J Vet Sci.  2017 Aug;18(S1):351-359. 10.4142/jvs.2017.18.S1.351.

Chimeric rabies glycoprotein with a transmembrane domain and cytoplasmic tail from Newcastle disease virus fusion protein incorporates into the Newcastle disease virion at reduced levels

• Yu GM
• Zu SL
• Zhou WW
• Wang XJ
• Shuai L
• Wang XL
• Ge JY
• Bu ZG

• KMID: 2412560
• DOI: http://doi.org/10.4142/jvs.2017.18.S1.351

Abstract

Rabies remains an important worldwide health problem. Newcastle disease virus (NDV) was developed as a vaccine vector in animals by using a reverse genetics approach. Previously, our group generated a recombinant NDV (LaSota strain) expressing the complete rabies virus G protein (RVG), named rL-RVG. In this study, we constructed the variant rL-RVGTM, which expresses a chimeric rabies virus G protein (RVGTM) containing the ectodomain of RVG and the transmembrane domain (TM) and a cytoplasmic tail (CT) from the NDV fusion glycoprotein to study the function of RVG's TM and CT. The RVGTM did not detectably incorporate into NDV virions, though it was abundantly expressed at the surface of infected BHK-21 cells. Both rL-RVG and rL-RVGTM induced similar levels of NDV virus-neutralizing antibody (VNA) after initial and secondary vaccination in mice, whereas rabies VNA induction by rL-RVGTM was markedly lower than that induced by rL-RVG. Though rL-RVG could spread from cell to cell like that in rabies virus, rL-RVGTM lost this ability and spread in a manner similar to the parental NDV. Our data suggest that the TM and CT of RVG are essential for its incorporation into NDV virions and for spreading of the recombinant virus from the initially infected cells to surrounding cells.

Keyword

Newcastle disease virus; antibody response; chimeric rabies glycoprotein; rabies virus; viral vector

MeSH Terms

Animals
Blotting, Western
Mice
Microscopy, Confocal
Newcastle disease virus/*genetics
Rabies/immunology/*prevention & control
Rabies virus/*genetics
Vaccines, Synthetic/*genetics/immunology/therapeutic use/ultrastructure
Virion/*genetics
Vaccines, Synthetic

Figure

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  Fig. 1 Construction of a viral vector for expressing the chimeric NDV/RV protein RVGTM. (A) NDV has two transmembrane glycoproteins; HN protein and F protein. The NDV F protein and rabies virus G protein (RVG) are type I transmembrane proteins. (B) Schematic of the generation of recombinant NDV rL bearing the RVGTM gene. RVGTM is a chimera of the RVG ectodomain fused with the sequence for the transmembrane domain and cytoplasmic tail of the NDV protein. The RVGTM sequence was inserted into the vector PBRN-rL at the PmeI site to produce prL-RVGTM. NDV, Newcastle disease virus; RV, rabies virus; RVGTM, chimeric rabies virus G protein.

• 

  Fig. 2 The rL-RVGTM expresses RV and NDV components. (A) Detection of RVGTM expression by immunofluorescence. BHK-21 cells were infected with rL, rL-RVG, or rL-RVGTM at a multiplicity of infection of 0.1. At 24 h post-infection, the cells were fixed and stained with chicken anti-NDV and dog anti-RV sera followed by fluorescein isothiocyanate (FITC)-conjugated rabbit anti-dog and tetramethyl rhodamine isocyanate (TRITC)-labeled goat anti-chicken antibodies. The cells were permeabilized with 0.02% Triton X-100 and cell nuclei were stained with DAPI. (B) Western blot analysis of recombinant NDV expressing RVGTM. Lysates of BHK-21 cells infected with rL, rL-RVG, or rL-RVGTM were separated by 12% SDS-PAGE and incubated with dog serum against RV, chicken serum against NDV, or anti-β-actin monoclonal antibody as a loading control. After incubation with peroxidase-conjugated secondary antibody, the proteins were visualized with 3,3-diaminobenzidine reagent. The locations of marker proteins are indicated on the left, and the antiserum or antibody used is indicated on the right. RVGTM, chimeric rabies virus G protein; RV, rabies virus; NDV, Newcastle disease virus; RVG, rabies virus G protein; MK, protein molecular marker.

• 

  Fig. 3 Comparison of viral growth kinetics and viral pathogenicity of rL, rL-RVG, and rL-RVGTM. (A) Ten-day-old embryonated eggs were inoculated with rL, rL-RVG, or rL-RVGTM (0.1 mL of 100 EID50), and the allantoic fluid from each group was harvested at different times (12, 24, 36, 48, 60, 72, 84, 96, or 108 h post-infection). The fluid from five eggs per time was pooled for TCID50 determination in BHK cells. The data shown are means of the results from five experiments, and the error bars indicate SD. (B) Pathogenicity of the recombinant viruses was determined in chicken embryos and chicks by determining the mean death time (MDT) in embryonated eggs, the intracerebral pathogenicity index (ICPI) in 1-day-old chickens, and the intravenous pathogenicity index (IVPI) in 6-week-old chickens. (C) Weight changes of mice inoculated with rL-RVG or rL-RVGTM. Two groups of 12 mice were inoculated intramuscularly (i.m.) in the thigh muscle with 5 × 107 EID50 rL-RVG or rL-RVGTM and were observed and weighed daily for 14 days. All mice survived and body weight changes of each group are shown as ratios of the body weight at day 0 (set as 100). RVG, rabies virus G protein; RVGTM, chimeric rabies virus G protein.

• 

  Fig. 4 RVGTM incorporates poorly into the NDV virion. (A) To determine whether the RVGTM incorporates into the NDV virion, the recombinant rL-RVG and rL-RVGTM viruses were purified through sucrose gradients. Equivalent amounts of rL, rL-RVG, and rL-RVGTM virions were analyzed by SDS-PAGE. (B and C) Top: ELISA analysis of the incorporation of RVGTM and RVG into the NDV virion was performed by testing for G protein. Bottom: ELISA analysis of NDV demonstrates that the amount of the two recombinant viruses is equivalent despite differences in G protein incorporation. RVGTM, chimeric rabies virus G protein; NDV, Newcastle disease virus; RVG, rabies virus G protein; MK, protein molecular marker; NP, nucleuo protein; M, matrixprotein; GP, Glycoprotein.

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  Fig. 5 rL-RVGTM shows a pattern of spread that is similar to the pattern of rL. (A) Immunofluorescence was used to examine the spread of the viruses. BHK-21 cells were infected with rL, rL-RVG, or rL-RVGTM for 1 h at a multiplicity of infection of 0.1, followed by washing in phosphate belanced solution (PBS) and incubation at 37℃. The infected cells were fixed at the indicated times (24, 36, 48, 60, 72, or 84 h) for immunofluorescent staining with chicken serum against NDV. As shown, rL-RVGTM lost the ability to spread from the initial infected cell to adjacent cells. (B) Supernatants were collected at the same times as in panel A to measure virus titers of 10-day-old embryonated chicken eggs. As shown, the peak titer of rL-RVGTM was similar to that of rL but was approximately one-fifth of a log lower than that of rL-RVG. RVGTM, chimeric rabies virus G protein; RVG, rabies virus G protein.

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  Fig. 6 rL-RVGTM elicits reduced levels of RV VNA. (A) Induction of serum antibodies specific to NDV and RV were assessed following intramuscularly (i.m.) immunization of mice with equivalent levels of rL-RVG and rL-RVGTM. Twelve mice from each group were given the first immunization at week 0 and a second dose 4 weeks later. Serum samples were collected at the third and fourth weeks after the first immunization and at appropriate times after second dose. Neutralization test of NDV VNA. Values represent the dilution ratio of NDV specific antibodies that can protect half of the cells from being infected by NDV. (B) Neutralization test of RV VNA. Values are presented as IU/mL. *p < 0.05, **p < 0.01. RVGTM, chimeric rabies virus G protein; RV, rabies virus; VNA, virus-neutralizing antibody; NDA, Newcastle disease virus; RVG, rabies virus G protein.

Reference

1. Albertini AA, Baquero E, Ferlin A, Gaudin Y. Molecular and cellular aspects of rhabdovirus entry. Viruses. 2012; 4:117–139.

2. Bukreyev A, Huang Z, Yang L, Elankumaran S, St Claire M, Murphy BR, Samal SK, Collins PL. Recombinant Newcastle disease virus expressing a foreign viral antigen is attenuated and highly immunogenic in primates. J Virol. 2005; 79:13275–13284.

3. Bukreyev A, Skiadopoulos MH, Murphy BR, Collins PL. Nonsegmented negative-strand viruses as vaccine vectors. J Virol. 2006; 80:10293–10306.

4. Chen L, Colman PM, Cosgrove LJ, Lawrence MC, Lawrence LJ, Tulloch PA, Gorman JJ. Cloning, expression, and crystallization of the fusion protein of Newcastle disease virus. Virology. 2001; 290:290–299.

5. Chen L, Gorman JJ, McKimm-Breschkin J, Lawrence LJ, Tulloch PA, Smith BJ, Colman PM, Lawrence MC. The structure of the fusion glycoprotein of Newcastle disease virus suggests a novel paradigm for the molecular mechanism of membrane fusion. Structure. 2001; 9:255–266.

6. Cleverley DZ, Lenard J. The transmembrane domain in viral fusion: essential role for a conserved glycine residue in vesicular stomatitis virus G protein. Proc Natl Acad Sci U S A. 1998; 95:3425–3430.

7. Della-Porta AJ, Spencer T. Newcastle disease. Aust Vet J. 1989; 66:424–426.

8. Dietzschold B, Schnell M, Koprowski H. Pathogenesis of rabies. Curr Top Microbiol Immunol. 2005; 292:45–56.

9. Dietzschold B, Wang HH, Rupprecht CE, Celis E, Tollis M, Ertl H, Heber-Katz E, Koprowski H. Induction of protective immunity against rabies by immunization with rabies virus ribonucleoprotein. Proc Natl Acad Sci U S A. 1987; 84:9165–9169.

10. Dietzschold B, Wiktor TJ, Trojanowski JQ, Macfarlan RI, Wunner WH, Torres-Anjel MJ, Koprowski H. Differences in cell-to-cell spread of pathogenic and apathogenic rabies virus in vivo and in vitro. J Virol. 1985; 56:12–18.

11. Dietzschold B, Wunner WH, Wiktor TJ, Lopes AD, Lafon M, Smith CL, Koprowski H. Characterization of an antigenic determinant of the glycoprotein that correlates with pathogenicity of rabies virus. Proc Natl Acad Sci U S A. 1983; 80:70–74.

12. DiNapoli JM, Kotelkin A, Yang L, Elankumaran S, Murphy BR, Samal SK, Collins PL, Bukreyev A. Newcastle disease virus, a host range-restricted virus, as a vaccine vector for intranasal immunization against emerging pathogens. Proc Natl Acad Sci U S A. 2007; 104:9788–9793.

13. DiNapoli JM, Ward JM, Cheng L, Yang L, Elankumaran S, Murphy BR, Samal SK, Collins PL, Bukreyev A. Delivery to the lower respiratory tract is required for effective immunization with Newcastle disease virus-vectored vaccines intended for humans. Vaccine. 2009; 27:1530–1539.

14. Doms RW, Keller DS, Helenius A, Balch WE. Role for adenosine triphosphate in regulating the assembly and transport of vesicular stomatitis virus G protein trimers. J Cell Biol. 1987; 105:1957–1969.

15. Faber M, Li J, Kean RB, Hooper DC, Alugupalli KR, Dietzschold B. Effective preexposure and postexposure prophylaxis of rabies with a highly attenuated recombinant rabies virus. Proc Natl Acad Sci U S A. 2009; 106:11300–11305.

16. Fu ZF. Rabies and rabies research: past, present and future. Vaccine. 1997; 15:Suppl. S20–S24.

17. Ge J, Deng G, Wen Z, Tian G, Wang Y, Shi J, Wang X, Li Y, Hu S, Jiang Y, Yang C, Yu K, Bu Z, Chen H. Newcastle disease virus-based live attenuated vaccine completely protects chickens and mice from lethal challenge of homologous and heterologous H5N1 avian influenza viruses. J Virol. 2007; 81:150–158.

18. Ge J, Tian G, Zeng X, Jiang Y, Chen H, Bua Z. Generation and evaluation of a Newcastle disease virus-based H9 avian influenza live vaccine. Avian Dis. 2010; 54:1 Suppl. S294–S296.

19. Ge J, Wang X, Tao L, Wen Z, Feng N, Yang S, Xia X, Yang C, Chen H, Bu Z. Newcastle disease virus-vectored rabies vaccine is safe, highly immunogenic, and provides long-lasting protection in dogs and cats. J Virol. 2011; 85:8241–8252.

20. Huang Z, Elankumaran S, Yunus AS, Samal SK. A recombinant Newcastle disease virus (NDV) expressing VP2 protein of infectious bursal disease virus (IBDV) protects against NDV and IBDV. J Virol. 2004; 78:10054–10063.

21. Jeetendra E, Ghosh K, Odell D, Li J, Ghosh HP, Whitt MA. The membrane-proximal region of vesicular stomatitis virus glycoprotein G ectodomain is critical for fusion and virus infectivity. J Virol. 2003; 77:12807–12818.

22. Khattar SK, Collins PL, Samal SK. Immunization of cattle with recombinant Newcastle disease virus expressing bovine herpesvirus-1 (BHV-1) glycoprotein D induces mucosal and serum antibody responses and provides partial protection against BHV-1. Vaccine. 2010; 28:3159–3170.

23. Maillard AP, Gaudin Y. Rabies virus glycoprotein can fold in two alternative, antigenically distinct conformations depending on membrane-anchor type. J Gen Virol. 2002; 83:1465–1476.

24. Morimoto K, Foley HD, McGettigan JP, Schnell MJ, Dietzschold B. Reinvestigation of the role of the rabies virus glycoprotein in viral pathogenesis using a reverse genetics approach. J Neurovirol. 2000; 6:373–381.

25. Morimoto K, Hooper DC, Spitsin S, Koprowski H, Dietzschold B. Pathogenicity of different rabies virus variants inversely correlates with apoptosis and rabies virus glycoprotein expression in infected primary neuron cultures. J Virol. 1999; 73:510–518.

26. Nakaya T, Cros J, Park MS, Nakaya Y, Zheng H, Sagrera A, Villar E, García-Sastre A, Palese P. Recombinant Newcastle disease virus as a vaccine vector. J Virol. 2001; 75:11868–11873.

27. Neumann G, Whitt MA, Kawaoka Y. A decade after the generation of a negative-sense RNA virus from cloned cDNA--what have we learned? J Gen Virol. 2002; 83:2635–2662.

28. Odell D, Wanas E, Yan J, Ghosh HP. Influence of membrane anchoring and cytoplasmic domains on the fusogenic activity of vesicular stomatitis virus glycoprotein G. J Virol. 1997; 71:7996–8000.

29. Park MS, Steel J, García-Sastre A, Swayne D, Palese P. Engineered viral vaccine constructs with dual specificity: avian influenza and Newcastle disease. Proc Natl Acad Sci U S A. 2006; 103:8203–8208.

30. Roche S, Gaudin Y. Characterization of the equilibrium between the native and fusion-inactive conformation of rabies virus glycoprotein indicates that the fusion complex is made of several trimers. Virology. 2002; 297:128–135.

31. Roche S, Rey FA, Gaudin Y, Bressanelli S. Structure of the prefusion form of the vesicular stomatitis virus glycoprotein G. Science. 2007; 315:843–848.

32. Veits J, Wiesner D, Fuchs W, Hoffmann B, Granzow H, Starick E, Mundt E, Schirrmeier H, Mebatsion T, Mettenleiter TC, Römer-Oberdörfer A. Newcastle disease virus expressing H5 hemagglutinin gene protects chickens against Newcastle disease and avian influenza. Proc Natl Acad Sci U S A. 2006; 103:8197–8202.

33. Weissenhorn W, Hinz A, Gaudin Y. Virus membrane fusion. FEBS Lett. 2007; 581:2150–2155.

34. Whitt MA, Buonocore L, Prehaud C, Rose JK. Membrane fusion activity, oligomerization, and assembly of the rabies virus glycoprotein. Virology. 1991; 185:681–688.

35. Wiktor TJ, Macfarlan RI, Reagan KJ, Dietzschold B, Curtis PJ, Wunner WH, Kieny MP, Lathe R, Lecocq JP, Mackett M. Protection from rabies by a vaccinia virus recombinant containing the rabies virus glycoprotein gene. Proc Natl Acad Sci U S A. 1984; 81:7194–7198.

36. Wyatt LS, Moss B, Rozenblatt S. Replication-deficient vaccinia virus encoding bacteriophage T7 RNA polymerase for transient gene expression in mammalian cells. Virology. 1995; 210:202–205.