Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1995 Nov;69(11):7006–7014. doi: 10.1128/jvi.69.11.7006-7014.1995

Effects of anti-E2 monoclonal antibody on sindbis virus replication in AT3 cells expressing bcl-2.

P Després 1, J W Griffin 1, D E Griffin 1
PMCID: PMC189620  PMID: 7474120

Abstract

Antibodies directed to Sindbis virus (SV) envelope protein E2 are able to control virus replication in vivo and in persistently infected cultures of neurons in vitro. We investigated the mechanisms by which anti-E2 monoclonal antibody (MAb) alters virus replication by using AT3 rat prostatic carcinoma cells expressing the inhibitor of apoptosis bcl-2. Treatment of SV-infected AT3-bcl-2 cells with anti-E2 MAb G5 for 2 h decreased the rate of virus release for 6 to 8 h after removal of the antibody. Electron microscopic analysis of MAb-treated cells revealed that failure of virus release was linked to a defect in the budding process. The decrease in extracellular virus particles occurred despite continued formation of nucleocapsids and synthesis of envelope glycoproteins. MAb treatment delayed the inhibition of K+ influx and shutoff of host cell protein synthesis by SV infection in a dose-dependent manner. Synthesis of host cell factors and of nonstructural polyprotein precursors required for the formation of initial replication complexes was also prolonged, causing a slower shutdown of overall viral RNA synthesis. We conclude that one mechanism by which anti-E2 MAb treatment down-regulates SV replication is by reestablishing certain critical host cell functions in infected cells.

Full Text

The Full Text of this article is available as a PDF (1.4 MB).

Selected References

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

  1. Bashford C. L., Alder G. M., Gray M. A., Micklem K. J., Taylor C. C., Turek P. J., Pasternak C. A. Oxonol dyes as monitors of membrane potential: the effect of viruses and toxins on the plasma membrane potential of animal cells in monolayer culture and in suspension. J Cell Physiol. 1985 Jun;123(3):326–336. doi: 10.1002/jcp.1041230306. [DOI] [PubMed] [Google Scholar]
  2. Desprès P., Frenkiel M. P., Deubel V. Differences between cell membrane fusion activities of two dengue type-1 isolates reflect modifications of viral structure. Virology. 1993 Sep;196(1):209–219. doi: 10.1006/viro.1993.1469. [DOI] [PubMed] [Google Scholar]
  3. Desprès P., Girard M., Bouloy M. Characterization of yellow fever virus proteins E and NS1 expressed in Vero and Spodoptera frugiperda cells. J Gen Virol. 1991 Jun;72(Pt 6):1331–1342. doi: 10.1099/0022-1317-72-6-1331. [DOI] [PubMed] [Google Scholar]
  4. Duffus W. A., Levy-Mintz P., Klimjack M. R., Kielian M. Mutations in the putative fusion peptide of Semliki Forest virus affect spike protein oligomerization and virus assembly. J Virol. 1995 Apr;69(4):2471–2479. doi: 10.1128/jvi.69.4.2471-2479.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Garry R. F., Bishop J. M., Parker S., Westbrook K., Lewis G., Waite M. R. Na+ and K+ concentrations and the regulation of protein synthesis in Sindbis virus-infected chick cells. Virology. 1979 Jul 15;96(1):108–120. doi: 10.1016/0042-6822(79)90177-6. [DOI] [PubMed] [Google Scholar]
  6. Garry R. F. Sindbis virus-induced inhibition of protein synthesis is partially reversed by medium containing an elevated potassium concentration. J Gen Virol. 1994 Feb;75(Pt 2):411–415. doi: 10.1099/0022-1317-75-2-411. [DOI] [PubMed] [Google Scholar]
  7. Griffin D. E., Levine B., Tyor W. R., Irani D. N. The immune response in viral encephalitis. Semin Immunol. 1992 Apr;4(2):111–119. [PubMed] [Google Scholar]
  8. Hardy W. R., Strauss J. H. Processing the nonstructural polyproteins of Sindbis virus: study of the kinetics in vivo by using monospecific antibodies. J Virol. 1988 Mar;62(3):998–1007. doi: 10.1128/jvi.62.3.998-1007.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jackson A. C., Moench T. R., Griffin D. E., Johnson R. T. The pathogenesis of spinal cord involvement in the encephalomyelitis of mice caused by neuroadapted Sindbis virus infection. Lab Invest. 1987 Apr;56(4):418–423. [PubMed] [Google Scholar]
  10. Jackson A. C., Moench T. R., Trapp B. D., Griffin D. E. Basis of neurovirulence in Sindbis virus encephalomyelitis of mice. Lab Invest. 1988 May;58(5):503–509. [PubMed] [Google Scholar]
  11. Johnson R. T., McFarland H. F., Levy S. E. Age-dependent resistance to viral encephalitis: studies of infections due to Sindbis virus in mice. J Infect Dis. 1972 Mar;125(3):257–262. doi: 10.1093/infdis/125.3.257. [DOI] [PubMed] [Google Scholar]
  12. Kail M., Hollinshead M., Ansorge W., Pepperkok R., Frank R., Griffiths G., Vaux D. The cytoplasmic domain of alphavirus E2 glycoprotein contains a short linear recognition signal required for viral budding. EMBO J. 1991 Sep;10(9):2343–2351. doi: 10.1002/j.1460-2075.1991.tb07773.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. LaStarza M. W., Lemm J. A., Rice C. M. Genetic analysis of the nsP3 region of Sindbis virus: evidence for roles in minus-strand and subgenomic RNA synthesis. J Virol. 1994 Sep;68(9):5781–5791. doi: 10.1128/jvi.68.9.5781-5791.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lee H., Ricker P. D., Brown D. T. The configuration of Sindbis virus envelope proteins is stabilized by the nucleocapsid protein. Virology. 1994 Oct;204(1):471–474. doi: 10.1006/viro.1994.1557. [DOI] [PubMed] [Google Scholar]
  15. Lemm J. A., Rice C. M. Assembly of functional Sindbis virus RNA replication complexes: requirement for coexpression of P123 and P34. J Virol. 1993 Apr;67(4):1905–1915. doi: 10.1128/jvi.67.4.1905-1915.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lemm J. A., Rice C. M. Roles of nonstructural polyproteins and cleavage products in regulating Sindbis virus RNA replication and transcription. J Virol. 1993 Apr;67(4):1916–1926. doi: 10.1128/jvi.67.4.1916-1926.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Levine B., Griffin D. E. Persistence of viral RNA in mouse brains after recovery from acute alphavirus encephalitis. J Virol. 1992 Nov;66(11):6429–6435. doi: 10.1128/jvi.66.11.6429-6435.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Levine B., Hardwick J. M., Trapp B. D., Crawford T. O., Bollinger R. C., Griffin D. E. Antibody-mediated clearance of alphavirus infection from neurons. Science. 1991 Nov 8;254(5033):856–860. doi: 10.1126/science.1658936. [DOI] [PubMed] [Google Scholar]
  19. Levine B., Huang Q., Isaacs J. T., Reed J. C., Griffin D. E., Hardwick J. M. Conversion of lytic to persistent alphavirus infection by the bcl-2 cellular oncogene. Nature. 1993 Feb 25;361(6414):739–742. doi: 10.1038/361739a0. [DOI] [PubMed] [Google Scholar]
  20. Li G. P., La Starza M. W., Hardy W. R., Strauss J. H., Rice C. M. Phosphorylation of Sindbis virus nsP3 in vivo and in vitro. Virology. 1990 Nov;179(1):416–427. doi: 10.1016/0042-6822(90)90310-n. [DOI] [PubMed] [Google Scholar]
  21. Liu N., Brown D. T. Phosphorylation and dephosphorylation events play critical roles in Sindbis virus maturation. Virology. 1993 Oct;196(2):703–711. doi: 10.1006/viro.1993.1527. [DOI] [PubMed] [Google Scholar]
  22. Liu N., Brown D. T. Transient translocation of the cytoplasmic (endo) domain of a type I membrane glycoprotein into cellular membranes. J Cell Biol. 1993 Feb;120(4):877–883. doi: 10.1083/jcb.120.4.877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lobigs M., Zhao H. X., Garoff H. Function of Semliki Forest virus E3 peptide in virus assembly: replacement of E3 with an artificial signal peptide abolishes spike heterodimerization and surface expression of E1. J Virol. 1990 Sep;64(9):4346–4355. doi: 10.1128/jvi.64.9.4346-4355.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lopez S., Yao J. S., Kuhn R. J., Strauss E. G., Strauss J. H. Nucleocapsid-glycoprotein interactions required for assembly of alphaviruses. J Virol. 1994 Mar;68(3):1316–1323. doi: 10.1128/jvi.68.3.1316-1323.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lustig S., Jackson A. C., Hahn C. S., Griffin D. E., Strauss E. G., Strauss J. H. Molecular basis of Sindbis virus neurovirulence in mice. J Virol. 1988 Jul;62(7):2329–2336. doi: 10.1128/jvi.62.7.2329-2336.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mendoza Q. P., Stanley J., Griffin D. E. Monoclonal antibodies to the E1 and E2 glycoproteins of Sindbis virus: definition of epitopes and efficiency of protection from fatal encephalitis. J Gen Virol. 1988 Dec;69(Pt 12):3015–3022. doi: 10.1099/0022-1317-69-12-3015. [DOI] [PubMed] [Google Scholar]
  27. Metsikkö K., Garoff H. Oligomers of the cytoplasmic domain of the p62/E2 membrane protein of Semliki Forest virus bind to the nucleocapsid in vitro. J Virol. 1990 Oct;64(10):4678–4683. doi: 10.1128/jvi.64.10.4678-4683.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mulvey M., Brown D. T. Formation and rearrangement of disulfide bonds during maturation of the Sindbis virus E1 glycoprotein. J Virol. 1994 Feb;68(2):805–812. doi: 10.1128/jvi.68.2.805-812.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mulvey M., Brown D. T. Involvement of the molecular chaperone BiP in maturation of Sindbis virus envelope glycoproteins. J Virol. 1995 Mar;69(3):1621–1627. doi: 10.1128/jvi.69.3.1621-1627.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nakhasi H. L., Cao X. Q., Rouault T. A., Liu T. Y. Specific binding of host cell proteins to the 3'-terminal stem-loop structure of rubella virus negative-strand RNA. J Virol. 1991 Nov;65(11):5961–5967. doi: 10.1128/jvi.65.11.5961-5967.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nakhasi H. L., Rouault T. A., Haile D. J., Liu T. Y., Klausner R. D. Specific high-affinity binding of host cell proteins to the 3' region of rubella virus RNA. New Biol. 1990 Mar;2(3):255–264. [PubMed] [Google Scholar]
  32. Novak J. E., Kirkegaard K. Improved method for detecting poliovirus negative strands used to demonstrate specificity of positive-strand encapsidation and the ratio of positive to negative strands in infected cells. J Virol. 1991 Jun;65(6):3384–3387. doi: 10.1128/jvi.65.6.3384-3387.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Petersen N. O., Johnson D. C., Schlesinger M. J. Scanning fluorescence correlation spectroscopy. II. Application to virus glycoprotein aggregation. Biophys J. 1986 Apr;49(4):817–820. doi: 10.1016/S0006-3495(86)83710-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rice C. M., Levis R., Strauss J. H., Huang H. V. Production of infectious RNA transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants. J Virol. 1987 Dec;61(12):3809–3819. doi: 10.1128/jvi.61.12.3809-3819.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sawicki D. L., Sawicki S. G. Alphavirus positive and negative strand RNA synthesis and the role of polyproteins in formation of viral replication complexes. Arch Virol Suppl. 1994;9:393–405. doi: 10.1007/978-3-7091-9326-6_39. [DOI] [PubMed] [Google Scholar]
  36. Sawicki D. L., Sawicki S. G., Keränen S., Käriäinen L. Specific Sindbis virus-coded function for minus-strand RNA synthesis. J Virol. 1981 Aug;39(2):348–358. doi: 10.1128/jvi.39.2.348-358.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sawicki D. L., Sawicki S. G. Short-lived minus-strand polymerase for Semliki Forest virus. J Virol. 1980 Apr;34(1):108–118. doi: 10.1128/jvi.34.1.108-118.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sherman L. A., Griffin D. E. Pathogenesis of encephalitis induced in newborn mice by virulent and avirulent strains of Sindbis virus. J Virol. 1990 May;64(5):2041–2046. doi: 10.1128/jvi.64.5.2041-2046.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shirako Y., Strauss J. H. Regulation of Sindbis virus RNA replication: uncleaved P123 and nsP4 function in minus-strand RNA synthesis, whereas cleaved products from P123 are required for efficient plus-strand RNA synthesis. J Virol. 1994 Mar;68(3):1874–1885. doi: 10.1128/jvi.68.3.1874-1885.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Stanley J., Cooper S. J., Griffin D. E. Monoclonal antibody cure and prophylaxis of lethal Sindbis virus encephalitis in mice. J Virol. 1986 Apr;58(1):107–115. doi: 10.1128/jvi.58.1.107-115.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Strauss E. G., Lenches E. M., Stamreich-Martin M. A. Growth and release of several alphaviruses in chick and BHK cells. J Gen Virol. 1980 Aug;49(2):297–307. doi: 10.1099/0022-1317-49-2-297. [DOI] [PubMed] [Google Scholar]
  42. Strauss J. H., Strauss E. G. The alphaviruses: gene expression, replication, and evolution. Microbiol Rev. 1994 Sep;58(3):491–562. doi: 10.1128/mr.58.3.491-562.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tyor W. R., Wesselingh S., Levine B., Griffin D. E. Long term intraparenchymal Ig secretion after acute viral encephalitis in mice. J Immunol. 1992 Dec 15;149(12):4016–4020. [PubMed] [Google Scholar]
  44. Ubol S., Levine B., Lee S. H., Greenspan N. S., Griffin D. E. Roles of immunoglobulin valency and the heavy-chain constant domain in antibody-mediated downregulation of Sindbis virus replication in persistently infected neurons. J Virol. 1995 Mar;69(3):1990–1993. doi: 10.1128/jvi.69.3.1990-1993.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Ubol S., Tucker P. C., Griffin D. E., Hardwick J. M. Neurovirulent strains of Alphavirus induce apoptosis in bcl-2-expressing cells: role of a single amino acid change in the E2 glycoprotein. Proc Natl Acad Sci U S A. 1994 May 24;91(11):5202–5206. doi: 10.1073/pnas.91.11.5202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Ulug E. T., Bose H. R., Jr Effect of tunicamycin on the development of the cytopathic effect in Sindbis virus-infected avian fibroblasts. Virology. 1985 Jun;143(2):546–557. doi: 10.1016/0042-6822(85)90393-9. [DOI] [PubMed] [Google Scholar]
  47. Ulug E. T., Garry R. F., Bose H. R., Jr The role of monovalent cation transport in Sindbis virus maturation and release. Virology. 1989 Sep;172(1):42–50. doi: 10.1016/0042-6822(89)90105-0. [DOI] [PubMed] [Google Scholar]
  48. Ulug E. T., Garry R. F., Waite M. R., Bose H. R., Jr Alterations in monovalent cation transport in Sindbis virus-infected chick cells. Virology. 1984 Jan 15;132(1):118–130. doi: 10.1016/0042-6822(84)90096-5. [DOI] [PubMed] [Google Scholar]
  49. Waite M. R., Pfefferkorn E. R. Inhibition of Sindbis virus production by media of low ionic strength: intracellular events and requirements for reversal. J Virol. 1970 Jan;5(1):60–71. doi: 10.1128/jvi.5.1.60-71.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wang Y. F., Sawicki S. G., Sawicki D. L. Alphavirus nsP3 functions to form replication complexes transcribing negative-strand RNA. J Virol. 1994 Oct;68(10):6466–6475. doi: 10.1128/jvi.68.10.6466-6475.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wesselingh S. L., Levine B., Fox R. J., Choi S., Griffin D. E. Intracerebral cytokine mRNA expression during fatal and nonfatal alphavirus encephalitis suggests a predominant type 2 T cell response. J Immunol. 1994 Feb 1;152(3):1289–1297. [PubMed] [Google Scholar]
  52. Zhao H., Garoff H. Role of cell surface spikes in alphavirus budding. J Virol. 1992 Dec;66(12):7089–7095. doi: 10.1128/jvi.66.12.7089-7095.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Zhong L. T., Sarafian T., Kane D. J., Charles A. C., Mah S. P., Edwards R. H., Bredesen D. E. bcl-2 inhibits death of central neural cells induced by multiple agents. Proc Natl Acad Sci U S A. 1993 May 15;90(10):4533–4537. doi: 10.1073/pnas.90.10.4533. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES