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. 1997 Oct;71(10):7361–7371. doi: 10.1128/jvi.71.10.7361-7371.1997

Encapsidation of adeno-associated virus type 2 Rep proteins in wild-type and recombinant progeny virions: Rep-mediated growth inhibition of primary human cells.

D M Kube 1, S Ponnazhagan 1, A Srivastava 1
PMCID: PMC192081  PMID: 9311814

Abstract

The adeno-associated virus type 2 (AAV) arrests the growth of primary human fibroblasts in vitro at high particle-to-cell ratios. To test the role of AAV gene expression in the observed growth inhibition, primary human cells were infected, under identical conditions, with wild-type (wt) AAV or with recombinant AAV that lacked all viral promoters and coding sequences. Significant, dose-dependent growth inhibition of primary human cells was observed with both wt and recombinant AAV at particle-to-cell ratios equal to or exceeding 10(4). In contrast, neither virus affected the growth of immortalized human cells even at a 10-fold-higher particle-to-cell ratio. AAV-induced growth arrest could be overcome by reculturing cells after treatment with trypsin. Even after reculturing, cells still harbored the proviral AAV genome. Thus, neither integration nor expression of the AAV genome appears to be required for the virus-induced growth-inhibitory effect on primary human cells. The growth-inhibitory effect of AAV was hypothesized to be mediated by virion-associated AAV Rep proteins, since these proteins have been reported to inhibit cellular DNA synthesis. Rep proteins tightly associated with wt as well as recombinant AAV could be detected on Western blots. Coinfection by adenovirus was necessary and sufficient for ample replication of recombinant AAV genomes lacking the rep gene. Although wt AAV-like particles arose during production of the recombinant AAV stocks, their low-titer levels were insufficient to cause the observed growth inhibition. AAV rep gene expression from these contaminating particles was not required for replication of the recombinant AAV genomes, which could be detected even in the absence of de novo Rep protein synthesis. Exposure of recombinant AAV to anti-AAV Rep protein antibodies did not abrogate viral infectivity. These results suggest that biologically active Rep proteins are encapsidated in mature progeny AAV particles. AAV Rep protein-mediated growth inhibition of primary human cells has implications in the use of AAV-based vectors in human gene therapy.

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Selected References

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  1. Ashktorab H., Srivastava A. Identification of nuclear proteins that specifically interact with adeno-associated virus type 2 inverted terminal repeat hairpin DNA. J Virol. 1989 Jul;63(7):3034–3039. doi: 10.1128/jvi.63.7.3034-3039.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bantel-Schaal U. Adeno-associated parvoviruses inhibit growth of cells derived from malignant human tumors. Int J Cancer. 1990 Jan 15;45(1):190–194. doi: 10.1002/ijc.2910450134. [DOI] [PubMed] [Google Scholar]
  3. Bantel-Schaal U. Growth properties of a human melanoma cell line are altered by adeno-associated parvovirus type 2. Int J Cancer. 1995 Jan 17;60(2):269–274. doi: 10.1002/ijc.2910600223. [DOI] [PubMed] [Google Scholar]
  4. Bantel-Schaal U., Stöhr M. Influence of adeno-associated virus on adherence and growth properties of normal cells. J Virol. 1992 Feb;66(2):773–779. doi: 10.1128/jvi.66.2.773-779.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berns K. I., Bohenzky R. A. Adeno-associated viruses: an update. Adv Virus Res. 1987;32:243–306. doi: 10.1016/s0065-3527(08)60479-0. [DOI] [PubMed] [Google Scholar]
  6. Berns K. I., Linden R. M. The cryptic life style of adeno-associated virus. Bioessays. 1995 Mar;17(3):237–245. doi: 10.1002/bies.950170310. [DOI] [PubMed] [Google Scholar]
  7. Botquin V., Cid-Arregui A., Schlehofer J. R. Adeno-associated virus type 2 interferes with early development of mouse embryos. J Gen Virol. 1994 Oct;75(Pt 10):2655–2662. doi: 10.1099/0022-1317-75-10-2655. [DOI] [PubMed] [Google Scholar]
  8. Chejanovsky N., Carter B. J. Replication of a human parvovirus nonsense mutant in mammalian cells containing an inducible amber suppressor. Virology. 1989 Jul;171(1):239–247. doi: 10.1016/0042-6822(89)90531-x. [DOI] [PubMed] [Google Scholar]
  9. Cotmore S. F., Tattersall P. A genome-linked copy of the NS-1 polypeptide is located on the outside of infectious parvovirus particles. J Virol. 1989 Sep;63(9):3902–3911. doi: 10.1128/jvi.63.9.3902-3911.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cotmore S. F., Tattersall P. The NS-1 polypeptide of minute virus of mice is covalently attached to the 5' termini of duplex replicative-form DNA and progeny single strands. J Virol. 1988 Mar;62(3):851–860. doi: 10.1128/jvi.62.3.851-860.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Groneberg J., Sutter D., Soboll H., Doerfler W. Morphological revertants of adenovirus type 12-transformed hamster cells. J Gen Virol. 1978 Sep;40(3):635–645. doi: 10.1099/0022-1317-40-3-635. [DOI] [PubMed] [Google Scholar]
  12. Heilbronn R., Bürkle A., Stephan S., zur Hausen H. The adeno-associated virus rep gene suppresses herpes simplex virus-induced DNA amplification. J Virol. 1990 Jun;64(6):3012–3018. doi: 10.1128/jvi.64.6.3012-3018.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hermonat P. L. Inhibition of H-ras expression by the adeno-associated virus Rep78 transformation suppressor gene product. Cancer Res. 1991 Jul 1;51(13):3373–3377. [PubMed] [Google Scholar]
  14. Hermonat P. L. Inhibition of bovine papillomavirus plasmid DNA replication by adeno-associated virus. Virology. 1992 Jul;189(1):329–333. doi: 10.1016/0042-6822(92)90710-7. [DOI] [PubMed] [Google Scholar]
  15. Hermonat P. L. The adeno-associated virus Rep78 gene inhibits cellular transformation induced by bovine papillomavirus. Virology. 1989 Sep;172(1):253–261. doi: 10.1016/0042-6822(89)90127-x. [DOI] [PubMed] [Google Scholar]
  16. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  17. Im D. S., Muzyczka N. The AAV origin binding protein Rep68 is an ATP-dependent site-specific endonuclease with DNA helicase activity. Cell. 1990 May 4;61(3):447–457. doi: 10.1016/0092-8674(90)90526-k. [DOI] [PubMed] [Google Scholar]
  18. Kampinga H. H., Brunsting J. F., Konings A. W. Acquisition of thermotolerance induced by heat and arsenite in HeLa S3 cells: multiple pathways to induce tolerance? J Cell Physiol. 1992 Feb;150(2):406–415. doi: 10.1002/jcp.1041500225. [DOI] [PubMed] [Google Scholar]
  19. Khleif S. N., Myers T., Carter B. J., Trempe J. P. Inhibition of cellular transformation by the adeno-associated virus rep gene. Virology. 1991 Apr;181(2):738–741. doi: 10.1016/0042-6822(91)90909-u. [DOI] [PubMed] [Google Scholar]
  20. Klein-Bauernschmitt P., zur Hausen H., Schlehofer J. R. Induction of differentiation-associated changes in established human cells by infection with adeno-associated virus type 2. J Virol. 1992 Jul;66(7):4191–4200. doi: 10.1128/jvi.66.7.4191-4200.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kotin R. M., Menninger J. C., Ward D. C., Berns K. I. Mapping and direct visualization of a region-specific viral DNA integration site on chromosome 19q13-qter. Genomics. 1991 Jul;10(3):831–834. doi: 10.1016/0888-7543(91)90470-y. [DOI] [PubMed] [Google Scholar]
  22. Kotin R. M., Siniscalco M., Samulski R. J., Zhu X. D., Hunter L., Laughlin C. A., McLaughlin S., Muzyczka N., Rocchi M., Berns K. I. Site-specific integration by adeno-associated virus. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2211–2215. doi: 10.1073/pnas.87.6.2211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Labow M. A., Graf L. H., Jr, Berns K. I. Adeno-associated virus gene expression inhibits cellular transformation by heterologous genes. Mol Cell Biol. 1987 Apr;7(4):1320–1325. doi: 10.1128/mcb.7.4.1320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McCarty D. M., Pereira D. J., Zolotukhin I., Zhou X., Ryan J. H., Muzyczka N. Identification of linear DNA sequences that specifically bind the adeno-associated virus Rep protein. J Virol. 1994 Aug;68(8):4988–4997. doi: 10.1128/jvi.68.8.4988-4997.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Muzyczka N. Use of adeno-associated virus as a general transduction vector for mammalian cells. Curr Top Microbiol Immunol. 1992;158:97–129. doi: 10.1007/978-3-642-75608-5_5. [DOI] [PubMed] [Google Scholar]
  26. Nahreini P., Woody M. J., Zhou S. Z., Srivastava A. Versatile adeno-associated virus 2-based vectors for constructing recombinant virions. Gene. 1993 Feb 28;124(2):257–262. doi: 10.1016/0378-1119(93)90402-o. [DOI] [PubMed] [Google Scholar]
  27. Ostrove J. M., Duckworth D. H., Berns K. I. Inhibition of adenovirus-transformed cell oncogenicity by adeno-associated virus. Virology. 1981 Sep;113(2):521–533. doi: 10.1016/0042-6822(81)90180-x. [DOI] [PubMed] [Google Scholar]
  28. Pereira D. J., McCarty D. M., Muzyczka N. The adeno-associated virus (AAV) Rep protein acts as both a repressor and an activator to regulate AAV transcription during a productive infection. J Virol. 1997 Feb;71(2):1079–1088. doi: 10.1128/jvi.71.2.1079-1088.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pereira D. J., Muzyczka N. The cellular transcription factor SP1 and an unknown cellular protein are required to mediate Rep protein activation of the adeno-associated virus p19 promoter. J Virol. 1997 Mar;71(3):1747–1756. doi: 10.1128/jvi.71.3.1747-1756.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ponnazhagan S., Erikson D., Kearns W. G., Zhou S. Z., Nahreini P., Wang X. S., Srivastava A. Lack of site-specific integration of the recombinant adeno-associated virus 2 genomes in human cells. Hum Gene Ther. 1997 Feb 10;8(3):275–284. doi: 10.1089/hum.1997.8.3-275. [DOI] [PubMed] [Google Scholar]
  31. Ponnazhagan S., Wang X. S., Woody M. J., Luo F., Kang L. Y., Nallari M. L., Munshi N. C., Zhou S. Z., Srivastava A. Differential expression in human cells from the p6 promoter of human parvovirus B19 following plasmid transfection and recombinant adeno-associated virus 2 (AAV) infection: human megakaryocytic leukaemia cells are non-permissive for AAV infection. J Gen Virol. 1996 Jun;77(Pt 6):1111–1122. doi: 10.1099/0022-1317-77-6-1111. [DOI] [PubMed] [Google Scholar]
  32. Prasad K. M., Trempe J. P. The adeno-associated virus Rep78 protein is covalently linked to viral DNA in a preformed virion. Virology. 1995 Dec 20;214(2):360–370. doi: 10.1006/viro.1995.0045. [DOI] [PubMed] [Google Scholar]
  33. Prasad K. M., Zhou C., Trempe J. P. Characterization of the Rep78/adeno-associated virus complex. Virology. 1997 Mar 3;229(1):183–192. doi: 10.1006/viro.1996.8431. [DOI] [PubMed] [Google Scholar]
  34. Salo R. J., Mayor H. D. Structural polypeptides of parvoviruses. Virology. 1977 May 1;78(1):340–345. doi: 10.1016/0042-6822(77)90107-6. [DOI] [PubMed] [Google Scholar]
  35. Samulski R. J., Berns K. I., Tan M., Muzyczka N. Cloning of adeno-associated virus into pBR322: rescue of intact virus from the recombinant plasmid in human cells. Proc Natl Acad Sci U S A. 1982 Mar;79(6):2077–2081. doi: 10.1073/pnas.79.6.2077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Samulski R. J., Chang L. S., Shenk T. Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression. J Virol. 1989 Sep;63(9):3822–3828. doi: 10.1128/jvi.63.9.3822-3828.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Samulski R. J., Zhu X., Xiao X., Brook J. D., Housman D. E., Epstein N., Hunter L. A. Targeted integration of adeno-associated virus (AAV) into human chromosome 19. EMBO J. 1991 Dec;10(12):3941–3950. doi: 10.1002/j.1460-2075.1991.tb04964.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schlehofer J. R. The tumor suppressive properties of adeno-associated viruses. Mutat Res. 1994 Mar 1;305(2):303–313. doi: 10.1016/0027-5107(94)90250-x. [DOI] [PubMed] [Google Scholar]
  39. Snyder R. O., Im D. S., Muzyczka N. Evidence for covalent attachment of the adeno-associated virus (AAV) rep protein to the ends of the AAV genome. J Virol. 1990 Dec;64(12):6204–6213. doi: 10.1128/jvi.64.12.6204-6213.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  41. Srivastava A., Lusby E. W., Berns K. I. Nucleotide sequence and organization of the adeno-associated virus 2 genome. J Virol. 1983 Feb;45(2):555–564. doi: 10.1128/jvi.45.2.555-564.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Srivastava C. H., Samulski R. J., Lu L., Larsen S. H., Srivastava A. Construction of a recombinant human parvovirus B19: adeno-associated virus 2 (AAV) DNA inverted terminal repeats are functional in an AAV-B19 hybrid virus. Proc Natl Acad Sci U S A. 1989 Oct;86(20):8078–8082. doi: 10.1073/pnas.86.20.8078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tratschin J. D., Tal J., Carter B. J. Negative and positive regulation in trans of gene expression from adeno-associated virus vectors in mammalian cells by a viral rep gene product. Mol Cell Biol. 1986 Aug;6(8):2884–2894. doi: 10.1128/mcb.6.8.2884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Trempe J. P., Carter B. J. Regulation of adeno-associated virus gene expression in 293 cells: control of mRNA abundance and translation. J Virol. 1988 Jan;62(1):68–74. doi: 10.1128/jvi.62.1.68-74.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Winocour E., Callaham M. F., Huberman E. Perturbation of the cell cycle by adeno-associated virus. Virology. 1988 Dec;167(2):393–399. [PubMed] [Google Scholar]
  46. Wistuba A., Weger S., Kern A., Kleinschmidt J. A. Intermediates of adeno-associated virus type 2 assembly: identification of soluble complexes containing Rep and Cap proteins. J Virol. 1995 Sep;69(9):5311–5319. doi: 10.1128/jvi.69.9.5311-5319.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wonderling R. S., Owens R. A. Binding sites for adeno-associated virus Rep proteins within the human genome. J Virol. 1997 Mar;71(3):2528–2534. doi: 10.1128/jvi.71.3.2528-2534.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yakobson B., Koch T., Winocour E. Replication of adeno-associated virus in synchronized cells without the addition of a helper virus. J Virol. 1987 Apr;61(4):972–981. doi: 10.1128/jvi.61.4.972-981.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Yalkinoglu A. O., Heilbronn R., Bürkle A., Schlehofer J. R., zur Hausen H. DNA amplification of adeno-associated virus as a response to cellular genotoxic stress. Cancer Res. 1988 Jun 1;48(11):3123–3129. [PubMed] [Google Scholar]
  50. Yang Q., Chen F., Ross J., Trempe J. P. Inhibition of cellular and SV40 DNA replication by the adeno-associated virus Rep proteins. Virology. 1995 Feb 20;207(1):246–250. doi: 10.1006/viro.1995.1072. [DOI] [PubMed] [Google Scholar]
  51. Yang Q., Chen F., Trempe J. P. Characterization of cell lines that inducibly express the adeno-associated virus Rep proteins. J Virol. 1994 Aug;68(8):4847–4856. doi: 10.1128/jvi.68.8.4847-4856.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Yang Q., Kadam A., Trempe J. P. Mutational analysis of the adeno-associated virus rep gene. J Virol. 1992 Oct;66(10):6058–6069. doi: 10.1128/jvi.66.10.6058-6069.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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