Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1996 Jan;70(1):580–584. doi: 10.1128/jvi.70.1.580-584.1996

Temperature-sensitive phenotype of the human parainfluenza virus type 3 candidate vaccine strain (cp45) correlates with a defect in the L gene.

R Ray, M S Galinski, B R Heminway, K Meyer, F K Newman, R B Belshe
PMCID: PMC189848  PMID: 8523574

Abstract

We have previously demonstrated that the temperature sensitivity of a human parainfluenza virus type 3 (HPIV-3) candidate vaccine strain (cp45), which is currently under evaluation in humans, is associated with poor transcriptional activity of the virus at the nonpermissive temperature (R. Ray, K. Meyer, F. Newman, and R. B. Belshe, J. Virol. 69:1959-1963, 1995). In this study, the temperature sensitivity of cp45 virus was further investigated by the complementation of a specific gene function. CV-1 cells were transfected with cloned genes from wild-type HPIV-3 encoding the large protein (L), phosphoprotein (P), and nucleocapsid protein (NP), alone or together, for the expression of biologically active proteins. Only cells expressing the L gene were able to rescue cp45 replication when incubated at the nonpermissive temperature (39.5 degrees C), whereas cells transiently expressing NP or P were incapable of rescuing the virus. The virus titers obtained following complementation of the L protein were 190 to 2,300 PFU/ml of culture medium, compared with the undetectable growth of the cp45 temperature-sensitive mutant at the nonpermissive temperature. Rescued progeny virus still maintained the temperature-sensitive phenotype. Results from this study suggest that the temperature sensitivity of the cp45 candidate vaccine strain is associated primarily with L-protein function and that the defect can be complemented by transient expression of the wild-type protein. This study underscores the importance of the L protein in RNA polymerase activity and its critical role in virus replication.

Full Text

The Full Text of this article is available as a PDF (309.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Armstrong K. M., Fairman R., Baldwin R. L. The (i, i + 4) Phe-His interaction studied in an alanine-based alpha-helix. J Mol Biol. 1993 Mar 5;230(1):284–291. doi: 10.1006/jmbi.1993.1142. [DOI] [PubMed] [Google Scholar]
  2. Belshe R. B., Hissom F. K. Cold adaptation of parainfluenza virus type 3: induction of three phenotypic markers. J Med Virol. 1982;10(4):235–242. doi: 10.1002/jmv.1890100403. [DOI] [PubMed] [Google Scholar]
  3. Belshe R. B., Karron R. A., Newman F. K., Anderson E. L., Nugent S. L., Steinhoff M., Clements M. L., Wilson M. H., Hall S. L., Tierney E. L. Evaluation of a live attenuated, cold-adapted parainfluenza virus type 3 vaccine in children. J Clin Microbiol. 1992 Aug;30(8):2064–2070. doi: 10.1128/jcm.30.8.2064-2070.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bouchard M. J., Lam D. H., Racaniello V. R. Determinants of attenuation and temperature sensitivity in the type 1 poliovirus Sabin vaccine. J Virol. 1995 Aug;69(8):4972–4978. doi: 10.1128/jvi.69.8.4972-4978.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Calain P., Curran J., Kolakofsky D., Roux L. Molecular cloning of natural paramyxovirus copy-back defective interfering RNAs and their expression from DNA. Virology. 1992 Nov;191(1):62–71. doi: 10.1016/0042-6822(92)90166-m. [DOI] [PubMed] [Google Scholar]
  6. Calain P., Roux L. The rule of six, a basic feature for efficient replication of Sendai virus defective interfering RNA. J Virol. 1993 Aug;67(8):4822–4830. doi: 10.1128/jvi.67.8.4822-4830.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Clements M. L., Belshe R. B., King J., Newman F., Westblom T. U., Tierney E. L., London W. T., Murphy B. R. Evaluation of bovine, cold-adapted human, and wild-type human parainfluenza type 3 viruses in adult volunteers and in chimpanzees. J Clin Microbiol. 1991 Jun;29(6):1175–1182. doi: 10.1128/jcm.29.6.1175-1182.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Crookshanks-Newman F. K., Belshe R. B. Protection of weanling hamsters from experimental infection with wild-type parainfluenza virus type 3 (para 3) by cold-adapted mutants of para 3. J Med Virol. 1986 Feb;18(2):131–137. doi: 10.1002/jmv.1890180205. [DOI] [PubMed] [Google Scholar]
  9. De B. P., Banerjee A. K. Rescue of synthetic analogs of genome RNA of human parainfluenza virus type 3. Virology. 1993 Sep;196(1):344–348. doi: 10.1006/viro.1993.1486. [DOI] [PubMed] [Google Scholar]
  10. De B. P., Galinski M. S., Banerjee A. K. Characterization of an in vitro system for the synthesis of mRNA from human parainfluenza virus type 3. J Virol. 1990 Mar;64(3):1135–1142. doi: 10.1128/jvi.64.3.1135-1142.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dimock K., Collins P. L. Rescue of synthetic analogs of genomic RNA and replicative-intermediate RNA of human parainfluenza virus type 3. J Virol. 1993 May;67(5):2772–2778. doi: 10.1128/jvi.67.5.2772-2778.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Enami M., Luytjes W., Krystal M., Palese P. Introduction of site-specific mutations into the genome of influenza virus. Proc Natl Acad Sci U S A. 1990 May;87(10):3802–3805. doi: 10.1073/pnas.87.10.3802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fuerst T. R., Niles E. G., Studier F. W., Moss B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8122–8126. doi: 10.1073/pnas.83.21.8122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Galinski M. S., Mink M. A., Lambert D. M., Wechsler S. L., Pons M. W. Molecular cloning and sequence analysis of the human parainfluenza 3 virus RNA encoding the nucleocapsid protein. Virology. 1986 Mar;149(2):139–151. doi: 10.1016/0042-6822(86)90116-9. [DOI] [PubMed] [Google Scholar]
  15. Galinski M. S., Mink M. A., Lambert D. M., Wechsler S. L., Pons M. W. Molecular cloning and sequence analysis of the human parainfluenza 3 virus mRNA encoding the P and C proteins. Virology. 1986 Nov;155(1):46–60. doi: 10.1016/0042-6822(86)90167-4. [DOI] [PubMed] [Google Scholar]
  16. Galinski M. S., Mink M. A., Pons M. W. Molecular cloning and sequence analysis of the human parainfluenza 3 virus gene encoding the L protein. Virology. 1988 Aug;165(2):499–510. doi: 10.1016/0042-6822(88)90594-6. [DOI] [PubMed] [Google Scholar]
  17. Graber M., Barry C., Dipaola J., Hasagawa A. Intracellular pH in OK cells. II. Effects of temperature on cell pH. Am J Physiol. 1992 May;262(5 Pt 2):F723–F730. doi: 10.1152/ajprenal.1992.262.5.F723. [DOI] [PubMed] [Google Scholar]
  18. Hall S. L., Sarris C. M., Tierney E. L., London W. T., Murphy B. R. A cold-adapted mutant of parainfluenza virus type 3 is attenuated and protective in chimpanzees. J Infect Dis. 1993 Apr;167(4):958–962. doi: 10.1093/infdis/167.4.958. [DOI] [PubMed] [Google Scholar]
  19. Hall S. L., Stokes A., Tierney E. L., London W. T., Belshe R. B., Newman F. C., Murphy B. R. Cold-passaged human parainfluenza type 3 viruses contain ts and non-ts mutations leading to attenuation in rhesus monkeys. Virus Res. 1992 Mar;22(3):173–184. doi: 10.1016/0168-1702(92)90049-f. [DOI] [PubMed] [Google Scholar]
  20. Harty R. N., Palese P. Mutations within noncoding terminal sequences of model RNAs of Sendai virus: influence on reporter gene expression. J Virol. 1995 Aug;69(8):5128–5131. doi: 10.1128/jvi.69.8.5128-5131.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Herlocher M. L., Maassab H. F., Webster R. G. Molecular and biological changes in the cold-adapted "master strain" A/AA/6/60 (H2N2) influenza virus. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6032–6036. doi: 10.1073/pnas.90.13.6032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Horikami S. M., Curran J., Kolakofsky D., Moyer S. A. Complexes of Sendai virus NP-P and P-L proteins are required for defective interfering particle genome replication in vitro. J Virol. 1992 Aug;66(8):4901–4908. doi: 10.1128/jvi.66.8.4901-4908.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hu X. L., Ray R., Compans R. W. Functional interactions between the fusion protein and hemagglutinin-neuraminidase of human parainfluenza viruses. J Virol. 1992 Mar;66(3):1528–1534. doi: 10.1128/jvi.66.3.1528-1534.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Krystal M., Li R., Lyles D., Pavlakis G., Palese P. Expression of the three influenza virus polymerase proteins in a single cell allows growth complementation of viral mutants. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2709–2713. doi: 10.1073/pnas.83.8.2709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lawson N. D., Stillman E. A., Whitt M. A., Rose J. K. Recombinant vesicular stomatitis viruses from DNA. Proc Natl Acad Sci U S A. 1995 May 9;92(10):4477–4481. doi: 10.1073/pnas.92.10.4477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lefkowitz E. J., Pattnaik A. K., Ball L. A. Complementation of a vesicular stomatitis virus glycoprotein G mutant with wild-type protein expressed from either a bovine papilloma virus or a vaccinia virus vector system. Virology. 1990 Oct;178(2):373–383. doi: 10.1016/0042-6822(90)90334-n. [DOI] [PubMed] [Google Scholar]
  27. Li R. A., Palese P., Krystal M. Complementation and analysis of an NP mutant of influenza virus. Virus Res. 1989 Feb;12(2):97–111. doi: 10.1016/0168-1702(89)90057-9. [DOI] [PubMed] [Google Scholar]
  28. Marks M. D., Kennedy-Morrow J., Lesnaw J. A. Assignment of the temperature-sensitive lesion in the replication mutant A1 of vesicular stomatitis virus to the N gene. J Virol. 1985 Jan;53(1):44–51. doi: 10.1128/jvi.53.1.44-51.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ongrádi J., Cunningham C., Szilágyi J. F. Temperature sensitivity of the transcriptase of mutants tsB1 and tsF1 of vesicular stomatitis virus New Jersey is a consequence of mutation affecting polypeptide L. J Gen Virol. 1985 Jul;66(Pt 7):1507–1513. doi: 10.1099/0022-1317-66-7-1507. [DOI] [PubMed] [Google Scholar]
  30. Park K. H., Huang T., Correia F. F., Krystal M. Rescue of a foreign gene by Sendai virus. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5537–5541. doi: 10.1073/pnas.88.13.5537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Park K. H., Krystal M. In vivo model for pseudo-templated transcription in Sendai virus. J Virol. 1992 Dec;66(12):7033–7039. doi: 10.1128/jvi.66.12.7033-7039.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Peden K. W., Spence S. L., Tack L. C., Cartwright C. A., Srinivasan A., Pipas J. M. A DNA replication-positive mutant of simian virus 40 that is defective for transformation and the production of infectious virions. J Virol. 1990 Jun;64(6):2912–2921. doi: 10.1128/jvi.64.6.2912-2921.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Peeples M. E., Bratt M. A. Virion functions of RNA+ temperature-sensitive mutants of Newcastle disease virus. J Virol. 1982 May;42(2):440–446. doi: 10.1128/jvi.42.2.440-446.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Peeples M. E., Rasenas L. L., Bratt M. A. RNA synthesis by Newcastle disease virus temperature-sensitive mutants in two RNA-negative complementation groups. J Virol. 1982 Jun;42(3):996–1006. doi: 10.1128/jvi.42.3.996-1006.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Portner A., Scroggs R. A., Marx P. S., Kingsbury D. W. A temperature-sensitive mutant of Sendai virus with an altered hemagglutinin-neuraminidase polypeptide: consequences for virus assembly and cytopathology. Virology. 1975 Sep;67(1):179–187. doi: 10.1016/0042-6822(75)90415-8. [DOI] [PubMed] [Google Scholar]
  36. Ray R., Brown V. E., Compans R. W. Glycoproteins of human parainfluenza virus type 3: characterization and evaluation as a subunit vaccine. J Infect Dis. 1985 Dec;152(6):1219–1230. doi: 10.1093/infdis/152.6.1219. [DOI] [PubMed] [Google Scholar]
  37. Ray R., Compans R. W. Glycoproteins of human parainfluenza virus type 3: affinity purification, antigenic characterization and reconstitution into lipid vesicles. J Gen Virol. 1987 Feb;68(Pt 2):409–418. doi: 10.1099/0022-1317-68-2-409. [DOI] [PubMed] [Google Scholar]
  38. Ray R., Compans R. W. Monoclonal antibodies reveal extensive antigenic differences between the hemagglutinin-neuraminidase glycoproteins of human and bovine parainfluenza 3 viruses. Virology. 1986 Jan 15;148(1):232–236. doi: 10.1016/0042-6822(86)90420-4. [DOI] [PubMed] [Google Scholar]
  39. Ray R., Meyer K., Newman F. K., Belshe R. B. Characterization of a live, attenuated human parainfluenza type 3 virus candidate vaccine strain. J Virol. 1995 Mar;69(3):1959–1963. doi: 10.1128/jvi.69.3.1959-1963.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sambrook J., Rodgers L., White J., Gething M. J. Lines of BPV-transformed murine cells that constitutively express influenza virus hemagglutinin. EMBO J. 1985 Jan;4(1):91–103. doi: 10.1002/j.1460-2075.1985.tb02322.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schnell M. J., Mebatsion T., Conzelmann K. K. Infectious rabies viruses from cloned cDNA. EMBO J. 1994 Sep 15;13(18):4195–4203. doi: 10.1002/j.1460-2075.1994.tb06739.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Shibata S., Yamamoto-Goshima F., Maeno K., Hanaichi T., Fujita Y., Nakajima K., Imai M., Komatsu T., Sugiura S. Characterization of a temperature-sensitive influenza B virus mutant defective in neuraminidase. J Virol. 1993 Jun;67(6):3264–3273. doi: 10.1128/jvi.67.6.3264-3273.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Shoemaker K. R., Fairman R., Schultz D. A., Robertson A. D., York E. J., Stewart J. M., Baldwin R. L. Side-chain interactions in the C-peptide helix: Phe 8 ... His 12+. Biopolymers. 1990 Jan;29(1):1–11. doi: 10.1002/bip.360290104. [DOI] [PubMed] [Google Scholar]
  44. Spriggs M. K., Collins P. L. Human parainfluenza virus type 3: messenger RNAs, polypeptide coding assignments, intergenic sequences, and genetic map. J Virol. 1986 Sep;59(3):646–654. doi: 10.1128/jvi.59.3.646-654.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Stokes A., Tierney E. L., Sarris C. M., Murphy B. R., Hall S. L. The complete nucleotide sequence of two cold-adapted, temperature-sensitive attenuated mutant vaccine viruses (cp12 and cp45) derived from the JS strain of human parainfluenza virus type 3 (PIV3). Virus Res. 1993 Oct;30(1):43–52. doi: 10.1016/0168-1702(93)90014-e. [DOI] [PubMed] [Google Scholar]
  46. Storey D. G., Dimock K., Kang C. Y. Structural characterization of virion proteins and genomic RNA of human parainfluenza virus 3. J Virol. 1984 Dec;52(3):761–766. doi: 10.1128/jvi.52.3.761-766.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sánchez A., Banerjee A. K. Studies on human parainfluenza virus 3: characterization of the structural proteins and in vitro synthesized proteins coded by mRNAs isolated from infected cells. Virology. 1985 May;143(1):45–54. doi: 10.1016/0042-6822(85)90095-9. [DOI] [PubMed] [Google Scholar]
  48. Takebe Y., Seiki M., Fujisawa J., Hoy P., Yokota K., Arai K., Yoshida M., Arai N. SR alpha promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol Cell Biol. 1988 Jan;8(1):466–472. doi: 10.1128/mcb.8.1.466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Thompson S. D., Portner A. Localization of functional sites on the hemagglutinin-neuraminidase glycoprotein of Sendai virus by sequence analysis of antigenic and temperature-sensitive mutants. Virology. 1987 Sep;160(1):1–8. doi: 10.1016/0042-6822(87)90037-7. [DOI] [PubMed] [Google Scholar]
  50. Tsipis J. E., Bratt M. A. Isolation and preliminary characterization of temperature-sensitive mutants of Newcastle disease virus. J Virol. 1976 Jun;18(3):848–855. doi: 10.1128/jvi.18.3.848-855.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Westrop G. D., Wareham K. A., Evans D. M., Dunn G., Minor P. D., Magrath D. I., Taffs F., Marsden S., Skinner M. A., Schild G. C. Genetic basis of attenuation of the Sabin type 3 oral poliovirus vaccine. J Virol. 1989 Mar;63(3):1338–1344. doi: 10.1128/jvi.63.3.1338-1344.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Whitt M. A., Chong L., Rose J. K. Glycoprotein cytoplasmic domain sequences required for rescue of a vesicular stomatitis virus glycoprotein mutant. J Virol. 1989 Sep;63(9):3569–3578. doi: 10.1128/jvi.63.9.3569-3578.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Zhao H., Banerjee A. K. Interaction between the nucleocapsid protein and the phosphoprotein of human parainfluenza virus 3. Mapping of the interacting domains using a two-hybrid system. J Biol Chem. 1995 May 26;270(21):12485–12490. doi: 10.1074/jbc.270.21.12485. [DOI] [PubMed] [Google Scholar]
  54. van Wyke Coelingh K. L., Winter C., Murphy B. R. Antigenic variation in the hemagglutinin-neuraminidase protein of human parainfluenza type 3 virus. Virology. 1985 Jun;143(2):569–582. doi: 10.1016/0042-6822(85)90395-2. [DOI] [PubMed] [Google Scholar]
  55. van Wyke Coelingh K., Tierney E. L. Antigenic and functional organization of human parainfluenza virus type 3 fusion glycoprotein. J Virol. 1989 Jan;63(1):375–382. doi: 10.1128/jvi.63.1.375-382.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES