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. 1994 Nov;68(11):6900–6909. doi: 10.1128/jvi.68.11.6900-6909.1994

Monoclonal antibodies against influenza virus PB2 and NP polypeptides interfere with the initiation step of viral mRNA synthesis in vitro.

J Bárcena 1, M Ochoa 1, S de la Luna 1, J A Melero 1, A Nieto 1, J Ortín 1, A Portela 1
PMCID: PMC237125  PMID: 7933070

Abstract

Two panels of monoclonal antibodies (MAbs) specific for the influenza A virus PA and PB2 polypeptides have been obtained from mice immunized with denatured proteins produced in Escherichia coli. All MAbs (13 specific for the PA polypeptide and 8 specific for the PB2 protein) reacted to the corresponding influenza virus protein in Western blotting (immunoblotting), immunoprecipitation, and immunofluorescence assays. To gain information about the roles of the nucleoprotein (NP) and PB2 and PA proteins during viral mRNA synthesis, the 21 anti-P antibodies and 3 anti-NP antibodies (J. A. López, M. Guillen, A. Sánchez-Fauquier, and J. A. Melero, J. Virol. Methods 13:255-264, 1986) were purified and tested for their ability to inhibit the transcriptase activity associated with viral cores purified from virions. Four of the antibodies (one anti-PB2 and the three anti-NP MAbs) inhibited transcription by more than 50% compared with unrelated control antibodies. The inhibitory effect was not due to a nonspecific effect of the antibody preparations, because these MAbs did not inhibit transcription when tested on influenza B virus nucleocapsids, which are not recognized by the antibodies. To determine whether the antibodies were acting on an early transcription step, transcription reactions were carried out in the presence of globin mRNA (a mixture of alpha- and beta-globin chains) and only one labeled nucleoside triphosphate (either GTP or CTP). The results obtained showed that MAbs to the PB2 and NP polypeptides interfered with the initiation step of mRNA-primed transcription. The implications of these results regarding initiation of viral mRNA synthesis are discussed.

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Selected References

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

  1. Akkina R. K., Chambers T. M., Londo D. R., Nayak D. P. Intracellular localization of the viral polymerase proteins in cells infected with influenza virus and cells expressing PB1 protein from cloned cDNA. J Virol. 1987 Jul;61(7):2217–2224. doi: 10.1128/jvi.61.7.2217-2224.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beaton A. R., Krug R. M. Synthesis of the templates for influenza virion RNA replication in vitro. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4682–4686. doi: 10.1073/pnas.81.15.4682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bishop D. H., Obijeski J. F., Simpson R. W. Transcription of the influenza ribonucleic acid genome by a virion polymerase. I. Optimal conditions for in vitro activity of the ribonucleic acid-dependent ribonucleic acid polymerase. J Virol. 1971 Jul;8(1):66–73. doi: 10.1128/jvi.8.1.66-73.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blaas D., Patzelt E., Kuechler E. Cap-recognizing protein of influenza virus. Virology. 1982 Jan 15;116(1):339–348. doi: 10.1016/0042-6822(82)90425-1. [DOI] [PubMed] [Google Scholar]
  5. Bouloy M., Morgan M. A., Shatkin A. J., Krug R. M. Cap and internal nucleotides of reovirus mRNA primers are incorporated into influenza viral complementary RNA during transcription in vitro. J Virol. 1979 Dec;32(3):895–904. doi: 10.1128/jvi.32.3.895-904.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bouloy M., Plotch S. J., Krug R. M. Both the 7-methyl and the 2'-O-methyl groups in the cap of mRNA strongly influence its ability to act as primer for influenza virus RNA transcription. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3952–3956. doi: 10.1073/pnas.77.7.3952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bouloy M., Plotch S. J., Krug R. M. Globin mRNAs are primers for the transcription of influenza viral RNA in vitro. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4886–4890. doi: 10.1073/pnas.75.10.4886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Braam J., Ulmanen I., Krug R. M. Molecular model of a eucaryotic transcription complex: functions and movements of influenza P proteins during capped RNA-primed transcription. Cell. 1983 Sep;34(2):609–618. doi: 10.1016/0092-8674(83)90393-8. [DOI] [PubMed] [Google Scholar]
  9. Detjen B. M., St Angelo C., Katze M. G., Krug R. M. The three influenza virus polymerase (P) proteins not associated with viral nucleocapsids in the infected cell are in the form of a complex. J Virol. 1987 Jan;61(1):16–22. doi: 10.1128/jvi.61.1.16-22.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. García-Barreno B., Palomo C., Peñas C., Delgado T., Perez-Breña P., Melero J. A. Marked differences in the antigenic structure of human respiratory syncytial virus F and G glycoproteins. J Virol. 1989 Feb;63(2):925–932. doi: 10.1128/jvi.63.2.925-932.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hagen M., Chung T. D., Butcher J. A., Krystal M. Recombinant influenza virus polymerase: requirement of both 5' and 3' viral ends for endonuclease activity. J Virol. 1994 Mar;68(3):1509–1515. doi: 10.1128/jvi.68.3.1509-1515.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Honda A., Uéda K., Nagata K., Ishihama A. RNA polymerase of influenza virus: role of NP in RNA chain elongation. J Biochem. 1988 Dec;104(6):1021–1026. doi: 10.1093/oxfordjournals.jbchem.a122569. [DOI] [PubMed] [Google Scholar]
  13. Horisberger M. A. Identification of a catalytic activity of the large basic P polypeptide of influenza virus. Virology. 1982 Jul 30;120(2):279–286. doi: 10.1016/0042-6822(82)90030-7. [DOI] [PubMed] [Google Scholar]
  14. Horisberger M. A. The large P proteins of influenza A viruses are composed of one acidic and two basic polypeptides. Virology. 1980 Nov;107(1):302–305. doi: 10.1016/0042-6822(80)90296-2. [DOI] [PubMed] [Google Scholar]
  15. Huang T. S., Palese P., Krystal M. Determination of influenza virus proteins required for genome replication. J Virol. 1990 Nov;64(11):5669–5673. doi: 10.1128/jvi.64.11.5669-5673.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ishihama A., Mizumoto K., Kawakami K., Kato A., Honda A. Proofreading function associated with the RNA-dependent RNA polymerase from influenza virus. J Biol Chem. 1986 Aug 5;261(22):10417–10421. [PubMed] [Google Scholar]
  17. Jones I. M., Reay P. A., Philpott K. L. Nuclear location of all three influenza polymerase proteins and a nuclear signal in polymerase PB2. EMBO J. 1986 Sep;5(9):2371–2376. doi: 10.1002/j.1460-2075.1986.tb04506.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kato A., Mizumoto K., Ishihama A. Purification and enzymatic properties of an RNA polymerase-RNA complex from influenza virus. Virus Res. 1985 Sep;3(2):115–127. doi: 10.1016/0168-1702(85)90002-4. [DOI] [PubMed] [Google Scholar]
  19. Kawakami K., Mizumoto K., Ishihama A. RNA polymerase of influenza virus. IV. Catalytic properties of the capped RNA endonuclease associated with the RNA polymerase. Nucleic Acids Res. 1983 Jun 11;11(11):3637–3649. doi: 10.1093/nar/11.11.3637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kimura N., Nishida M., Nagata K., Ishihama A., Oda K., Nakada S. Transcription of a recombinant influenza virus RNA in cells that can express the influenza virus RNA polymerase and nucleoprotein genes. J Gen Virol. 1992 Jun;73(Pt 6):1321–1328. doi: 10.1099/0022-1317-73-6-1321. [DOI] [PubMed] [Google Scholar]
  21. Kingsbury D. W., Webster R. G. Some properties of influenza virus nucleocapsids. J Virol. 1969 Sep;4(3):219–225. doi: 10.1128/jvi.4.3.219-225.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Krug R. M., Broni B. A., LaFiandra A. J., Morgan M. A., Shatkin A. J. Priming and inhibitory activities of RNAs for the influenza viral transcriptase do not require base pairing with the virion template RNA. Proc Natl Acad Sci U S A. 1980 Oct;77(10):5874–5878. doi: 10.1073/pnas.77.10.5874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Krug R. M. Influenza viral RNPs newly synthesized during the latent period of viral growth in MDCK cells. Virology. 1971 Apr;44(1):125–136. doi: 10.1016/0042-6822(71)90159-0. [DOI] [PubMed] [Google Scholar]
  24. Köhler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7;256(5517):495–497. doi: 10.1038/256495a0. [DOI] [PubMed] [Google Scholar]
  25. Lopez J. A., Guillen M., Sanchez-Fauquier A., Melero J. A. An antigen-binding assay to determine the specificity of monoclonal antibodies against influenza virus and mapping of epitopes. J Virol Methods. 1986 Jun;13(3):255–264. doi: 10.1016/0166-0934(86)90019-4. [DOI] [PubMed] [Google Scholar]
  26. McGeoch D., Kitron N. Influenza virion RNA-dependent RNA polymerase: stimulation by guanosine and related compounds. J Virol. 1975 Apr;15(4):686–695. doi: 10.1128/jvi.15.4.686-695.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mena I., de la Luna S., Albo C., Martín J., Nieto A., Ortín J., Portela A. Synthesis of biologically active influenza virus core proteins using a vaccinia virus-T7 RNA polymerase expression system. J Gen Virol. 1994 Aug;75(Pt 8):2109–2114. doi: 10.1099/0022-1317-75-8-2109. [DOI] [PubMed] [Google Scholar]
  28. Nieto A., de la Luna S., Bárcena J., Portela A., Valcárcel J., Melero J. A., Ortín J. Nuclear transport of influenza virus polymerase PA protein. Virus Res. 1992 Jun;24(1):65–75. doi: 10.1016/0168-1702(92)90031-4. [DOI] [PubMed] [Google Scholar]
  29. Parvin J. D., Palese P., Honda A., Ishihama A., Krystal M. Promoter analysis of influenza virus RNA polymerase. J Virol. 1989 Dec;63(12):5142–5152. doi: 10.1128/jvi.63.12.5142-5152.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Plotch S. J., Bouloy M., Krug R. M. Transfer of 5'-terminal cap of globin mRNA to influenza viral complementary RNA during transcription in vitro. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1618–1622. doi: 10.1073/pnas.76.4.1618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Plotch S. J., Bouloy M., Ulmanen I., Krug R. M. A unique cap(m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription. Cell. 1981 Mar;23(3):847–858. doi: 10.1016/0092-8674(81)90449-9. [DOI] [PubMed] [Google Scholar]
  32. Plotch S. J., Krug R. M. Influenza virion transcriptase: synthesis in vitro of large, polyadenylic acid-containing complementary RNA. J Virol. 1977 Jan;21(1):24–34. doi: 10.1128/jvi.21.1.24-34.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pons M. W., Schulze I. T., Hirst G. K., Hauser R. Isolation and characterization of the ribonucleoprotein of influenza virus. Virology. 1969 Oct;39(2):250–259. doi: 10.1016/0042-6822(69)90045-2. [DOI] [PubMed] [Google Scholar]
  34. Portela A., Jones L. D., Nuttall P. Identification of viral structural polypeptides of Thogoto virus (a tick-borne orthomyxo-like virus) and functions associated with the glycoprotein. J Gen Virol. 1992 Nov;73(Pt 11):2823–2830. doi: 10.1099/0022-1317-73-11-2823. [DOI] [PubMed] [Google Scholar]
  35. Robertson H. D., Dickson E., Plotch S. J., Krug R. M. Identification of the RNA region transferred from a representative primer, beta-globin mRNA, to influenza mRNA during in vitro transcription. Nucleic Acids Res. 1980 Mar 11;8(5):925–942. doi: 10.1093/nar/8.5.925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rochovansky O. M. RNA synthesis by ribonucleoprotein-polymerase complexes isolated from influenza virus. Virology. 1976 Sep;73(2):327–338. doi: 10.1016/0042-6822(76)90394-9. [DOI] [PubMed] [Google Scholar]
  37. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  38. Schreier E., Ladhoff A. M., Stompor S., Michel S. Interaction between anti-influenza viral polymerase antibodies and RNP particles using the in vitro transcription process and an immunogold labelling technique. Acta Virol. 1988 Sep;32(5):403–408. [PubMed] [Google Scholar]
  39. Seong B. L., Brownlee G. G. Nucleotides 9 to 11 of the influenza A virion RNA promoter are crucial for activity in vitro. J Gen Virol. 1992 Dec;73(Pt 12):3115–3124. doi: 10.1099/0022-1317-73-12-3115. [DOI] [PubMed] [Google Scholar]
  40. Shapiro G. I., Krug R. M. Influenza virus RNA replication in vitro: synthesis of viral template RNAs and virion RNAs in the absence of an added primer. J Virol. 1988 Jul;62(7):2285–2290. doi: 10.1128/jvi.62.7.2285-2290.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Shulman M., Wilde C. D., Köhler G. A better cell line for making hybridomas secreting specific antibodies. Nature. 1978 Nov 16;276(5685):269–270. doi: 10.1038/276269a0. [DOI] [PubMed] [Google Scholar]
  42. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  43. Ulmanen I., Broni B. A., Krug R. M. Role of two of the influenza virus core P proteins in recognizing cap 1 structures (m7GpppNm) on RNAs and in initiating viral RNA transcription. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7355–7359. doi: 10.1073/pnas.78.12.7355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Ulmanen I., Broni B., Krug R. M. Influenza virus temperature-sensitive cap (m7GpppNm)-dependent endonuclease. J Virol. 1983 Jan;45(1):27–35. doi: 10.1128/jvi.45.1.27-35.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. de la Luna S., Martín J., Portela A., Ortín J. Influenza virus naked RNA can be expressed upon transfection into cells co-expressing the three subunits of the polymerase and the nucleoprotein from simian virus 40 recombinant viruses. J Gen Virol. 1993 Mar;74(Pt 3):535–539. doi: 10.1099/0022-1317-74-3-535. [DOI] [PubMed] [Google Scholar]
  46. de la Luna S., Martínez C., Ortín J. Molecular cloning and sequencing of influenza virus A/Victoria/3/75 polymerase genes: sequence evolution and prediction of possible functional domains. Virus Res. 1989 Jun;13(2):143–155. doi: 10.1016/0168-1702(89)90012-9. [DOI] [PubMed] [Google Scholar]
  47. del Río L., Martínez C., Domingo E., Ortín J. In vitro synthesis of full-length influenza virus complementary RNA. EMBO J. 1985 Jan;4(1):243–247. doi: 10.1002/j.1460-2075.1985.tb02342.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. van Wyke K. L., Bean W. J., Jr, Webster R. G. Monoclonal antibodies to the influenza A virus nucleoprotein affecting RNA transcription. J Virol. 1981 Jul;39(1):313–317. doi: 10.1128/jvi.39.1.313-317.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]

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