The 3h untranslated regions (UTRs) of the three genomic RNAs of alfalfa mosaic virus consist of a 3h homologous sequence of 145 nt and upstream unique sequences 18-34 nt in length. Mutations were made in the 3h UTR of a cDNA clone of RNA3. Point mutations in five AUGC motifs which interfere with specific binding of coat protein to the 3h UTR had no effect on template activity of RNA3 for minus-strand RNA synthesis in vitro by purified viral RNA-dependent RNA polymerase (RdRp). Deletion analysis showed that the 3h homologous sequence of 145 nt was sufficient for a low level of template activity in the in vitro RdRp assay and a similarly low level of RNA3 accumulation in plants. The presence of an additional sequence of nucleotides 145-165 from the 3h end of RNA3 enhanced template recognition by RdRp in vitro and accumulation of RNA3 in vivo to wild-type levels.Alfalfa mosaic virus (AMV) has a tripartite single-stranded RNA genome of messenger-sense polarity. RNAs 1 and 2 encode proteins P1 and P2, which are part of the viral replicase. RNA3 encodes the viral movement protein, P3, and the coat protein (CP), which is translated from a subgenomic messenger RNA, RNA4. The 3h untranslated regions (UTRs) of the AMV RNAs comprise a 3h sequence of 145 nt that is 80 % homologous in the three RNAs and upstream unique sequences of 18 (RNA1), 21 (RNA2) or 34 nt (RNA3). The 3h UTRs can be folded into a number of stem-loop structures that are flanked by AUGC motifs and contain specific binding sites for viral CP. Previously, we have shown that the 3h UTR of RNA3 contains a minimum of two independent binding sites for CP, site I being located in the homologous sequence of 145 nt and site II requiring at least part of the region that is unique to the Author for correspondence : John Bol.Fax j31 71 527 4469. e-mail J.BOL!chem.LeidenUniv.nl 3h UTR of RNA3 (Reusken et al., 1994). A CP binding site in the 3h-terminal 39 nt of RNA3 has been analysed in most detail (Houser-Scott et al., 1994Reusken & Bol, 1996). Binding of CP to the three genomic AMV RNAs is required to initiate infection (Bol et al., 1971 ;Smit et al., 1981).In addition to binding sites for CP, the 3h UTRs of AMV RNAs contain cis-acting sequences involved in template activity for minus-strand RNA synthesis by purified AMV RNA-dependent RNA polymerase (RdRp) in vitro and replication of the RNAs in vivo. Previously, we have shown that the full-length 3h UTRs of AMV RNAs 1, 2 and 3 are sufficient for wild-type (wt) levels of in vitro template activity and in vivo replication of the RNAs, whereas the 3h 127 nt are not (van Rossum et al., 1997). Here, we have further analysed sequences in the 3h UTR of RNA3 required for in vitro and in vivo RNA synthesis.Except for plasmids pTE208 and pBRAC35, the construction of all plasmids, shown schematically in Fig. 1 (a) and Fig. 2 (a), has been described previously (Reusken et al., 1994). Plasmid pBRWT contains an insert consisting of the T7 promoter, a pBR-derived sequence of 783 nt (thin line in Figs 1 a and 2 a) and a sequence ...

The 5' untranslated regions (UTRs) of the genomic RNAs 1, 2, and 3 of alfalfa mosaic virus (AMV) are 100, 54, and 345 nucleotides (nt) long, respectively, and lack extensive sequence similarity to each other. RNA 3 encodes the movement protein P3 and the coat protein and can be replicated in transgenic tobacco plants expressing the replicase proteins P1 and P2 (P12 plants). 5' Cis-acting sequences involved in RNA 3 replication have been shown to be confined to the 5' UTR. When the 5' UTR of RNA 3 was replaced by the 5' UTRs of RNAs 1 or 2, the recombinant RNA was not infectious to P12 plants. Also, when the P3 gene in RNA 3 was put under the control of a subgenomic promoter and the 5' UTR of this RNA was replaced by 5' terminal RNA 1 sequences of 103 to 860 nt long or RNA 2 sequences of 57 to 612 nt long, no accumulation of the hybrid RNAs was observed. Deletion of the 5' 22 nucleotides of RNA 3 resulted in the accumulation of a major progeny that lacked the 5' 79 nt. However, when the 5' 22 nucleotides of RNA 3 were replaced by the complete 5' UTR of RNA 1 or 5' sequences of RNAs 1, 2, or 3 with a length of 5 to 15 nt, accumulation of the full-length mutant RNAs was observed. The effect of mutations in the 5' viral sequences of 5 to 15 nt was analyzed. It is concluded that although elements within nucleotides 80-345 of the 5' UTR of RNA 3 are sufficient for replication, a specific sequence of 3 to 5 nt is required to target the replicase to an initiation site corresponding to the 5' end of the RNA.

The genome of Alfalfa mosaic virus (AMV) is divided among three plus-strand RNAs which are separately encapsidated. The RNA 1-encoded P1 protein with putative methyltransferase and helicase activity and the RNA 2-encoded P2 protein containing the GDD motif of viral plus-strand RNA polymerases have been identified as subunits of the purified AMV RNA-dependent RNA polymerase complex (RdRp ; Quadt et al., 1991). The P3 protein encoded by RNA 3 is involved in virus movement, and the subgenomic RNA 4, which is synthesized from the 3' half of minus-strand RNA 3,

The 3 untranslated regions (UTRs) of alfalfa mosaic virus (AMV) RNAs 1, 2, and 3 consist of a common 3-terminal sequence of 145 nucleotides (nt) and upstream sequences of 18 to 34 nt that are unique for each RNA. The common sequence can be folded into five stem-loop structures, A to E, despite the occurrence of 22 nt differences between the three RNAs in this region. Exchange of the common sequences or full-length UTRs between the three genomic RNAs did not affect the replication of these RNAs in vivo, indicating that the UTRs are functionally equivalent. Mutations that disturbed base pairing in the stem of hairpin E reduced or abolished RNA replication, whereas compensating mutations restored RNA replication. In vitro, the 3 UTRs of the three RNAs were recognized with similar efficiencies by the AMV RNA-dependent RNA polymerase (RdRp). A deletion analysis of template RNAs indicated that a 3-terminal sequence of 127 nt in each of the three AMV RNAs was not sufficient for recognition by the RdRp. Previously, it has been shown that this 127-nt sequence is sufficient for coat protein binding. Apparently, sequences required for recognition of AMV RNAs by the RdRp are longer than sequences required for CP binding.

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