Comparison of the Role of 5′ Terminal Sequences of Alfalfa Mosaic Virus RNAs 1, 2, and 3 in Viral RNA Replication

Rossum, C. M. A. van; Neeleman, Lyda; Bol, John F.

doi:10.1006/viro.1997.8707

Cited by 12 publications

(15 citation statements)

References 37 publications

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“…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 present study, the 5Ј UTR of RNA 1 (L1) was replaced with the 5Ј UTR of RNA 2 (L2) or RNA 3 (L3).…”

mentioning

confidence: 97%

“…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).…”

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confidence: 97%

“…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.…”

mentioning

confidence: 99%

“…To that end, sequences containing L2 or L3 downstream of a 35S promoter were transferred to pBSR1 from pCA27T-Nco or pCA35T-Nco using KpnI and NcoI. pCA27T-Nco and pCa35T-Nco contain DNA copies of RNAs 2 and 3, respectively (26,27). "Nco" refers to an NcoI site engineered into the cDNA across the position of the start codon of either P2 or P3.…”

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confidence: 99%

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

Vlot

Bol

2003

J Virol

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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 ...

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mentioning

confidence: 97%

“…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).…”

mentioning

confidence: 97%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Vlot

Bol

2003

J Virol

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Cis-Acting Functions of Alfalfa Mosaic Virus Proteins Involved in Replication and Encapsidation of Viral RNA

Neeleman

Bol

1999

Virology

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cDNA clones of RNAs 1 and 2 of alfalfa mosaic virus (AMV) were slightly modified to permit transcription of infectious RNAs with T7 RNA polymerase. Together with transcripts of an available clone of AMV RNA 3, these transcripts were used to study cis- and trans-acting functions of AMV proteins in protoplasts from nontransgenic tobacco plants and from plants transformed with the P1 and P2 genes, encoded by RNAs 1 and 2, respectively. Transgenic P1 was unable to complement mutations in the P1 gene in RNA 1, pointing to a cis-acting function of P1 in RNA 1 replication. A study of the replication of RNA 3 mutants in nontransgenic protoplasts revealed that coat protein (CP) expressed from RNA 3 in the inoculum is required in trans for replication and encapsidation of RNAs 1 and 2 but is required in cis for replication and encapsidation of RNA 3. CP is required in the inoculum to initiate infection of nontransgenic plants and protoplasts. When protoplasts expressing both P1 and P2 (P12 protoplasts) were infected with RNAs 1, 2, and 3, initiation of replication of RNAs 1 and 2 required the presence of CP in the inoculum, whereas the initiation of replication of RNA 3 did not. This demonstrated that CP expressed from RNA 3 cannot substitute for the early function of CP in the inoculum. The results showed that CP in the inoculum is required to permit viral minus-strand RNA synthesis, whereas CP expressed from RNA 3 after the initiation of infection is required for plus-strand RNA synthesis.

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Analysis of cis-Acting Sequences Involved in Plus-Strand Synthesis of a Turnip Crinkle Virus-Associated Satellite RNA Identifies a New Carmovirus Replication Element

2000

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Satellite RNA C (satC) is a 356-base subviral RNA associated with turnip crinkle virus (TCV). A 3'-proximal element (3'-UCCCAAAGUAU) located 11 bases from the 3' terminus of satC minus strands can function as an independent promoter in an in vitro RNA-dependent RNA polymerase (RdRp) transcription system. Furthermore, in the absence of a 5'-proximal element, the 3'-proximal element is required for complementary strand synthesis in vitro. Site-directed mutagenesis was conducted to investigate the functional significance of this element and the 3' minus-strand terminal sequence "3'-OH-CCCUAU," which contains the minus-strand 3'-end sequence "3'-OH-CC(1-2)(A/U)(A/U)(A/U)" found in all carmovirus RNAs. Single mutations in the 3'-terminal sequence, which we have named the carmovirus consensus sequence (CCS), suppressed satC plus-strand synthesis to undetectable levels in protoplasts while still permitting some minus-strand synthesis. However, single and multiple mutations introduced into the 3'-proximal element had little or no effect on satC accumulation in protoplasts. In vivo genetic selection (SELEX) of the minus-strand 3'-terminal 21 bases revealed that all satC species accumulating in plants contained the 3' CCS. In addition, the 3'-proximal element preferentially contained a sequence similar to the CCS and/or polypurines, suggesting that this element may also contribute to accumulation of satC in vivo.

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Comparison of the Role of 5′ Terminal Sequences of Alfalfa Mosaic Virus RNAs 1, 2, and 3 in Viral RNA Replication

Cited by 12 publications

References 37 publications

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

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

Cis-Acting Functions of Alfalfa Mosaic Virus Proteins Involved in Replication and Encapsidation of Viral RNA

Analysis of cis-Acting Sequences Involved in Plus-Strand Synthesis of a Turnip Crinkle Virus-Associated Satellite RNA Identifies a New Carmovirus Replication Element

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