Archives of Virology

, Volume 141, Issue 2, pp 315–329 | Cite as

Nucleotide sequence and expression inE. coli of the complete P4 type VP4 from a G2 serotype human rotavirus

  • N. P. Mahajan
  • C. Durga Rao
Original Papers


The complete sequence of a P4 type VP4 gene from a G2 serotype human rotavirus, IS2, isolated in India has been determined. Although the IS2 VP4 is highly homologous to the other P4 type alleles, it contained acidic amino acid substitutions at several positions that make it acidic among the P4 type alleles that are basic. Moreover, comparative sequence analysis revealed unusual polymorphism in members of the P4 type at amino acid position 393 which is highly conserved in members of other VP4 types. To date, expression of complete VP4 inE. coli has not been achieved. In this study we present successful expression inE. coli of the complete VP4 as well as VP8* and VP5* cleavage subunits in soluble form as fusion proteins of the maltose-binding protein (MBP) and their purification by single-step affinity chromatography. The hemagglutinating activity exhibited by the recombinant protein was specifically inhibited by the antiserum raised against it. Availability of pure VP4 proteins should facilitate development of polyclonal and monoclonal antibodies (MAbs) for P serotyping of rotaviruses.


Recombinant Protein Amino Acid Substitution Affinity Chromatography Complete Sequence Amino Acid Position 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Brosius J, Holy A (1984) Regulation of ribosomal RNA promoters with a synthetic lac promoter. Proc Natl Acad Sci USA 81: 6929–6933Google Scholar
  2. 2.
    Browning GF, Fitzgerald TA, Chalmers RM, Snodgrass DR (1991) A novel group A rotavirus G serotype: serological and genomic characterization of equine isolate FI23. J Clin Microbiol 29: 2043–2046Google Scholar
  3. 3.
    Burke B, Bridger JC, Desselberger U (1994) Temporal correlation between a single amino acid change in the VP4 of a porcine rotavirus and a marked change in pathogenicity. Virology 202: 754–759Google Scholar
  4. 4.
    Burke B, McCrae MA, Desselberger U (1994) Sequence analysis of two porcine rotaviruses differing in growth in vitro and in pathogenicity: distinct VP4 sequences and conservation of NS53, VP6 and VP7 genes. J Gen Virol 75: 2205–2212Google Scholar
  5. 5.
    Coulson BS (1993) Typing of human rotavirus VP4 by an enzyme immunoassay using monoclonal antibodies. J Clin Mircobiol 31: 1–8Google Scholar
  6. 6.
    Das M, Dunn SJ, Woode GN, Greenberg HB, Rao CD (1993) Both surface proteins VP4 and VP7 of an asymptomatic neonatal rotavirus strain (I321) have high levels of sequence identity with the homologous proteins of serotype 10 bovine rotaviruses. Virology 194: 374–379Google Scholar
  7. 7.
    Dunn SJ, Burns JW, Cross TL, Vo PT, Ward RL, Bremont M, Greenberg HB (1994) Comparison of VP4 and VP7 of five murine rotavirus strains. Virology 203: 250–259Google Scholar
  8. 8.
    Estes MK, Cohen J (1989) Rotavirus gene structure and function. Microbiol Rev 53: 410–449Google Scholar
  9. 9.
    Fukudome K, Yoshie O, Konno T (1989) Comparison of human, simian and bovine rotaviruses for requirement of sialic acid in hemagglutination and cell adsorption. Virology 172: 196–205Google Scholar
  10. 10.
    Gerna G, Sears J, Hoshino Y, Steele AD, Nakagomi O, Sarasini A, Flores J (1994) Identification of a new VP4 serotype of human rotaviruses. Virology 200: 66–71Google Scholar
  11. 11.
    Gorziglia M, Green KY, Nishikawa K, Taniguchi K, Jones R, Kapikian AZ, Chanock RM (1988) Sequence of the fourth gene of human rotavirus recovered from asymptomatic or symptomatic infections. J Virol 62: 2978–2983Google Scholar
  12. 12.
    Gorziglia M, Larralde G, Ward RL (1990) Neutralization epitopes on rotavirus SA11 4fM outer capsid proteins. J Virol 64: 4534–4539Google Scholar
  13. 13.
    Hardy ME, Gorziglia M, Woode GN (1993) The outer capsid protein VP4 of equine rotavirus strain H2 represents a unique type by amino acid sequence analysis. Virology 193: 492–497Google Scholar
  14. 14.
    Hoshino Y, Kapikian AZ (1994) Rotavirus antigens. In: Ramig RF (ed) Rotaviruses. Springer, Berlin Heidelberg New York Tokyo, pp 179–227Google Scholar
  15. 15.
    Huang J, Nagesha HS, Holmes IH (1993) Comparative sequence analysis of VP4s from five Australian porcine rotaviruses: implication of an apparent new P type. Virology 196: 319–327Google Scholar
  16. 16.
    Isa P, Snodgrass DR (1994) Serological and genomic characterization of equine rotavirus VP4 proteins identifies three different P serotypes. Virology 201: 364–372Google Scholar
  17. 17.
    Isegawa Y, Nakagomi O, Brussow H, Minamoto N, Nakagomi T, Ueda S (1994) A unique VP4 gene allele carried by an unusual bovine rotavirus strain, 993/83. Virology 198: 366–369Google Scholar
  18. 18.
    Isegawa Y, Nakagomi O, Nakagomi T, Ueda S (1992) VP4 sequence highly conserved in human rotavirus strain AU-1 and feline rotavirus strain FRV-1. J Gen Virol 73: 1939–1946Google Scholar
  19. 19.
    Kalica AR, Flores J, Greenberg HB (1983) Identification of rotaviral gene that codes for the hemagglutinin and protease enhanced plaque formation. Virology 125: 194–205Google Scholar
  20. 20.
    Kantharidis P, Dyall-Smith ML, Holmes IH (1987) Marked sequence variation between segment 4 genes of human RV5 and simian SA11 rotaviruses. Arch Virol 93: 111–121Google Scholar
  21. 21.
    Kantharidis P, Dyall-Smith ML, Tregear GW, Holmes IH (1988) Nucleotide sequence of UK bovine rotavirus segment 4: possible host restriction of VP3 genes. Virology 168: 308–315Google Scholar
  22. 22.
    Kapikian AZ, Chanock RM (1990) Rotaviruses. In: Fields BN, Knipe DN, Melnick JL, Chanock RM, Roizman B, Shope RE (eds) Virology, vol 2. Raven Press, New York, pp 1353–1404Google Scholar
  23. 23.
    Kobayashi N, Taniguchi K, Urasawa S (1990) Identification of operationally overlapping and independent cross-reactive neutralization regions on human rotavirus VP4. J Gen Virol 71: 2615–2623Google Scholar
  24. 24.
    Larralde G, Gorziglia M (1992) Distribution of conserved and serotype-specific epitopes on the VP8 subunit of rotavirus VP4 proteins. J Virol 66: 7438–7443Google Scholar
  25. 25.
    Lizano M, Lopez S, Arias C (1991) The amino-terminal half of rotavirus SA11 4fM VP4 protein contains a hemagglutination domain and primes for neutralizing antibodies to the virus. J Virol 65: 1983–1991Google Scholar
  26. 26.
    Maina CV, Riggs PD, Grandea AG III, Slatko BE, Moran LS, Taglia-Monte JA, McReynolds LA, Guan C (1988) A vector to express and purify foreign proteins inEscherichia coli by fusion to and separated from maltose-binding protein. Gene 74: 365–373Google Scholar
  27. 27.
    Mackow ER, Barnett JW, Chan H, Greenberg HB (1989) The rhesus rotavirus outer capsid protein VP4 functions as a hemagglutinin and is antigenically conserved when expressed by a baculovirus recombinant. J Virol 63: 1661–1668Google Scholar
  28. 28.
    Mackow ER, Shaw RD, Matsui SM, Vo PT, Dang MN, Greenberg HB (1988) The rhesus rotavirus gene encoding protein VP3: location of amino acids involved in homologous and heterologous rotavirus neutralization and identification of a putative fusion region. Proc Natl Acad Sci USA 85: 645–649Google Scholar
  29. 29.
    Mitchell DB, Both GW (1988) Complete nucleotide sequence of the simian rotavirus SA11 VP4 gene. Nucleic Acids Res 17: 2122Google Scholar
  30. 30.
    Nishikawa K, Gorziglia M (1989) Complete nucleotide sequence of the VP3 gene of porcine rotavirus OSU. Nucleic Acids Res 16: 11847Google Scholar
  31. 31.
    Nishikawa K, Taniguchi K, Torres A, Hoshino Y, Green K, Kapikian AZ, Chanock RM, Gorziglia M (1988) Comparative analysis of VP3 gene of divergent strains of rotaviruses simian SA11 and bovine Nebraska calf diarrhea virus. J Virol 62: 4022–4026Google Scholar
  32. 32.
    Offit PA, Blavat G, Greenberg HB, Clark HF (1986) Molecular basis for rotavirus virulence: role of rotavirus gene segment 4. J Virol 57: 46–49Google Scholar
  33. 33.
    Okayama H, Berg P (1983) A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells. Mol Cell Biol 3: 280–289Google Scholar
  34. 34.
    Prasad BVV, Burns JW, Marietta E, Estes MK, Chiu W (1990) Localization of VP4 neutralization sites in rotavirus by three-dimensional cryoelectron microscopy. Nature 343: 476–479Google Scholar
  35. 35.
    Quian Y, Green KY (1991) Human rotavirus strain 69M has a unique VP4 as determined by amino acid sequence analysis. Virology 182: 407–412Google Scholar
  36. 36.
    Ramig RF, Galle KL (1990) Rotavirus genome segment 4 determines viral replication phenotype in cultured liver cells (Help G2). J Virol 64: 1044–1049Google Scholar
  37. 37.
    Saiki R, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N (1985) Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230: 1350–1354Google Scholar
  38. 38.
    Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467Google Scholar
  39. 39.
    Schnappenhein R, Rantenberg P (1987) A luminescence western blot with enhanced sensitivity for antibodies to human immunodeficiency virus. Eur J Clin Mircobiol 6: 49–51Google Scholar
  40. 40.
    Sereno M, Gorziglia M (1994) The outer capsid protein VP4 of murine rotavirus strain Eb represents a tentative new P type. Virology 199: 500–504Google Scholar
  41. 41.
    Shaw AL, Rothnagel R, Chen D, Ramig RF, Chiu W, Prasad BVV (1993) Three-dimensional visualization of the rotavirus hemagglutinin structure. Cell 74: 693–701Google Scholar
  42. 42.
    Shaw RD, Vo PT, Offit PA, Coulson BS, Greenberg HB (1986) Antigenic mapping of the surface protein of rhesus rotavirus. Virology 155: 434–451Google Scholar
  43. 43.
    Shen S, Burke B, Desselberger U (1993) Nucleotide sequence of the VP4 and VP7 genes of a chinese lamb rotavirus: evidence for a new P type in a G10 type virus. Virology 197: 497–500Google Scholar
  44. 44.
    Sippel A (1973) Purification and characterization of adenosine triphosphate: ribonucleic acid adenyl-transferase fromE. coli. Eur J Biochem 37: 31–40Google Scholar
  45. 45.
    Studier W, Rosenberg AM, Dunn JJ, Dubendorff JW (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185: 61–89Google Scholar
  46. 46.
    Sukumaran M, Gowda K, Maiya PP, Srinivas TP, Kumar MS, Aijaz S, Reddy RR, Padilla L, Greenberg HB, Rao CD (1992) Exclusive asymptomatic neonatal infections by human rotavirus strains having subgroup I specificity and long RNA electropherotype. Arch Virol 126: 239–251Google Scholar
  47. 47.
    Taniguchi K, Maloy WL, Nishikawa K, Green KY, Hoshino Y, Urasawa S, Kapikian AZ, Chanock RM, Gorziglia M (1988) Identification of cross-reactive and serotype 2-specific neutralization epitopes on VP3 of human rotavirus. J Virol 62: 2421–2426Google Scholar
  48. 48.
    Taniguchi K, Nishikawa K, Kobayashi N, Urasawa T, Wu HX, Gorziglia M, Urasawa S (1994) Differences in plaque size and VP4 sequence found in SA11 virus clones having simian authentic VP4. Virology 198: 325–330Google Scholar
  49. 49.
    Taniguchi K, Urasawa T, Urasawa S (1994) Species specificity and interspecies relatedness in VP4 genotype demonstrated by VP4 sequence analysis of equine, feline and canine rotavirus strains. Virology 200: 390–400Google Scholar
  50. 50.
    Taniguchi K, Urasawa T, Kobayashi N, Gorziglia M, Urasawa S (1990) Nucleotide sequence of VP4 and VP7 genes of human rotaviruses with subgroup I specificity and long RNA pattern: implication for new G serotype specificity. J Virol 64: 5640–5644Google Scholar
  51. 51.
    Urasawa T, Taniguchi K, Kobayashi N, Mise K, Hasegawa A, Yamazi Y, Urasawa S (1993) Nucleotide sequence of VP4 and VP7 genes of a unique human rotavirus strain Mc35 with subgroup I and serotype 10 specificity. Virology 195: 766–771Google Scholar
  52. 52.
    Zhang S, Zubay G, Goldman E (1991) Low-usage codons inEscherichia coli, yeast, fruit fly and primates. Gene 105: 61–72Google Scholar

Copyright information

© Springer-Verlag 1996

Authors and Affiliations

  • N. P. Mahajan
    • 1
  • C. Durga Rao
    • 1
    • 2
  1. 1.Department of Microbiology and Cell BiologyIndian Institute of ScienceBangaloreIndia
  2. 2.Centre for Genetic EngineeringIndian Institute of ScienceBangaloreIndia

Personalised recommendations