1887

Abstract

Sequences encoding the green fluorescent protein (GFP) were inserted into the envelope protein (Env) of ecotropic Moloney murine leukaemia virus, MoMLV. Insertion of these sequences into the proline-rich region (PRR) of Env resulted in a chimeric GFP-Env protein that allowed retrovirus vector transduction of murine cells with titres similar to wild-type Env. However, N-terminal extension with GFP did not result in a functional Env protein. GFP sequences were then inserted into the Env PRR of E-MO virus, a MoMLV that carries epidermal growth factor sequences at the N terminus of its Env protein. The resulting virus, GFP-EMO1, replicates to the same titres as the parental virus. In a chronically infected cell culture, GFP-EMO1 was genetically stable. However, additional insertions of sequences that led to recombination or that may have been incompatible with virus replication were deleted and decreased virus titre. In summary, Env PRR can be used to tag individual virus particles with GFP, which leaves other regions available for modification in studies aimed at altering virus tropism.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.18761-0
2003-02-01
2019-10-17
Loading full text...

Full text loading...

/deliver/fulltext/jgv/84/2/vir840369.html?itemId=/content/journal/jgv/10.1099/vir.0.18761-0&mimeType=html&fmt=ahah

References

  1. Albritton, L. M., Tseng, L., Scadden, D. & Cunningham, J. M. ( 1989; ). A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection. Cell 57, 659–666.[CrossRef]
    [Google Scholar]
  2. Allured, V. S., Collier, R. J., Carroll, S. F. & McKay, D. B. ( 1986; ). Structure of exotoxin A of Pseudomonas aeruginosa at 3·0 Å resolution. Proc Natl Acad Sci U S A 83, 1320–1324.[CrossRef]
    [Google Scholar]
  3. Buchholz, C. J., Peng, K. W., Morling, F. J., Zhang, J., Cosset, F.-L. & Russell, S. J. ( 1998; ). In vivo selection of protease cleavage sites from retrovirus display libraries. Nat Biotechnol 16, 951–954.[CrossRef]
    [Google Scholar]
  4. Bupp, K. & Roth, M. J. ( 2002; ). Altering retroviral tropism using a random-display envelope library. Mol Ther 5, 329–335.[CrossRef]
    [Google Scholar]
  5. Cosset, F.-L., Morling, F. J., Takeuchi, Y., Weiss, R. A., Collins, M. K. & Russell, S. J. ( 1995; ). Retroviral retargeting by envelopes expressing an N-terminal binding domain. J Virol 69, 6314–6322.
    [Google Scholar]
  6. Erlwein, O., Wels, W. & Schnierle, B. S. ( 2002; ). Chimeric ecotropic MLV envelope proteins that carry EGF receptor specific ligands and the pseudomonas exotoxin A translocation domain to target gene transfer to human cancer cells. Virology 302, 333–341.[CrossRef]
    [Google Scholar]
  7. Hunter, E. ( 1997; ). Viral entry and receptors. In Retroviruses, pp 71–120. Edited by J. M. Coffin, S. H. Hughes & H. E. Varmus. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  8. Hwang, J., Fitzgerald, D. J., Adhya, S. & Pastan, I. ( 1987; ). Functional domains of Pseudomonas exotoxin identified by deletion analysis of the gene expressed in E. coli. Cell 48, 129–136.[CrossRef]
    [Google Scholar]
  9. Kayman, S. C., Park, H., Saxon, M. & Pinter, A. ( 1999; ). The hypervariable domain of the murine leukemia virus surface protein tolerates large insertions and deletions, enabling development of a retroviral particle display system. J Virol 73, 1802–1808.
    [Google Scholar]
  10. Kizhatil, K., Gromley, A. & Albritton, L. M. ( 2001; ). Two point mutations produce infectious retrovirus bearing a green fluorescent protein–SU fusion protein. J Virol 75, 11881–11885.[CrossRef]
    [Google Scholar]
  11. Lavillette, D., Maurice, M., Roche, C., Russell, S. J., Sitbon, M. & Cosset, F.-L. ( 1998; ). A proline-rich motif downstream of the receptor binding domain modulates conformation and fusogenicity of murine retroviral envelopes. J Virol 72, 9955–9965.
    [Google Scholar]
  12. Lindemann, D., Bock, M., Schweizer, M. & Rethwilm, A. ( 1997; ). Efficient pseudotyping of murine leukemia virus particles with chimeric human foamy virus envelope proteins. J Virol 71, 4815–4820.
    [Google Scholar]
  13. Pear, W. S., Nolan, G. P., Scott, M. L. & Baltimore, D. ( 1993; ). Production of high-titre helper-free retroviruses by transient transfection. Proc Natl Acad Sci U S A 90, 8392–8396.[CrossRef]
    [Google Scholar]
  14. Sambrook, J., Fritsch, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  15. Schnierle, B. S. & Groner, B. ( 1996; ). Retroviral targeted delivery. Gene Ther 3, 1069–1073.
    [Google Scholar]
  16. Schnierle, B. S., Moritz, D., Jeschke, M. & Groner, B. ( 1996; ). Expression of chimeric envelope proteins in helper cell lines and integration into Moloney murine leukemia virus particles. Gene Ther 3, 334–342.
    [Google Scholar]
  17. Schnierle, B. S., Stitz, J., Bosch, V., Nocken, F., Merget-Millitzer, H., Engelstädter, M., Kurth, R., Groner, B. & Cichutek, K. ( 1997; ). Pseudotyping of murine leukemia virus with the envelope glycoproteins of HIV generates a retroviral vector with specificity of infection for CD4-expressing cells. Proc Natl Acad Sci U S A 94, 8640–8645.[CrossRef]
    [Google Scholar]
  18. Wu, B. W., Cannon, P. M., Gordon, E. M., Hall, F. L. & Anderson, W. F. ( 1998; ). Characterization of the proline-rich region of murine leukemia virus envelope protein. J Virol 72, 5383–5391.
    [Google Scholar]
  19. Wu, B. W., Lu, J., Gallaher, T. K., Anderson, W. F. & Cannon, P. M. ( 2000; ). Identification of regions in the Moloney murine leukemia virus SU protein that tolerate the insertion of an integrin-binding peptide. Virology 269, 7–17.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.18761-0
Loading
/content/journal/jgv/10.1099/vir.0.18761-0
Loading

Data & Media loading...

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error