UDC 636.4:619:616.98:578:577.2.08:51-76

doi: 10.15389/agrobiology.2015.6.785eng

Acknowledgements:
Supported by Russian Foundation for Basic Research, science project «Mol-a-ved» (15-34-20995).

AFRICAN SWINE FEVER VIRUS GLYCOPROTEINS
p54 AND CD2v IN THE CONTEXT OF IMMUNE RESPONSE
MODULATION: BIOINFORMATIC ANALYSIS OF GENETIC
VARIABILITY AND HETEROGENEITY

K.A. Mima, G.S. Burmakina, I.A. Titov, A.S. Malogolovkin

All-Russian Institute of Veterinary Virology and Microbiology, Federal Agency of Scientific Organizations,
Pokrov, Petushinskii Region, Vladimir Province, 601120 Russia,
e-mail mima89@ya.ru

Received June 30, 2015

The African swine fever virus (ASFV) is a unique representative of Asfaviridae family, which still remains the sole member of genus Asfarvirus. ASF virus is the causative agent of one of the most dangerous diseases of the animals from Suidae family, and moreover, it is capable of infecting soft ticks of the genus Ornithodoros. Genetic and phenotypic heterogeneity of ASF virus is one of the main reasons for the lack of vaccines against this dangerous transboundary disease. In this work we present the analysis of structure and functions of the most variable glycoproteins ASFV p54 and CD2v using bioinformatics analysis and recombinant constructs expressed in mammalian cell cultures, the African green monkey cell culture COS-I and the human embryonal kidney cell culture HEK-293. The index of variability of amino acid sequences for P54 and CD2v proteins was calculated by Simpson’s method. The CD2v protein has variable region (N-terminal domain), which is highly glycosylated (28-30 sites) and located in the outer surface of the cell membrane. This region also contains immunoglobulin domain (amino acids at positions 1-225), which is responsible for CD2v interaction with antibodies. The revealed differences in post-translational modifications and genetic variations of CD2v protein might explain the diversity of the hemadsorption phenomenon among ASF virus isolates. In contrast, p54 protein has variable glycosylated extracellular and intracellular parts. High level of differences in the nucleotide sequences of p54gene (E183L) for various ASFV isolates may be the result of random mutations during virus evolution. Characteristic antigenic properties of ASF virus isolates can obviously be due to found peculiar post-translational processing and genetic variations on СD2v protein. Herein we report the first bioinformatic analysis of post-traslation N- and O-glycosylation in most variable ASF virus proteins, p54 and СD2v. A transient expression of gene constructions used to obtain the recombinant products, p54-EGFP and CD2v-HA, allowed us to demonstrate the evidence for different localization of viral proteins p54-EGFP and CD2v-HA in the transfected cells. Particularly, the fluorescence caused by p54-EGFP was observed in the cytoplasm of the COS-I cells, transfected with recombinant plasmid р54-pEGFP-N1, whereas recombinant CD2v-НА protein was detected only in cell membrane. According to immunoblotting analysis, the CD2v molecular weight was 90 kDa against calculated 65 kDa indicating about 30 % of carbohydrate component in this surface glycoprotein. Moreover, 25 kDa and 90 kDa CD2v molecules, the probable differently glycosylated forms, were revealed in immonoblotting test that is in line with other published data. Thus, bioinformatic analysis and in vitro studies using transient expression in COS-I и HEK-293 cell cultures have shown that protein CD2v is the most likely candidate to define the interaction of ASF virus with the virus-specific antibodies. 

Keywords: African swine fever, glycoprotein, variability, glycosylation, transient expression, immunotypes.

 

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REFERENCES

  1. Dixon L.K., Rock D.L., Vinuela E. African swine fever-like viruses. Virus taxonomy: classification and nomenclature of viruses. Arch. Virol., 1995, 69(10): 92-94.
  2. Michaud V., Randriamparany T., Albina E. Comprehensive phylogenetic reconstructions of African swine fever virus: proposal for a new classification and molecular dating of the virus. PLoS ONE, 2013, 8(7): 1-14 CrossRef
  3. Hubalek Z., Rudolf I., Nowotny N. Arboviruses pathogenic for domestic and wild animals. Adv. Virus Res., 2014, 89: 201-275 CrossRef
  4. Porterfield J.S. The basis of arbovirus classification. Med. Biol., 1975, 53(5): 400-405.
  5. Burrage T.G. African swine fever virus infection in Ornithodoros ticks. Virus Res., 2013, 173(1): 131-139 CrossRef
  6. Boinas F., Ribeiro R., Madeira S., Palma M., de Carvalho I.L., Nuncio S., Wilson A.J. The medical and veterinary role of Ornithodoros erraticus complex ticks (Acari: Ixodida) on the Iberian Peninsula. Journal of Vector Ecology, 2014, 39(2): 238-248 CrossRef
  7. Wilkinson P.J. African swine fever virus. In: Virus infections of porcines. M. Pensaert (ed.). Elsevier Science Publishers, Amsterdam, The Netherlands, 1989: 17-35.
  8. Chapman D.A., Tcherepanov V., Upton C., Dixon L.K. Comparison of the genome sequences of non-pathogenic and pathogenic African swine fever virus. J. Gen. Virol., 2008, 89(2): 397-408 CrossRef
  9. Nix R.J., Gallardo C., Hutchings G., Blanco E., Dixon L.K. Molecular epidemiology of African swine fever virus studied by analysis of four variable genome regions. Arch. Virol., 2006, 151(12): 2475-2494 CrossRef
  10. Yanez R.J., Rodriguez J.M., Nogal M.L., Yuste L., Enriquez C., Rodriguez J.F., Vinuela E. Analysis of the complete nucleotide sequence of African swine fever virus. Virology, 1995, 208(1): 249-278.
  11. Villiers E.P., Gallardo C., Arias M., Silva M., Upton C., Martin R., Bishop R.P. Phylogenomic analysis of 11 complete African swine fever virus genome sequences. Virology, 2010, 400(1): 128-136 CrossRef
  12. Chacon M.R., Almazan F., Nogal M.L., Vinuela E., Rodriguez J.F. The African swine fever. Virology, 1995, 214(2): 670-674.
  13. Martinez P.L., Simon M.C., Lopez-Otín C., Vinuela E. Characterization of the African swine fever virus protein p14.5: a DNA binding protein. Virology, 1997, 229(1): 201-211.
  14. Dixon L.K., Chapman D.A., Netherton C.L., Upton C. African swine fever virus replication and genomics. Virus Res., 2013, 173(1): 3-14 CrossRef
  15. Simon M.C., Freije J.M., Andres G., Lopez-Otín C., Vinuela E. Mapping and sequence of the gene encoding protein p17, a major African swine fever virus structural protein. Virology, 1995, 206(2): 1140-1144.
  16. Simon M.C., Andres G., Almazan F., Vinuela E. Proteolytic processing in African swine fever virus: evidence for a new structural polyprotein, pp62. J. Virol., 1997, 71(8): 5799-5804.
  17. Carrascosa J.L., Carazo J.M., Carrascosa A.L., Garcia N., Santisteban A., Vinuela E. General morphology and capsid fine structure of African swine fever virus particles. Virology, 1984, 132(1): 160-172.
  18. Andres G., Simon M.C., Vinuela E. Assembly of African swine fever virus: role of polyprotein pp220. J. Virol., 1966, 71(3): 2331-2341.
  19. Breese S.S. Jr., DeBoer C.J. Electron microscope observation of African swine fever virus in tissue culture cells. Virology, 1966, 28(3): 420-428.
  20. Karalova E.M., Voskanian G.E., Sarkisian Kh.V., Abroian L.O., Ave-
    tisian A.S., Akopian L.A., Semerdzhian Z.B., Zakarian O.S., Arzumanian G.A., Karalian Z.A. Voprosy virusologii, 2011, 56(1): 33-37 (in Russian).
  21. Salas M.L., Andres G. African swine fever virus morphogenesis. Virus Res., 2013, 173(1): 29-41 CrossRef
  22. Suarez C., Salas M.L., Rodriguez J.M. African swine fever virus polyprotein pp62 is essential for viral core development. J. Virol., 2010, 84(1): 176-187 CrossRef
  23. Gomez-Puertas P., Rodriguez F., Oviedo J.M., Brun A., Alonso C., Escribano J.M. The African swine fever virus proteins p54 and p30 are involved in two distinct steps of virus attachment and both contribute to the antibody-mediated protective immune response. Virology, 1998, 243(2): 461-471.
  24. Sereda A.D., Balyshev V.M. Voprosy virusologii, 2011, 4: 38-42.
  25. Borca M.V., Carrillo C., Zsak L., Laegreid W.W., Kutish G.F., Neilan J.G., Burrage T.G., Rock D.L. Deletion of a CD2-like gene, 8-DR, from African swine fever virus affects viral infection in domestic swine. J. Virol., 1998, 72(4): 2881-2889.
  26. Quintero J.C., Wesley R.D., Whyard T.C., Gregg D., Mebus C.A. In vitro and in vivo association of African swine fever virus with swine erythrocytes. Am. J. Vet. Res., 1986, 47(5): 1125-1131.
  27. Rodriguez J.M., Yanez R.J., Almazan F., Vinuela E., Rodriguez J.F. African swine fever virus encodes a Cd2 homolog responsible for the adhesion of erythrocytes to infected cells. J. Virol., 1993, 67(9): 5312-5320.
  28. Kazakova A.S. Konstruirovanie produtsentov rekombinantnykh belkov R72, R30 i R54 virusa afrikanskoi chumy svinei. Kandidatskaya dissertatsiya [Design of producers of Р72, Р30 and Р54 African swine fever virus proteins. PhD Thesis]. Pokrov, 2013.
  29. Garcia-Boronat M., Diez-Rivero C.M., Reinherz E.L., Reche P.A. PVS: a web server for protein sequence variability analysis tuned to facilitate conserved epitope discovery. Nucl. Acids Res., 2008, 36: 35-41 CrossRef
  30. Díez-Rivero C.M., Reche P. Discovery of conserved epitopes through sequence variability analyses. Bioinformatics for Immunomics, 2010, 3: 95-101 CrossRef
  31. Maurisse R., De Semir D., Emamekhoo H., Bedayat B., Abdolmohammadi A., Parsi H., Gruenert D.C. Comparative transfection of DNA into primary and transformed mammalian cells from different lineages. BioMed Central Biotechnol., 2010, 10(9): 2-9 CrossRef
  32. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, 227(5259): 680-685.

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