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Journal of Virology, August 2000, p. 7422-7430, Vol. 74, No. 16
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Use of a Transient Assay for Studying the Genetic Determinants of Fv1 Restriction

Michael Bock, Kate N. Bishop, Greg Towers,dagger and Jonathan P. Stoye*

Division of Virology, National Institute for Medical Research, London NW7 1AA, United Kingdom

Received 22 February 2000/Accepted 23 May 2000

To probe the genetic determinants controlling the interaction between the retroviral restriction gene Fv1 and its murine leukemia virus target, we set out to develop rapid, transient assays for Fv1 function. Cells were transfected or transduced with Fv1 expression plasmids which can produce green fluorescent protein via an internal ribosome entry site positioned between the Fv1 and green fluorescent protein coding sequences. Fv1 function was then assessed by comparing virus replication in green fluorescent protein-positive and -negative cells, using retroviral vectors encoding a second fluorescent marker, yellow fluorescent protein, or beta -galactosidase. Using this assay, we could show that Fv1 specificities were not as absolute as previously thought, since the Fv1b allele was capable of interacting with "nonrestricted" B- and NB-tropic viruses and by shuffling the n- and b-alleles of Fv1, it was possible to generate a Fv1 molecule capable of restricting N-, B-, and NB-tropic viruses equally efficiently. Further, we could show that the presence of nonrestricting Fv1 in the same cell as restrictive Fv1 abrogates restriction, implying competition for binding to the retroviral target.

* Corresponding author. Mailing address: Division of Virology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom. Phone: 44-208/959-3666, ext. 2140. Fax: 44-208/906-4477. E-mail: jstoye{at}nimr.mrc.ac.uk.

dagger Present address: Wohl Virion Centre, Windeyer Institute of Medical Sciences, University College London, London W1P 6DB, United Kingdom.

Journal of Virology, August 2000, p. 7422-7430, Vol. 74, No. 16
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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