The 5′ Untranslated Region of Alfalfa Mosaic Virus RNA 1 Is Involved in Negative-Strand RNA Synthesis

Abstract

The three genomic RNAs of alfalfa mosaic virus each contain a unique 5 untranslated region (5 UTR). Replacement of the 5 UTR of RNA 1 by that of RNA 2 or 3 yielded infectious replicons. The sequence of a putative 5 stem-loop structure in RNA 1 was found to be required for negative-strand RNA synthesis. A similar putative 5 stem-loop structure is present in RNA 2 but not in RNA 3.Alfalfa mosaic virus (AMV) is a positive-strand tripartite RNA virus belonging to the family Bromoviridae (reviewed in reference 2). RNAs 1 and 2 encode the replicase proteins P1 and P2, respectively. P1 contains a methyltransferase-like domain in its N-terminal half, as well as a helicase-like domain in its C-terminal half (6,18). Residues in P1 that are highly conserved among alphaviral methyltransferase proteins or superfamily I helicases are required for AMV RNA replication (29,30). P2 is the viral RNA-dependent RNA polymerase (RdRp) protein (16). RNA 3 encodes the movement protein P3, as well as the coat protein, CP, which is translated from subgenomic RNA 4. The three genomic RNAs of AMV are not infectious unless either CP or CP-mRNA 4 is present in the inoculum (2). It has been found that addition of a poly(A) tail to the 3Ј ends of the AMV RNAs partially replaces the requirement for CP in the inoculum (13). Moreover, binding of CP to the 3Ј untranslated regions (UTRs) of RNAs 3 and 4 strongly enhanced translation of these RNAs (13; L. Neeleman, H. J. M. Linthorst, and J. F. Bol, submitted for publication).AMV RNAs are flanked by 5Ј and 3Ј UTRs that are highly structured and presumed to contain cis-acting elements involved in translation and replication (14,15,22,23,24,25,27,28). The 3Ј UTRs of the three RNAs consist of a common 3Ј-terminal sequence of 145 nucleotides (nt) preceded by 18 to 34 nt that are unique for each RNA. It has been shown that RNA 3 with its 3Ј UTR replaced with that of either RNA 1 or 2 is replicated in P12 plants at the level of wild-type (wt) RNA 3 (25). P12 plants express P1 and P2 from nuclear transgenes and support replication of RNA 3 and synthesis of RNA 4 (21). In AMV strain 425, the 5Ј UTRs of RNAs 1 and 2 consist of 100 and 54 nt, respectively, while the 5Ј UTR of RNA 3 consists of 345 nt that are predicted to fold into a complex structure (24). The 5Ј UTR of RNA 3 contains several 27-nt repeats that encompass ICR 2-like motifs (24). These motifs are homologous to box B elements, which are present in brome mosaic virus (BMV) RNAs and mediate recruitment of the BMV RNAs to viral replication complexes (3,19,20,24). The 5Ј UTRs of AMV RNAs 1 and 2 do not contain recognizable ICR 2-like motifs. However, the 5Ј-terminal 11 nt of RNAs 1 and 2 are identical, and the 5Ј UTRs of both RNAs contain a predicted 5Ј-terminal stem-loop structure consisting of a 12-bp stem and a 4-nt loop (M fold) ( Fig. 1A and B). Interestingly, replication of RNA 3 with its 5Ј UTR replaced with that of either RNA 1 or 2 was not supported in P12 plants (27).Replication of AMV RNA 1 carrying the 5 UTR of either RNA 2 or 3. In the ...

Crucial Role of the 5′ Conserved Structure ofBamboo Mosaic VirusSatellite RNA in Downregulation of Helper Viral RNA Replication

“…The conserved RNA structures also commonly occur in the 5Ј UTR, or even within coding regions (8,35), involved in RNA replication (5,7,32,36,37,50,52,54,58), translation (28,30,58), virion assembly (7), adaption (5), virulence (15), or even small interfering RNA-or microRNA-mediated destruction (34). The HV region in the 5Ј UTR of satBaMV isolates identified phylogenetic groups (57) whose divergence may be a result of independent evolution in a distinct host-helper-satBaMV relationship.…”

Repression and Derepression of Minus-Strand Synthesis in a Plus-Strand RNA Virus Replicon

“…Our present results also suggest the involvement of the satC 5ЈGG in initiation of minus-strand synthesis in vitro. Published studies using a variety of viral genomic RNA and subviral RNA replicons also support a role for the 5Ј end in minus-strand synthesis, through either protein-protein bridges, direct RNA-RNA interactions, or proposed 5Ј stabilization of 3Ј elements (4,14,23,31,61,66,69). Deletion of the satC 5ЈGG resulted in the synthesis of many additional products in the absence of any other alterations.…”

“…3Ј-proximal sequences comprising diverse forms such as tRNA-like structures, poly(A) tails, pseudoknots, hairpins, and short primary sequences without high-order structures have been identified as core promoters for minus-strand synthesis (11). Involvement of 5Ј-proximal sequences and structural elements in minusstrand synthesis has also been found for an increasing number of viruses (4,14,23,31,61,66,69). In addition, since plus strands cannot be templates for further translation during active replication (15), viral RNAs must contain the necessary information to switch between replication and translation as well as to support asymmetric synthesis of plus and minus strands, requirements that may include transcriptional enhancers (36,39,42,43) and repressors that function via RNA-RNA (39) or protein-RNA (11,73) interactions.…”

Structural Properties of a Multifunctional T-Shaped RNA Domain That Mediate Efficient Tomato Bushy Stunt Virus RNA Replication

“…For instance, targeting of Brome mosaic virus RNA2 to replication complexes requires a hairpin in its 5Ј UTR (4). In Poliovirus (1,11), Sindbis virus (7), and Alfalfa mosaic virus (26), 5Ј UTR elements promote negative-strand synthesis, and for Potato virus X (14), an RNA structure in its 5Ј UTR facilitates positive-strand synthesis. For TBSV, analyses of positive-and negative-strand DI RNA accumulation levels in this and other studies have not revealed any strand-specific functions for the TSD, SL5, or the DSD (23).…”