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Journal of Virology, July 2001, p. 6440-6449, Vol. 75, No. 14
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.14.6440-6449.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Role of the 3'-Untranslated Regions of Alfalfa Mosaic Virus RNAs in the Formation of a Transiently Expressed Replicase in Plants and in the Assembly of Virions

A. Corina Vlot, Lyda Neeleman, Huub J. M. Linthorst, and John F. Bol*

Institute of Molecular Plant Sciences, Gorlaeus Laboratories, Leiden University, Leiden, The Netherlands

Received 18 January 2001/Accepted 16 April 2001

Alfalfa mosaic virus (AMV) RNAs 1 and 2 encode the replicase proteins P1 and P2, respectively, whereas RNA 3 encodes the movement protein and the coat protein (CP). When RNAs 1 and 2 were transiently expressed from a T-DNA vector (R12 construct) by agroinfiltration of Nicotiana benthamiana, the infiltrated leaves accumulated minus-strand RNAs 1 and 2 and relatively small amounts of plus-strand RNAs. In addition, RNA-dependent RNA polymerase (RdRp) activity could be detected in extracts of the infiltrated leaves. After transient expression of RNAs 1 and 2 with the 3'-untranslated regions (UTRs) of both RNAs deleted (R1Delta /2Delta construct), no replication of RNAs 1 and 2 was observed, while the infiltrated leaves supported replication of RNA 3 after inoculation of the leaves with RNA 3 or expression of RNA 3 from a T-DNA vector (R3 construct). No RdRp activity could be isolated from leaves infiltrated with the R1Delta /2Delta construct, although P1 and P2 sedimented in a region of a glycerol gradient where active RdRp was found in plants infiltrated with R12. RdRp activity could be isolated from leaves infiltrated with constructs R1Delta /2 (3'-UTR of RNA 1 deleted), R1/2Delta (3'-UTR of RNA 2 deleted), or R1Delta /2Delta plus R3. This demonstrates that the 3'-UTR of AMV RNAs is required for the formation of a complex with in vitro enzyme activity. RNAs 1 and 2 with the 3'-UTRs deleted were encapsidated into virions by CP expressed from RNA 3. This shows that the high-affinity binding site for CP at the 3'-termini of AMV RNAs is not required for assembly of virus particles.

* Corresponding author. Mailing address: Institute of Molecular Plant Sciences, Gorlaeus Laboratories, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands. Phone: (31) 71-5274749. Fax: (31) 71-5274469. E-mail: j.bol{at}chem.leidenuniv.nl.

Journal of Virology, July 2001, p. 6440-6449, Vol. 75, No. 14
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.14.6440-6449.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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