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
Ac. Vlot et al., 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, J VIROLOGY, 75(14), 2001, pp. 6440-6449
Alfalfa mosaic virus (AMV) RNAs 1 and 2 encode the replicase proteins P1 an
d 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 ve
ctor (R12 construct) by agroinfiltration of Nicotinana benthamiana, the inf
iltrated leaves accumulated minus-strand RNAs 1 and 2 and relatively small
amounts of plus strand RNAs. In addition, RNA-dependent RNA polymerase (RdR
p) activity could be detected in extracts of the infiltrated leaves. After
transient expression of RNAs 1 and 2 with the 3 ' -untranslated regions (UT
Rs) of both RNAs deleted (R1 Delta /2 Delta construct), no replication of R
NAs 1 and 2 was observed, while the infiltrated leaves supported replicatio
n 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 fr
om 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 fou
nd in plants infiltrated with R12. RdRp activity could be isolated from lea
ves 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 de
monstrates 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 del
eted 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.