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

doi:10.1128/JVI.74.16.7422-7430.2000

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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, 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 leukemiavirus target, we set out to develop rapid, transient assays forFv1 function. Cells were transfected or transduced with Fv1 expressionplasmids which can produce green fluorescent protein via an internalribosome entry site positioned between the Fv1 and green fluorescentprotein coding sequences. Fv1 function was then assessed by comparingvirus replication in green fluorescent protein-positive and -negativecells, using retroviral vectors encoding a second fluorescentmarker, yellow fluorescent protein, or $beta$ -galactosidase. Usingthis assay, we could show that Fv1 specificities were not as absoluteas previously thought, since the Fv1^b allele was capable of interacting with "nonrestricted" B- andNB-tropic viruses and by shuffling the n- and b-alleles of Fv1,it was possible to generate a Fv1 molecule capable of restrictingN-, B-, and NB-tropic viruses equally efficiently. Further, wecould show that the presence of nonrestricting Fv1 in the samecell as restrictive Fv1 abrogates restriction, implying competitionfor binding to the retroviraltarget.

^* 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.

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

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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