Fig 1.
Charge of HN protein and its relationship with NDV thermostability.
(A) Schematic representation showing the construction of chimeric NDVs. Grey and blue bars represent the genes of TS09-C and LaSota strain, respectively. Corresponding nucleotide numbers where the HN fragments are fused using In-fusion cloning technology are depicted. For each chimeric virus, the viral thermostability (Time for 90% infectivity loss, min) at 56°C is indicated at right side of the diagram. (B) Percentage of amino acids with different characteristic in HN proteins from thermostable and thermolabile NDV strains. (C) Negatively charged amino acids composition of HN proteins from thermostable and thermolabile NDV strains. (D) Theoretical charge of HN proteins from thermostable and thermolabile NDV strains. The four thermostable strains are TS09-C, V4, I-2, and Ulster. The four thermolabile strains are LaSota, Mukteswar, HB1103, and HN1007. The theoretical charge of protein at pH 7.4 is determined by using IPC software (http://isoelectric.ovh.org), based on its amino acid sequence. (E) Scatter diagram showing the relationship between charge of HN protein and thermostability of NDV at 56°C. Each point represents the data from one NDV isolate. (F) Effect of each charged amino acid substitution on the charge of HN protein of NDV strain TS09-C. (G) Mapping of charge-associated amino acid substitutions onto the surface of HN protein of NDV strain TS09-C. The 23 charge-associated amino acid substitutions are labeled on the protein surface and colored red. (H-J) Molecular surfaces of thermostable and thermolabile NDV HN proteins are colored according to electrostatic potentials with a range of red (- 5.0 V) to blue (+ 5.0 V). The structures of HN proteins from NDVs are obtained by homology modeling, using the HN structure of AV strain (PBD ID code 3T1E) as a template.
Fig 2.
Effect of charge-associated amino acid mutations on the surface charge of NDV HN protein and viral thermostability.
(A) Schematic representation showing the construction of recombinant NDVs mutated in HN proteins. In the HN protein of NDV mutants, the red and green lines indicate the amino acid mutations lead to the increase and decrease of the negative charge of HN protein, respectively. The uncharged, positively-charged, and negatively-charged amino acid residues are colored black, green and red, respectively. For each NDV mutant, the theoretical charge of HN protein are indicated at right side of the diagram. (B) Scatter diagram summarizes the relationship between the charge of HN protein and viral thermostability (Time for 90% infectivity loss, min) at 56°C. Each point represents the data from one NDV mutant. (C) Molecular surfaces of mutated HN proteins are colored according to electrostatic potentials with a range of red (- 5.0 V) to blue (+ 5.0 V). The names of NDV strains are indicated at the bottom of structures. The structures of mutated HN proteins are obtained by homology modeling, using the HN structure of AV strain (PBD ID code 3T1E) as a template.
Fig 3.
Effect of the surface charge of HN proteins on the cell-binding stability of NDV.
Heat-inactivation kinetics of HA activity (A) and NA activity (B) of the NDV mutants are determined at 56°C. (C) Electron micrographs of heat-treated rNDVs. NDV mutants are heat-treated for 10 min at 56°C, then negatively stained with phosphotungstic acid. (D) R18-labeled NDV mutants are heat-treated for 10 min at 56°C, then inoculated into BHK-21 cells (moi 50) at 37°C. At 1 hpi, cells are directly observed using fluorescence microscopy. At 7 hpi, HN expression in BHK-21 cells is analyzed by IFA using rabbit serum against HN protein. (E) NDV mutants are heat-treated for the indicated times at 56°C, then incubated with BHK-21 cells (moi 10) at 4°C for 1 hour. The lysates of cells bound with NDV and equal volumes of the virus input are resolved by SDS-PAGE. Western blot is performed with rabbit serum against NP protein. (F) BHK-21 cells are infected with NDV mutants heat-treated for 10 min at 56°C (moi 1.0). Cells are harvested and lysed at the indicated time points, then analyzed for the replication level of NDV genomic RNA by using a real-time RT-PCR assay. (G) BHK-21 cells are infected with NDV mutants heat-treated for 10 min at 56°C (moi 1.0). Media from infected cells are harvested at the indicated time points and virus titers are determined by TCID50 titration in BHK-21 cells.
Fig 4.
Effect of the surface charge of HN protein on its structural stability.
(A) SDS-PAGE analysis of HN proteins cleaved from viral particles of NDV rTS-HN-N3 and rTS-HN-P4. (B) Heat-inactivation kinetics of NA activity of cHN proteins and NDV mutants. After heat-treatment at the indicated temperature for 10 min, the proteins and viruses are tested for their NA activity. The inactivated fractions of NA activity are represented on a percent scale as a function of heat-treatment temperature. The stability of protein/virus is shown as the temperature for a 50% decrease in NA activity of protein/virus heat-treated for 10 min (Tm-NA). (C, D) Far UV CD thermal unfolding profiles of protein cHN-N3 (C) and cHN-P4 (D). The CD spectra at different temperature ranging from 40°C (purple) to 75°C (blue) are measured using a CD spectrophotometer J-1500 (JASCO). The legend on the right shows the line colors and their corresponding temperatures. (E) Temperature-induced transition of HN proteins as monitored by the changes in ellipticity at 222 nm. The protein stability is shown as the temperature for a 50% decrease in Δ Ellipticity at 222 nm (Tm-CD). (F, G) Secondary structure contents of protein cHN-N3 (F) and cHN-P4 (G) under different heat-treatment temperature.
Fig 5.
Effect of the surface charge of HN protein on its temperature-induced aggregation.
(A) The DLS spectra of HN proteins heat-treated for 10 min at 56°C are measured using Zetasizer Nano ZS (Malvern). (B) Temperature-induced aggregation of cHN as monitored by protein size. After heat-treatment at the indicated temperature for 10 min, the proteins are tested for size. The protein stability is shown as the temperature for a 50% increase in the size of protein heat-treated for 10 min (Tm-SZ). (C) Electron micrographs of heat-treated HN proteins. cHNs are heat-treated at the indicated temperature for 10 min, then negatively stained with uranyl formate. (D) Effect of pH on the stability of cHN proteins. Tm-NA of protein at the indicated pH is shown as the temperature for a 50% decrease in NA activity of protein heat-treated for 10 min. (E) Zeta potentials of HN proteins at the indicated pH are measured using Zetasizer Nano S (Malvern). (F-H) Negative and positive iso-potential contours of HN proteins are displayed at a level of -5 kT/e (red) and +5 kT/e (blue), respectively. The names of HN proteins are indicated at the bottom of structures. The structures of HN proteins are obtained by homology modeling, using the HN structure of AV strain (PBD ID code 3T1E) as a template. (I) Proposed model for the effect of surface charge of HN protein on its temperature-induced aggregation.
Fig 6.
Improvement in the thermal stability of NDV LaSota vaccines by surface charge engineering.
(A) Schematic representation showing the construction of recombinant NDVs with an enhanced negative charge of HN protein via introducing charge-associated amino acid mutations. (B) Scatter diagram showing the relationship between thermostability of rNDVs at 56°C and their corresponding HN charge. (C, D) Heat-inactivation kinetics of infectivity of live NDV vaccines are determined at 37°C (C) and 42°C (D). The live liquid vaccines are prepared by diluting the allantoic fluids infected with rNDVs, to a final viral concentration of 107.0 EID50/ml with Tris-HCl (pH 7.8) containing 3% gelatin. (E) HI antibody response induced in chickens by live liquid NDV vaccines before or after storage at 37°C. The birds are immunized with the indicated vaccines at a volume of 0.1 ml, and challenged with 105.0 EID50 NDV strain F48E8 at 2 weeks post-vaccination. The protection rates are calculated and indicated at the bottom of bar. HI titers from each group are determined prior to the challenge (n = 5). (F, G) Heat-inactivation kinetics of HA activities of inactivated NDV vaccines are determined at 37°C (F) and 42°C (G). The inactivated vaccines are prepared by inactivating the allantoic fluids infected with rNDVs by using 0.05% BPL at 37°C for 2 h, followed by diluting to 107.5 EID50/ml with Tris-HCl (pH 7.8). (H) HI antibody response induced in chickens by inactivated NDV vaccines before or after storage at 37°C. The birds are immunized with the indicated vaccines at a volume of 0.5 ml, and challenged with 105.0 EID50 NDV strain F48E8 at 4 weeks post-vaccination. The protection rates are calculated and indicated at the bottom of bar. HI titers from each group are determined prior to the challenge (n = 5).
Fig 7.
Improvement in the thermal stability of H1N1 IAV vaccine by surface charge engineering.
(A) Heat map showing the percentage distribution of attachment glycoprotein sequences by charge (2.0 per unit, horizontal axis) for each enveloped virus (vertical axis). (B) Multi-sequence alignment of HA proteins from H1N1 IAV strains SC18, PR8-E, and FAV09, representing high, middle, and low negative charge at pH 7.4, respectively. The amino acid substitutions that can greatly influence the charge of HA protein are indicated with color letters. The dots represent the identical amino acid residues, or the substitutions that do not greatly influence the charge of HA protein. (C) Schematic representation showing the construction of recombinant H1N1 IAVs with changed negative charge of HA proteins via introducing charge-associated amino acid mutations. (D) Scatter diagram showing the relationship between thermostability of rIAVs at 56°C and their corresponding HA charges. (E-G) Molecular surfaces of HA proteins are colored according to electrostatic potentials with a range of red (- 5.0 V) to blue (+ 5.0 V). The names of IAV strains are indicated at the bottom of structures. The structures of mutated HA proteins are obtained by homology modeling, using the HN structure of P1/1951 strain (PBD ID code 6N41) as a template. (H) Survival of inactivated IAV vaccines. The inactivated vaccines are prepared by inactivating the allantoic fluids infected with rIAVs by using 0.05% BPL at 37°C for 2 h, followed by diluting to 107.5 EID50/ml with Tris-HCl (pH 7.8). (I, J) Animal test of stored vaccines. The inactivated IAV vaccines are stored at 37°C for indicated times, and used to immunize BALB/c mice with a volume of 0.1 ml. At 4 weeks post-vaccination, mice are challenged with 103.0 EID50 IAV strain rPR8-E, then monitored daily for clinical signs and mortality for 14 days. HI titers from each group (I) are determined prior to the challenge (n = 5). The percentages of survival of mice from each group (J) is calculated.
Fig 8.
Proposed model for the surface charge of HN protein as “the key determinant” for the thermostability of NDV.
The negative surface charge of HN from thermostable NDV is considerably higher than that of HN from thermolabile NDV. The first step of NDV infection is the binding of HN with the sialic acid receptor of the host cells. When thermolabile virion is subjected to environmental heat, HN proteins with low negative surface charge tend to aggregate, and detach from viral particles. Then the naked viral particles can-not bind to the cell receptors and the viral infection does not occur. However, when the thermostable virion is subjected to heat, HN proteins with high negative surface charge do not aggregate, and remain evenly distributed on the viral particles. This allows the initiation of infection and complete cycle of viral replication.