Department of Molecular and Structural Biology1, Department of Medical Microbiology and Immunology2, C. F. Moellers Allé, Bldg 130, DK-8000 Aarhus, University of Aarhus, Denmark
Author for correspondence: Finn Skou Pedersen. Fax +45 86196500. e-mail fsp{at}mbio.aau.dk
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
Main text |
---|
![]() ![]() ![]() ![]() |
---|
Fv1 is found in two main allelic versions, Fv1n and Fv1b, present in NIH Swiss and BALB/c mice respectively. The Fv1n allele restricts replication of B-tropic MLV while allowing replication of N-tropic viruses and vice versa. NB-tropic viruses infect mice having either allele. The amino acid at CA position 110 determines if a virus is N- (Arg) or B-tropic (Glu) (Kozak & Chakraborti, 1996 ). It has been shown that the Fv1 alleles are codominant in such a way that Fv1n/b mice restrict both N- and B-tropic viruses. Likewise, mixed virus particles harbouring both N- and B-tropic determinants will be sensitive to both Fv1 alleles. The restriction of infection in non-permissive cells is not absolute, but leads to a 501000 decrease in virus titre. It is believed that Fv1 restriction can be relieved, or abrogated, if the same cell is hit by two or more virus particles (Duran-Troise et al., 1977
). However, the mechanism underlying Fv1 restriction remains unclear.
Recently, Towers et al. (2000) published the intriguing observation that a wide range of non-murine cells restricts N-tropic viruses in an Fv1-like manner. This study used single-round transfer of vesicular stomatitis virus G protein (or amphotropic) pseudotyped retrovirus vectors to infect mammalian cells using both N- and B-tropic packaging constructs. It was shown that the restriction was dependent on CA position 110 and that mixed virus particles were sensitized. The gene responsible for the restriction was dubbed REF1 (restriction factor 1). Using a different approach, we could detect this restriction in the context of full-length ecotropic replication-competent MLV infecting mCAT-1-expressing human cells as compared to infections with pseudotyped vector preparations. We also included NB-tropic (as well as N- and B-tropic) determinants in our study. Part of our work took advantage of an EGFP-expressing virus that allowed us to detect the infection of a virus harbouring a full-length genome. We here present data that support and strengthen the existence of Fv1-like restriction in human cells.
In an initial series of experiments we observed that N-tropic Akv MLV failed to replicate in a human cell line (U2OS; Ponten & Saksela, 1967 ) modified to stably express the ecotropic mCAT-1 receptor, whereas the NB-tropic Moloney MLV replicated efficiently. We tested this more rigorously by using SL3-3 and SL3-3NB MLV. The parental SL3-3 virus is N-tropic whereas the derived SL3-3NB was turned NB-tropic by insertion of Moloney sequence at the N-terminal region of CA (position 1135) leading to five amino acid substitutions conferring the NB tropism (Thomas et al., 1993
). Target cells were infected with equal amounts of these four viruses (normalized by reverse transcriptase assay) in the presence of 6 µg polybrene per ml and passaged under standard conditions for 18 days in order to allow virus spread. Virus production was quantified from supernatant by reverse transcriptase assay (Lovmand et al., 1998
) and normalized to the total number of cells (Fig. 1
). Similar results were obtained when using 10-fold more or less virus (data not shown). Direct transfection of infectious N-tropic Akv DNA into the mCAT-1-expressing U2OS cells likewise did not lead to spread of virus even after prolonged cell culturing (>50 days, data not shown). To rule out contamination of cell stocks we amplified the GADPH gene by PCR from U2OS genomic DNA and verified by sequencing the gene of human origin. Sequencing of RNA (cDNA) isolated from virus particles likewise verified that no virus contamination nor spontaneous mutations had occurred within the CA region during the experiment.
|
To further address the escape requirements of viruses using the ecotropic entry pathway in human cells, we took advantage of an EGFP-expressing virus (AkvU3-EGFP) that allows direct titre measurement (Jespersen et al., 1999 ; Bachrach et al.,2002
). This N-tropic virus was modified in the CA region to alter the tropism. The B-tropic variant, AkvBU3-EGFP, was changed at genome position 16081609 from AG to GA resulting in an Arg to Glu substitution at CA position 110. This point mutation was introduced into a PCR fragment which was subsequently cloned into the flanking SacII and DraIII sites (Fig. 2A
). The NB-tropic variant, AkvNBU3-EGFP, had a Moloney fragment inserted at position 12811685, resulting in the following amino acid changes in the N-terminal region of CA: position 4, Leu to Ala; position 46, Thr to Ile; position 82, Asn to Asp; position 110, Arg to Ala; and position 117, Leu to His. The substitution was generated by extending a Moloney PCR fragment encompassing CA position 1135 (forward primer harbouring an Akv-derived linker from SacII to the p12/CA border) with an Akv PCR fragment (with the DraIII site) overlapping at position 16751695. The PCR product was cloned into the SacII and DraIII sites of Akv-U3EGFP.
|
|
We have thus confirmed the Gag-defined post-entry block to N-tropic virus infection in human cells as observed by Towers et al. (2000) , and further extended the analogy between REF1 and Fv1 in two important ways: (i) by finding that NB-tropic determinants also escape restriction and (ii) by using full-length ecotropic MLV instead of pseudotyped vectors (Towers et al., 2000
).
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() |
---|
Bénit, L., Parseval, N. D., Casella, J.-F., Callebaut, I., Cordonnier, A. & Heidmann, T. (1997). Cloning of a new murine endogenous retrovirus, MuERV-L, with strong similarity to the human HERV-L element and with the gag coding sequence closely related to the Fv1 restriction gene. Journal of Virology 71, 5652-5657.[Abstract]
Best, S., Le Tissier, P., Towers, G. & Stoye, J. P. (1996). Positional cloning of the mouse retrovirus restriction gene Fv1. Nature 382, 826-829.[Medline]
Celis, J. E., Justesen, J., Madsen, P. S., Lovmand, J., Ratz, G. P. & Celis, A. (1987). Major proteins induced and down-regulated by interferons in human cultured cells: identification of a unique set of proteins induced by interferon- in epithelial, fibroblast, and lymphoid cells. Leukemia 1, 800-813.[Medline]
Duran-Troise, G., Bassin, R. H., Rein, A. & Gerwin, B. I. (1977). Loss of Fv1 restriction in BALB/3T3 cells following infection with a single N-tropic murine leukemia virus particle. Cell 10, 479-488.[Medline]
Genome International Sequencing Consortium (2001). Initial sequencing and analysis of the human genome. Nature 409, 860921.[Medline]
Goff, S. P. (1996). Operating under Gag order: a block against incoming virus by the Fv1 gene. Cell 86, 691-693.[Medline]
Hartley, J. W. & Rowe, W. P. (1975). Clonal cell lines from a feral mouse embryo which lack host-range restrictions for murine leukemia viruses. Virology 65, 128-134.[Medline]
Jespersen, T., Duch, M., Carrasco, M. L., Warming, S. & Pedersen, F. S. (1999). Expression of heterologous genes from an IRES trans lational cassette in replication competent murine leukemia virus vectors. Gene 239, 227-235.[Medline]
Kozak, C. A. & Chakraborti, A. (1996). Single amino acid changes in the murine leukemia virus capsid protein gene define the target of Fv1 resistance. Virology 225, 300-305.[Medline]
Lilly, F. (1970). Fv2: identification and location of a second gene governing the spleen focus response to Friend leukemia virus in mice. Journal of the National Cancer Institute 45, 163-169.[Medline]
Lovmand, J., Lund, A. H. & Pedersen, F. S. (1998). Growth and purification of murine leukemia virus. In Cell Biology: A Laboratory Handbook , pp. 528-533. Edited by E. J. Celis. London:Academic Press.
Ponten, J. & Saksela, E. (1967). Two established in vitro cell lines from human mesenchymal tumours. International Journal of Cancer 2, 434-447.
Pryciak, P. M. & Varmus, H. E. (1992). Fv1 restriction and its effects on murine leukemia virus integration in vivo and in vitro. Journal of Virology 66, 5959-5966.[Abstract]
Qi, C.-F., Bonhomme, F., Buckler-White, A., Buckler, C., Orth, A., Lander, M. R., Chattopadhyay, S. K. & Morse, H. C.III (1998). Molecular phylogeny of Fv1. Mammalian Genome 9, 1049-1055.[Medline]
Sitbon, M., Nishio, J., Wehrly, K., Lodmell, D. & Chesebro, B. (1985). Use of a focal immunofluorescence assay on live cells for quantification of retroviruses: distinction of host range classes in virus mixtures and biological cloning of dual-tropic murine leukemia viruses. Virology 141, 110-118.[Medline]
Thomas, C. Y., Nucklos, J. D., Murphy, C. & Innes, D. (1993). Generation and pathogenicity of an NB-tropic SL3-3 murine leukemia virus. Virology 193, 1013-1017.[Medline]
Towers, G., Bock, M., Martin, S., Takeuchi, Y., Stoye, J. P. & Danos, O. (2000). A conserved mechanism of retrovirus restriction in mammals. Proceedings of the National Academy of Sciences, USA 97, 12295-12299.
Received 29 August 2001;
accepted 25 October 2001.