction of mutant NDVs. (A) Multiple amino acid sequence alignment of HN proteins. The black box denotes the point variation in identity between thermostable and thermolabile strains. (B) Schematic representation of the construction of mutant viruses. The mutated ICs containing the S315P, I369V and/or V329A substitution(s) were designated as cHR-La-HN-A, cHR-La-HN-B, and cHR-La-HN-C, respectively, according to the mutation sites. The triangles with different colors indicates the different mutation sites.

FIGURE 6

The thermostability and neuraminidase (NA) activity tests of the mutant viruses. Heat-inactivation kinetics of infectivity of different viruses were evaluated by EID₅₀ assay (A) at 56°C. p-values are represented as follow: * < 0.05; ^## < 0.01, compared to cHR-La-HN-A. Heat-inactivation kinetics of HA activity (B) and NA activity (C) of different viruses were determined by HA and NA tests at 56°C. p-values are represented as follow: * < 0.05; ** < 0.01; *** < 0.001; ^### < 0.001, compared to cHR-La-HN-A. Means and standard deviations are shown for three independent experiments.

FIGURE 7

Syncytium formation induced by different NDVs. (A) Vero cells were infected with different NDVs that were heat-treated at 56°C for 10 min. Different groups of cells were stained with Giemsa solution at 36 h post-infection. Images were immediately acquired under an inverted microscope at × 100 magnification. The syncytia formation were indicated by red arrows. (B) The fusion index values for the different NDVs were calculated as the ratio of the total number of nuclei to the number of cells in which the nuclei were observed. All values are expressed relative to the value for HR09 (100%). Means and standard deviations are shown for three independent experiments. p-values are represented as follow: * < 0.05; ** < 0.01; *** < 0.001.

FIGURE 8

The 3D structure of the HN protein was generated by the homology model of the crystal structure of the HN ectodomain of the NDV/Victoria/32 (PDB ID code: 3t1e.1.A). Schematic drawing of the HN dimer showing the location of the residues Ser315, Ile369 and Val329. The main sialic acid receptor binding sites (Arg416, Glu401, and Tyr526) are indicated in green.