Early and Late Functions of Alfalfa Mosaic Virus Coat Protein Can Be Mutated Separately

Vossen, E.A.G. van der; Neeleman, Lyda; Bol, John F.

doi:10.1006/viro.1994.1411

Cited by 90 publications

(89 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the 3h UTR of RNA3 upstream elements that are unique to RNA3 are required to enhance the activity of this ' core ' promoter to wt levels. Mutant 3∆KB was found to accumulate at wt levels in P12 plants (van der Vossen et al, 1994) and showed wt template activity in the in vitro RdRp assay (this study). This shows that the sequence of nucleotides 145-165 from the 3h end of RNA3 is sufficient to enhance activity of the core promoter to give wt levels.…”

Section: M a Van Rossum And Others C M A Van Rossum And Othersmentioning

confidence: 51%

“…Previously, we showed that mutations in motifs 2 and 3 affected CP binding to site I in the 3h UTR of RNA3 in vitro whereas mutations in motifs 4 and 5 affected CP binding to site II (Reusken et al, 1994 ;Reusken & Bol, 1996). In addition, we observed that replacing AUGC motif 2 or 3 by AGGC reduced or abolished accumulation of RNA3 in plants, respectively (van der Vossen et al, 1994). Apparently, the defect in these mutants in accumulation in vivo correlates with a defect in CP binding to RNA3 rather than with a defect in RdRp recognition in the in vitro assays.…”

Section: M a Van Rossum And Others C M A Van Rossum And Othersmentioning

confidence: 74%

“…accumulation in vivo (van der Vossen et al, 1994). Here, we tested the effect of these mutations on template activity in the in vitro RdRp assay.…”

Section: M a Van Rossum And Others C M A Van Rossum And Othersmentioning

confidence: 99%

“…It has been shown by van der Vossen et al (1994) that mutant 3∆KB accumulated at wt levels in transgenic tobacco plants transformed with the AMV P1 and P2 genes (P12 plants) whereas no accumulation of mutants 3∆BD and 3∆D in these plants was observed. Here, we analysed the accumulation of mutants 3∆KK and 3∆KK-C in P12 plants.…”

Section: M a Van Rossum And Others C M A Van Rossum And Othersmentioning

confidence: 99%

“…3 (a) were produced in pAL3, a full-length infectious clone of AMV RNA3. The introduction of a KpnI site downstream of the UGA stop codon of the CP gene (mutant p3Kpn) and deletion of the KpnI-Bst XI fragment [p3∆KB, deletion (∆) of nucleotides 166-178 from the 3h end], the Bst XI-DraIII fragment (p3∆BD, ∆128-169) and 3 nt at the DraIII site (p3∆D, ∆128-130) have been described previously (van der Vossen et al, 1994). Deletion of the KpnI-DraIII fragment (p3∆KD, ∆128-178) was described by van Rossum et al (1997).…”

mentioning

confidence: 99%

See 4 more Smart Citations

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

Rossum

Reusken²,

Brederode³

et al. 1997

Journal of General Virology

View full text Add to dashboard Cite

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 ...

show abstract

Section: M a Van Rossum And Others C M A Van Rossum And Othersmentioning

confidence: 51%

Section: M a Van Rossum And Others C M A Van Rossum And Othersmentioning

confidence: 74%

“…accumulation in vivo (van der Vossen et al, 1994). Here, we tested the effect of these mutations on template activity in the in vitro RdRp assay.…”

Section: M a Van Rossum And Others C M A Van Rossum And Othersmentioning

confidence: 99%

Section: M a Van Rossum And Others C M A Van Rossum And Othersmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

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

Rossum

Reusken²,

Brederode³

et al. 1997

Journal of General Virology

View full text Add to dashboard Cite

show abstract

A plant viral coat protein RNA binding consensus sequence contains a crucial arginine.

et al. 1996

View full text Add to dashboard Cite

A defining feature of alfalfa mosaic virus (AMV) and ilarviruses [type virus: tobacco streak virus (TSV)] is that, in addition to genomic RNAs, viral coat protein is required to establish infection in plants. AMV and TSV coat proteins, which share little primary amino acid sequence identity, are functionally interchangeable in RNA binding and initiation of infection. The lysinerich amino-terminal RNA binding domain of the AMV coat protein lacks previously identified RNA binding motifs. Here, the AMV coat protein RNA binding domain is shown to contain a single arginine whose specific side chain and position are crucial for RNA binding. In addition, the putative RNA binding domain of two ilarvirus coat proteins, TSV and citrus variegation virus, is identified and also shown to contain a crucial arginine. AMV and ilarvirus coat protein sequence alignment centering on the key arginine revealed a new RNA binding consensus sequence. This consensus may explain in part why heterologous viral RNA-coat protein mixtures are infectious.

show abstract

Base‐pairing potential identified by in vitro selection predicts the kinked RNA backbone observed in the crystal structure of the alfalfa mosaic virus RNA‐coat protein complex

Boyce

Scott

Guogas

et al. 2005

J of Molecular Recognition

View full text Add to dashboard Cite

The three-dimensional structure of the 3' terminus of alfalfa mosaic virus RNA in complex with an amino-terminal coat protein peptide revealed an unusual RNA fold with inter-AUGC basepairing stabilized by key arginine residues (Guogas, et al., 2004). To probe viral RNA interactions with the full-length coat protein, we have used in vitro genetic selection to characterize potential folding patterns among RNAs isolated from a complex randomized pool. Nitrocellulose filter retention, electrophoretic mobility bandshift analysis, and hydroxyl radical footprinting techniques were used to define binding affinities and to localize the potential RNA-protein interaction sites. Minimized binding sites were identified that included both the randomized domain and a portion of the constant regions of the selected RNAs. The selected RNAs, identified by their ability to bind full-length coat protein, have the potential to form the same unusual inter-AUGC Watson-Crick base pairs observed in the crystal structure, although the primary sequences diverge from the wild-type RNA. A constant feature of both the wild-type RNA and the selected RNAs is a G ribonucleotide in the third position of an AUGC-like repeat. Competitive binding assays showed that substituting adenosine for the constant guanosine in either the wild-type or selected RNAs impaired coat protein binding. These data suggest that the interactions observed in the RNA-peptide structure are likely recapitulated when the full-length protein binds. Further, the results underscore the power of in vitro genetic selection for probing RNA-protein structure and function.

show abstract

Early and Late Functions of Alfalfa Mosaic Virus Coat Protein Can Be Mutated Separately

Cited by 90 publications

References 0 publications

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

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

A plant viral coat protein RNA binding consensus sequence contains a crucial arginine.

Base‐pairing potential identified by in vitro selection predicts the kinked RNA backbone observed in the crystal structure of the alfalfa mosaic virus RNA‐coat protein complex

Contact Info

Product

Resources

About