The 3' untranslated region of alfalfa mosaic virus RNA3 contains a core promoter for minus-strand RNA synthesis and an enhancer element.

Defective interfering (DI) RNAs of Tomato bushy stunt virus (TBSV), a plus-sense RNA virus, comprise four conserved noncontiguous regions (I through IV) derived from the viral genome. Region III, a 70-nucleotidelong sequence corresponding to a genomic segment located 378 nucleotides upstream of the 3 terminus of the genome, has been found to enhance DI RNA accumulation by approximately 10-fold in an orientationindependent manner (D. Ray and K. A. White, Virology 256:162-171, 1999). In this study, a more detailed structure-function analysis of region III was conducted. RNA secondary-structure analyses indicated that region III contains stem-loop structures in both plus and minus strands. Through deletion analyses of a DI RNA, a primary determinant of region III activity was mapped to the 5-proximal 35-nucleotide segment. Compensatory-type mutational analyses showed that a stem-loop structure in the minus strand of this subregion was required for enhanced DI RNA replication. The same stem-loop structure was also found to function in a position-independent manner in a DI RNA (albeit at reduced levels) and to be important for efficient accumulation within the context of the TBSV genome. Taken together, these observations suggest that the 5-proximal segment of region III is a modular RNA replication element that functions primarily through the formation of an RNA hairpin structure in the minus strand.Genome replication represents a fundamental step in virus reproduction. In plus-sense RNA viruses, this is a two-step process whereby minus strands are synthesized from the genome and are used subsequently as templates for the synthesis of progeny genomes. This process is asymmetrical, with viral plus strands accumulating to approximately 10-to 1,000-fold excess over minus strands (4).Both cis-acting RNA elements and trans-acting factors (i.e., viral RNA-dependent RNA polymerase [RdRp] and/or host factors) are required for viral genome replication (4). Usually, cis-acting replication elements reside in 5Ј-and 3Ј-terminal untranslated regions of genomes (4,8,9), however, several internally positioned cis-acting elements have also been identified. These include RNA replication signals that are located in (i) coding regions of rhinovirus type 14 (25), poliovirus (12) and bacteriophage Q␤ (22) Tomato bushy stunt virus (TBSV), the type member of the genus Tombusvirus (33), is a plus-sense single-stranded RNA plant virus whose ϳ4.8-kb genome encodes five proteins with defined functions (Fig. 1) (18,28,31,34,35,37). Prototypical DI RNAs associated with TBSV comprise four regions derived entirely from the genome (Fig. 1). Based on their correspondence to the genomic 5Ј and 3Ј termini and mutational analysis in vivo, regions I and IV likely contain cis-acting RNA elements important for initiation of RNA synthesis (5,13,14,40,42). This concept is consistent with in vitro studies that have identified core promoter elements necessary for plus and minus strand synthesis within regions I and IV, respectively (27,29). Region II, whic...

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confidence: 99%

An Internally Located RNA Hairpin Enhances Replication of Tomato Bushy Stunt Virus RNAs

Ray

White

2003

“…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.…”

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confidence: 99%

“…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.…”

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confidence: 99%

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

Vlot

Bol

2003

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 ...

“…These RNA replication enhancers, which can be found in both plus and minus strands of viral RNAs, are located at variable positions in relation to the initiation site for RNA transcription and are thought to facilitate the recruitment of RdRp to the template (17). Replication enhancers have been characterized for a number of RNA viruses such as bacteriophage Q␤ (2), Alfalfa mosaic virus (46), Tomato bushy stunt virus (37), Brome mosaic virus (10,35,36), and Sindbis virus (11).…”

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confidence: 99%

Fitness of a Turnip Crinkle Virus Satellite RNA Correlates with a Sequence-Nonspecific Hairpin and Flanking Sequences That Enhance Replication and Repress the Accumulation of Virions

Sun

Simon

2003

Most plant viruses possess plus-sense single-stranded RNA genomes. Replication of these genomes is mediated by a replicase complex that in general comprises a virus-encoded RNA-dependent RNA polymerase (RdRp), other virally encoded proteins, and possibly host factors (17). The replicase must recognize its cognate RNA through direct or indirect interaction with specific cis-acting elements in the RNA template (4, 9), which for Brome mosaic virus are functional at various locations relative to the minus-strand initiation site (36). Replicases can accurately identify the correct start site for transcription of the genomic RNA, even when the site is artificially placed distal to the 3Ј end of the template (24,40,43). In addition, many viral RdRps synthesize 3Ј-coterminal subgenomic RNAs by using internal promoters located on minusstrand replication intermediates (18, 25, 48) that do not necessarily contain sequence or structural similarity to promoters that mediate full-length complementary-strand synthesis. Some plant viruses also provide the replication machinery for associated satellite RNAs (satRNAs), most of which share little sequence similarity with the viral genomic RNA (38). Thus, a single viral replicase must recognize a variety of seemingly different promoter elements.In addition to core promoter elements, many RNA virus genomes contain additional cis-acting elements that enhance but are not required for basal level transcription (29, 37). These RNA replication enhancers, which can be found in both plus and minus strands of viral RNAs, are located at variable positions in relation to the initiation site for RNA transcription and are thought to facilitate the recruitment of RdRp to the template (17). Replication enhancers have been characterized for a number of RNA viruses such as bacteriophage Q␤ (2), Alfalfa mosaic virus (46), Tomato bushy stunt virus (37), Brome mosaic virus (10,35,36), and Sindbis virus (11).Since most satRNAs associated with plant viruses are fewer than 500 bases in size and do not encode proteins, they are ideal templates for examining the structure and function of cis replication elements. The Turnip crinkle virus (TCV) (family Tombusviridae, genus Carmovirus) isolate TCV-M, a singlestranded RNA virus of 4,054 bases, was originally found to be associated with three related satRNAs, including satD (194 bases) and satC (356 bases) (1,22,39). satD appears to have originated from numerous short noncontiguous stretches of TCV genomic RNA sequence (5), while satC is a chimeric molecule containing nearly full-length satD at its 5Ј end and two discontinuous segments from TCV genomic RNA at its 3Ј end (Fig. 1A) (39). satC is also an unusually virulent satRNA, strongly intensifying the symptoms of TCV on hosts that display discernible symptoms when infected with TCV alone (20).Most satRNAs are considered to be parasitic agents, selfishly coopting the replication machinery of their helper virus without conferring any obvious advantage to the virus. satC, by interfering with the replication of ...