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. 1996 Dec;70(12):8752–8757. doi: 10.1128/jvi.70.12.8752-8757.1996

Amplification, expression, and packaging of a foreign gene by giardiavirus in Giardia lamblia.

D C Yu 1, A L Wang 1, C C Wang 1
PMCID: PMC190971  PMID: 8971003

Abstract

Giardia lamblia is an intestinal protozoan parasite and one of the earliest eukaryotic divergents. The trophozoite multiplies via asexual binary fission and lacks all natural means of lateral gene transfer. A system is developed here for long-term expression of a foreign gene in this organism by exploiting recombinant virions derived from the giardiavirus (GLV), a double-stranded RNA virus that infects many Giardia isolates. An in vitro transcript of the cloned GLV cDNA, comprising the firefly luciferase-encoding region flanked by 5' and 3' fragments of GLV positive-strand RNA, was electroporated into GLV-infected trophozoites. Luciferase activity in electroporated cells peaked on day 2 at levels 6 orders of magnitude above background. Expression of this foreign gene remained at 80% of its peak level after 30 days in the absence of selective pressure. The chimeric RNA was replicated as double-stranded RNA and packaged into virus-like particles. The recombinant virions were partially purified from the wild-type helper virus by CsCl equilibrium density-gradient centrifugation and used to superinfect Giardia trophozoites. At multiplicities of infection of 100 or higher, these chimeric virions were able to initiate new rounds of expression of luciferase activity in the superinfected cells. Thus, the engineered virion can be successfully used to introduce and efficiently express a heterologous gene in this eukaryotic microorganism.

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Selected References

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  1. Adam R. D. The biology of Giardia spp. Microbiol Rev. 1991 Dec;55(4):706–732. doi: 10.1128/mr.55.4.706-732.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cavalier-Smith T. Kingdom protozoa and its 18 phyla. Microbiol Rev. 1993 Dec;57(4):953–994. doi: 10.1128/mr.57.4.953-994.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dubensky T. W., Jr, Driver D. A., Polo J. M., Belli B. A., Latham E. M., Ibanez C. E., Chada S., Brumm D., Banks T. A., Mento S. J. Sindbis virus DNA-based expression vectors: utility for in vitro and in vivo gene transfer. J Virol. 1996 Jan;70(1):508–519. doi: 10.1128/jvi.70.1.508-519.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Furfine E. S., Wang C. C. Transfection of the Giardia lamblia double-stranded RNA virus into giardia lamblia by electroporation of a single-stranded RNA copy of the viral genome. Mol Cell Biol. 1990 Jul;10(7):3659–3662. doi: 10.1128/mcb.10.7.3659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Furfine E. S., White T. C., Wang A. L., Wang C. C. A single-stranded RNA copy of the Giardia lamblia virus double-stranded RNA genome is present in the infected Giardia lamblia. Nucleic Acids Res. 1989 Sep 25;17(18):7453–7467. doi: 10.1093/nar/17.18.7453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Glorioso J. C., DeLuca N. A., Fink D. J. Development and application of herpes simplex virus vectors for human gene therapy. Annu Rev Microbiol. 1995;49:675–710. doi: 10.1146/annurev.mi.49.100195.003331. [DOI] [PubMed] [Google Scholar]
  7. Hashimoto T., Nakamura Y., Kamaishi T., Nakamura F., Adachi J., Okamoto K., Hasegawa M. Phylogenetic place of mitochondrion-lacking protozoan, Giardia lamblia, inferred from amino acid sequences of elongation factor 2. Mol Biol Evol. 1995 Sep;12(5):782–793. doi: 10.1093/oxfordjournals.molbev.a040256. [DOI] [PubMed] [Google Scholar]
  8. Hashimoto T., Nakamura Y., Nakamura F., Shirakura T., Adachi J., Goto N., Okamoto K., Hasegawa M. Protein phylogeny gives a robust estimation for early divergences of eukaryotes: phylogenetic place of a mitochondria-lacking protozoan, Giardia lamblia. Mol Biol Evol. 1994 Jan;11(1):65–71. doi: 10.1093/oxfordjournals.molbev.a040093. [DOI] [PubMed] [Google Scholar]
  9. Miller R. L., Wang A. L., Wang C. C. Identification of Giardia lamblia isolates susceptible and resistant to infection by the double-stranded RNA virus. Exp Parasitol. 1988 Jun;66(1):118–123. doi: 10.1016/0014-4894(88)90056-2. [DOI] [PubMed] [Google Scholar]
  10. Miller R. L., Wang A. L., Wang C. C. Purification and characterization of the Giardia lamblia double-stranded RNA virus. Mol Biochem Parasitol. 1988 Apr;28(3):189–195. doi: 10.1016/0166-6851(88)90003-5. [DOI] [PubMed] [Google Scholar]
  11. Morgenstern J. P., Land H. A series of mammalian expression vectors and characterisation of their expression of a reporter gene in stably and transiently transfected cells. Nucleic Acids Res. 1990 Feb 25;18(4):1068–1068. doi: 10.1093/nar/18.4.1068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Mushegian A. R., Shepherd R. J. Genetic elements of plant viruses as tools for genetic engineering. Microbiol Rev. 1995 Dec;59(4):548–578. doi: 10.1128/mr.59.4.548-578.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Richards C. D., Braciak T., Xing Z., Graham F., Gauldie J. Adenovirus vectors for cytokine gene expression. Ann N Y Acad Sci. 1995 Jul 21;762:282–293. doi: 10.1111/j.1749-6632.1995.tb32333.x. [DOI] [PubMed] [Google Scholar]
  14. Sepp T., Wang A. L., Wang C. C. Giardiavirus-resistant Giardia lamblia lacks a virus receptor on the cell membrane surface. J Virol. 1994 Mar;68(3):1426–1431. doi: 10.1128/jvi.68.3.1426-1431.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Sogin M. L., Gunderson J. H., Elwood H. J., Alonso R. A., Peattie D. A. Phylogenetic meaning of the kingdom concept: an unusual ribosomal RNA from Giardia lamblia. Science. 1989 Jan 6;243(4887):75–77. doi: 10.1126/science.2911720. [DOI] [PubMed] [Google Scholar]
  16. Tai J. H., Wang A. L., Ong S. J., Lai K. S., Lo C., Wang C. C. The course of giardiavirus infection in the Giardia lamblia trophozoites. Exp Parasitol. 1991 Nov;73(4):413–423. doi: 10.1016/0014-4894(91)90065-5. [DOI] [PubMed] [Google Scholar]
  17. Wang A. L., Wang C. C. Discovery of a specific double-stranded RNA virus in Giardia lamblia. Mol Biochem Parasitol. 1986 Dec;21(3):269–276. doi: 10.1016/0166-6851(86)90132-5. [DOI] [PubMed] [Google Scholar]
  18. Wang A. L., Yang H. M., Shen K. A., Wang C. C. Giardiavirus double-stranded RNA genome encodes a capsid polypeptide and a gag-pol-like fusion protein by a translation frameshift. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8595–8599. doi: 10.1073/pnas.90.18.8595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Wu C. H., Wang C. C., Yang H. M., Wang A. L. Sequences at four termini of the giardiavirus double-stranded RNA. Gene. 1995 May 26;158(1):129–131. doi: 10.1016/0378-1119(95)00092-k. [DOI] [PubMed] [Google Scholar]
  20. Yee J., Nash T. E. Transient transfection and expression of firefly luciferase in Giardia lamblia. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5615–5619. doi: 10.1073/pnas.92.12.5615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Yu D. C., Wang A. L., Wu C. H., Wang C. C. Virus-mediated expression of firefly luciferase in the parasitic protozoan Giardia lamblia. Mol Cell Biol. 1995 Sep;15(9):4867–4872. doi: 10.1128/mcb.15.9.4867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Yu D. C., Wang C. C. Identification of cis-acting signals in the giardiavirus (GLV) genome required for expression of firefly luciferase in Giardia lamblia. RNA. 1996 Aug;2(8):824–834. [PMC free article] [PubMed] [Google Scholar]
  23. Yu D., Wang C. C., Wang A. L. Maturation of giardiavirus capsid protein involves posttranslational proteolytic processing by a cysteine protease. J Virol. 1995 May;69(5):2825–2830. doi: 10.1128/jvi.69.5.2825-2830.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]

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