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

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 Nicotinana 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 (R1 Delta /2 Delta 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 R1 Delta /2 Delta 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 R1 Delta /2 (3 ' -UTR of RNA 1 deleted), R1/2 Delta (3 ' -UTR of RNA 2 deleted), or R1 Delta /2 Delta 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.