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Package of NDV-pseudotyped HIV-Luc virus and its application in the neutralization assay for NDV infection.

Wang B, Wang B, Liu P, Li T, Si W, Xiu J, Liu H - PLoS ONE (2014)

Bottom Line: It has been a great threat for the poultry industry all around the world.In this report, we successfully produced infectious pseudotyped pNL4-3-Luc-R(-)E(-) (HIV-Luc) viruses with the HN and F envelope proteins of NDV.Further investigation revealed the cytoplasmic domains of HN and F, especially HN, plays a significant role in the infection efficiency of these pseudotyped HIV-Luc viruses.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, China; College of Animal Sciences, Fujian Agriculture and Forestry University, Fuzhou, China.

ABSTRACT
Newcastle disease virus (NDV) is a member of the Paramyxovirinae subfamily and can infect most species of birds. It has been a great threat for the poultry industry all around the world. In this report, we successfully produced infectious pseudotyped pNL4-3-Luc-R(-)E(-) (HIV-Luc) viruses with the HN and F envelope proteins of NDV. Further investigation revealed the cytoplasmic domains of HN and F, especially HN, plays a significant role in the infection efficiency of these pseudotyped HIV-Luc viruses. Replacement of, or direct fusion to the cytoplasmic domain of the HN protein by that of vesicular stomatitis virus G (VSV-G) could greatly enhance or destroy the infective potential of HN and F-pseudotyped (NDV-pseudotyped) HIV-Luc virus. We further established a novel neutralization assay to evaluate neutralizing antibodies against NDV with the NDV-pseudotyped HIV-Luc viruses. Comparative neutralization data indicate that the results determined by using the NDV-pseudotyped HIV-Luc viruses are as reliable as those by the conventional virus-neutralization assay (VN test) with native NDV. Moreover, the results show that the novel neutralization assay is more sensitive than the VN test.

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Analysis of factors impacting the infection efficiency of NDV-pseudotyped virus.A panel of mutant and wild-type HN and F combinations were co-transfected into 293 T cells with the HIV-Luc vector. Pseudoviruses in the culture medium were harvested 48 h later and used to infect 293 T cells in 96-well plates. The luciferase activity of the infected cells was detected 48 h post infection and normalized with the titer of p24 in the pseudoviruses. All results are shown as means ± SD from four independent experiments. “**” indicated P<0.01.
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pone-0099905-g003: Analysis of factors impacting the infection efficiency of NDV-pseudotyped virus.A panel of mutant and wild-type HN and F combinations were co-transfected into 293 T cells with the HIV-Luc vector. Pseudoviruses in the culture medium were harvested 48 h later and used to infect 293 T cells in 96-well plates. The luciferase activity of the infected cells was detected 48 h post infection and normalized with the titer of p24 in the pseudoviruses. All results are shown as means ± SD from four independent experiments. “**” indicated P<0.01.

Mentions: Although the NDV-pseudotyped HIV-Luc viral particle was successfully produced, the infection efficiency was found to be over 34-fold lower than with the VSV-G-pseudotyped HIV-Luc virus (P<0.01) (Figure 2C). We hypothesized that this may be due to the impact of the cytoplasmic domain of the envelope proteins [33], [34]. To address this, four plasmids containing mutant HN or F were constructed (Figure 1A and B). After confirming their expression on 293 T cells (Figure 1C), nine combinations of wild-type and mutant HN and F were co-transfected into 293 T cells with HIV-Luc vector to produce corresponding pseudotyped viruses. As shown in figure 3, of the nine combinations, six produce infectious pseudotyped viruses including HN plus F, HN plus FΔCT, HN plus F/VCT, VCT/HN plus F, VCT/HN plus FΔCT and VCT/HN plus F/VCT. Among the infectious pseudotyped viruses, the infection efficiencies of VCT/HN plus F and VCT/HN plus F/VCT were the highest, reaching from 8 to 50 folds higher than all other groups (P<0.01). The infection efficiency between the two combinations was not significantly different (P>0.05). This indicated that replacement of the cytoplasmic domain of HN by VCT greatly enhanced the infection efficiency of NDV-pseudotyped HIV-Luc viruses. However, fusion of VCT to the N-terminus of the cytoplasmic domain of HN resulted in the complete loss of infectivity as shown in the combinations of VCT-HN plus F, VCT-HN plus FΔCT and VCT-HN plus F/VCT (Figure 3). These results revealed the important role of the cytoplasmic domain of HN on the infection efficiency of NDV-pseudotyped HIV-Luc viruses. Moreover, when we compared VCT/HN plus FΔCT with the combinations of VCT/HN plus F and VCT/HN plus F/VCT, we saw a significant reduction in the infection efficiency with the pseudotyped virus containing FΔCT (P<0.01). This result revealed that the cytoplasmic domain of F also plays an important role in the infection efficiency of NDV-pseudotyped HIV-Luc viruses. This was further confirmed by comparison among the combinations of HN plus F, HN plus FΔCT and HN plus F/VCT, where both the deletion and VCT replacement of the cytoplasmic domain of F significantly reduced infectivity (P<0.01) (Figure 3). Altogether, these results indicated that the cytoplasmic domain of HN was the dominant factor behind the infection efficiency of the NDV-pseudotyped HIV-Luc virus, and VCT/HN plus F or F/VCT is the optimal recombination for the production of highly infectious pseudoviruses.


Package of NDV-pseudotyped HIV-Luc virus and its application in the neutralization assay for NDV infection.

Wang B, Wang B, Liu P, Li T, Si W, Xiu J, Liu H - PLoS ONE (2014)

Analysis of factors impacting the infection efficiency of NDV-pseudotyped virus.A panel of mutant and wild-type HN and F combinations were co-transfected into 293 T cells with the HIV-Luc vector. Pseudoviruses in the culture medium were harvested 48 h later and used to infect 293 T cells in 96-well plates. The luciferase activity of the infected cells was detected 48 h post infection and normalized with the titer of p24 in the pseudoviruses. All results are shown as means ± SD from four independent experiments. “**” indicated P<0.01.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4061091&req=5

pone-0099905-g003: Analysis of factors impacting the infection efficiency of NDV-pseudotyped virus.A panel of mutant and wild-type HN and F combinations were co-transfected into 293 T cells with the HIV-Luc vector. Pseudoviruses in the culture medium were harvested 48 h later and used to infect 293 T cells in 96-well plates. The luciferase activity of the infected cells was detected 48 h post infection and normalized with the titer of p24 in the pseudoviruses. All results are shown as means ± SD from four independent experiments. “**” indicated P<0.01.
Mentions: Although the NDV-pseudotyped HIV-Luc viral particle was successfully produced, the infection efficiency was found to be over 34-fold lower than with the VSV-G-pseudotyped HIV-Luc virus (P<0.01) (Figure 2C). We hypothesized that this may be due to the impact of the cytoplasmic domain of the envelope proteins [33], [34]. To address this, four plasmids containing mutant HN or F were constructed (Figure 1A and B). After confirming their expression on 293 T cells (Figure 1C), nine combinations of wild-type and mutant HN and F were co-transfected into 293 T cells with HIV-Luc vector to produce corresponding pseudotyped viruses. As shown in figure 3, of the nine combinations, six produce infectious pseudotyped viruses including HN plus F, HN plus FΔCT, HN plus F/VCT, VCT/HN plus F, VCT/HN plus FΔCT and VCT/HN plus F/VCT. Among the infectious pseudotyped viruses, the infection efficiencies of VCT/HN plus F and VCT/HN plus F/VCT were the highest, reaching from 8 to 50 folds higher than all other groups (P<0.01). The infection efficiency between the two combinations was not significantly different (P>0.05). This indicated that replacement of the cytoplasmic domain of HN by VCT greatly enhanced the infection efficiency of NDV-pseudotyped HIV-Luc viruses. However, fusion of VCT to the N-terminus of the cytoplasmic domain of HN resulted in the complete loss of infectivity as shown in the combinations of VCT-HN plus F, VCT-HN plus FΔCT and VCT-HN plus F/VCT (Figure 3). These results revealed the important role of the cytoplasmic domain of HN on the infection efficiency of NDV-pseudotyped HIV-Luc viruses. Moreover, when we compared VCT/HN plus FΔCT with the combinations of VCT/HN plus F and VCT/HN plus F/VCT, we saw a significant reduction in the infection efficiency with the pseudotyped virus containing FΔCT (P<0.01). This result revealed that the cytoplasmic domain of F also plays an important role in the infection efficiency of NDV-pseudotyped HIV-Luc viruses. This was further confirmed by comparison among the combinations of HN plus F, HN plus FΔCT and HN plus F/VCT, where both the deletion and VCT replacement of the cytoplasmic domain of F significantly reduced infectivity (P<0.01) (Figure 3). Altogether, these results indicated that the cytoplasmic domain of HN was the dominant factor behind the infection efficiency of the NDV-pseudotyped HIV-Luc virus, and VCT/HN plus F or F/VCT is the optimal recombination for the production of highly infectious pseudoviruses.

Bottom Line: It has been a great threat for the poultry industry all around the world.In this report, we successfully produced infectious pseudotyped pNL4-3-Luc-R(-)E(-) (HIV-Luc) viruses with the HN and F envelope proteins of NDV.Further investigation revealed the cytoplasmic domains of HN and F, especially HN, plays a significant role in the infection efficiency of these pseudotyped HIV-Luc viruses.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, China; College of Animal Sciences, Fujian Agriculture and Forestry University, Fuzhou, China.

ABSTRACT
Newcastle disease virus (NDV) is a member of the Paramyxovirinae subfamily and can infect most species of birds. It has been a great threat for the poultry industry all around the world. In this report, we successfully produced infectious pseudotyped pNL4-3-Luc-R(-)E(-) (HIV-Luc) viruses with the HN and F envelope proteins of NDV. Further investigation revealed the cytoplasmic domains of HN and F, especially HN, plays a significant role in the infection efficiency of these pseudotyped HIV-Luc viruses. Replacement of, or direct fusion to the cytoplasmic domain of the HN protein by that of vesicular stomatitis virus G (VSV-G) could greatly enhance or destroy the infective potential of HN and F-pseudotyped (NDV-pseudotyped) HIV-Luc virus. We further established a novel neutralization assay to evaluate neutralizing antibodies against NDV with the NDV-pseudotyped HIV-Luc viruses. Comparative neutralization data indicate that the results determined by using the NDV-pseudotyped HIV-Luc viruses are as reliable as those by the conventional virus-neutralization assay (VN test) with native NDV. Moreover, the results show that the novel neutralization assay is more sensitive than the VN test.

Show MeSH
Related in: MedlinePlus