Abstract
We have recently shown that live attenuated virus vaccines produced on chicken-derived cells contain low levels of particle-associated reverse transcriptase (RT). In both virus and corresponding control harvests produced on chicken embryo fibroblasts, these activities were present at significantly higher concentrations than in the vaccines. In order to identify the putative retrovirus sequence responsible for this activity, a novel method for the selective PCR amplification of particle-associated retrovirus RNA that uses DNA primers complementary to the primer binding sites of the known exogenous retroviruses in combination with an anchor primer was applied. A product of the endogenous avian retrovirus family EAV-0, termed EAV-0(B1), was reproducibly generated with a tRNA(Trp)-derived primer from the RT peak fraction of a sucrose density gradient run with a harvest of a live attenuated measles vaccine. In contrast, no products were detected with primers derived from tRNA(Pro), tRNA(Lys)1,2 or tRNA(Lys)3. In the same fraction, genomic RNA of EAV-0(B1) was demonstrated by long PCR. Analysis of several sucrose density gradients from different harvests of various manufacturers demonstrated accumulation of, and colocalization with, RT activity for the EAV-0(B1) RNA but not for a chicken cellular mRNA. Synthesis of cDNA from EAV-0(B1) RNA was shown by endogenous RT reaction. Furthermore, complexes of naturally primed EAV-0(B1) RNA with RT were demonstrated. Taken together, these data strongly suggest that EAV-0 is able to produce virus-like particles with an active RT.
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- Baltimore D. RNA-dependent DNA polymerase in virions of RNA tumour viruses. Nature. 1970 Jun 27;226(5252):1209–1211. doi: 10.1038/2261209a0. [DOI] [PubMed] [Google Scholar]
- Barnes W. M. PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc Natl Acad Sci U S A. 1994 Mar 15;91(6):2216–2220. doi: 10.1073/pnas.91.6.2216. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bauer G., Friis R. R., Jilek G., Hofschneider P. H. Purification and characterization of particles containing RNA-dependent DNA polymerase, in the allantoic fluid of uninfected leukosis virus-free chicken eggs. Biochim Biophys Acta. 1978 Mar 29;518(1):125–137. doi: 10.1016/0005-2787(78)90122-3. [DOI] [PubMed] [Google Scholar]
- Bauer G., Friis R. R., Mattersberger H., Hofschneider P. H. Controlled release of particle-associated RNA-dependent DNA polymerase by primary chick embryo cell cultures. Exp Cell Res. 1978 Dec;117(2):383–392. doi: 10.1016/0014-4827(78)90151-9. [DOI] [PubMed] [Google Scholar]
- Bauer G., Hofschneider P. H. An RNA-dependent DNA polymerase, different from the known viral reverse transcriptases, in the chicken system. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3025–3029. doi: 10.1073/pnas.73.9.3025. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Boyce-Jacino M. T., O'Donoghue K., Faras A. J. Multiple complex families of endogenous retroviruses are highly conserved in the genus Gallus. J Virol. 1992 Aug;66(8):4919–4929. doi: 10.1128/jvi.66.8.4919-4929.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Boyce-Jacino M. T., Resnick R., Faras A. J. Structural and functional characterization of the unusually short long terminal repeats and their adjacent regions of a novel endogenous avian retrovirus. Virology. 1989 Nov;173(1):157–166. doi: 10.1016/0042-6822(89)90231-6. [DOI] [PubMed] [Google Scholar]
- Böni J., Pyra H., Schüpbach J. Sensitive detection and quantification of particle-associated reverse transcriptase in plasma of HIV-1-infected individuals by the product-enhanced reverse transcriptase (PERT) assay. J Med Virol. 1996 May;49(1):23–28. doi: 10.1002/(SICI)1096-9071(199605)49:1<23::AID-JMV4>3.0.CO;2-M. [DOI] [PubMed] [Google Scholar]
- Böni J., Stalder J., Reigel F., Schüpbach J. Detectionof reverse transcriptase activity in live attenuated virus vaccines. Clin Diagn Virol. 1996 Feb;5(1):43–53. doi: 10.1016/0928-0197(95)00159-x. [DOI] [PubMed] [Google Scholar]
- Cheng S., Chang S. Y., Gravitt P., Respess R. Long PCR. Nature. 1994 Jun 23;369(6482):684–685. doi: 10.1038/369684a0. [DOI] [PubMed] [Google Scholar]
- Cheng S., Fockler C., Barnes W. M., Higuchi R. Effective amplification of long targets from cloned inserts and human genomic DNA. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5695–5699. doi: 10.1073/pnas.91.12.5695. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dunwiddie C. T., Resnick R., Boyce-Jacino M., Alegre J. N., Faras A. J. Molecular cloning and characterization of gag-, pol-, and env-related gene sequences in the ev- chicken. J Virol. 1986 Sep;59(3):669–675. doi: 10.1128/jvi.59.3.669-675.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dunwiddie C., Faras A. J. Presence of retrovirus reverse transcriptase-related gene sequences in avian cells lacking endogenous avian leukosis viruses. Proc Natl Acad Sci U S A. 1985 Aug;82(15):5097–5101. doi: 10.1073/pnas.82.15.5097. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Frohman M. A., Dush M. K., Martin G. R. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8998–9002. doi: 10.1073/pnas.85.23.8998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Junghans R. P., Duesberg P. H., Knight C. A. In vitro synthesis of full-length DNA transcripts of Rous sarcoma virus RNA by viral DNA polymerase. Proc Natl Acad Sci U S A. 1975 Dec;72(12):4895–4899. doi: 10.1073/pnas.72.12.4895. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Loh E. Y., Elliott J. F., Cwirla S., Lanier L. L., Davis M. M. Polymerase chain reaction with single-sided specificity: analysis of T cell receptor delta chain. Science. 1989 Jan 13;243(4888):217–220. doi: 10.1126/science.2463672. [DOI] [PubMed] [Google Scholar]
- Mahy B. W., Hadler S. C. Detection of an RNA-dependent DNA polymerase activity. Clin Diagn Virol. 1996 Feb;5(1):1–2. [PubMed] [Google Scholar]
- Pyra H., Böni J., Schüpbach J. Ultrasensitive retrovirus detection by a reverse transcriptase assay based on product enhancement. Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1544–1548. doi: 10.1073/pnas.91.4.1544. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Resnick R. M., Boyce-Jacino M. T., Fu Q., Faras A. J. Phylogenetic distribution of the novel avian endogenous provirus family EAV-0. J Virol. 1990 Oct;64(10):4640–4653. doi: 10.1128/jvi.64.10.4640-4653.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Saluz H., Jost J. P. A simple high-resolution procedure to study DNA methylation and in vivo DNA-protein interactions on a single-copy gene level in higher eukaryotes. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2602–2606. doi: 10.1073/pnas.86.8.2602. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Temin H. M., Mizutani S. RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Nature. 1970 Jun 27;226(5252):1211–1213. doi: 10.1038/2261211a0. [DOI] [PubMed] [Google Scholar]
- Yong W. H., Wyman S., Levy J. A. Optimal conditions for synthesizing complementary DNA in the HIV-1 endogenous reverse transcriptase reaction. AIDS. 1990 Mar;4(3):199–206. doi: 10.1097/00002030-199003000-00004. [DOI] [PubMed] [Google Scholar]