scholarly journals Minimal Template Requirements for Initiation of Minus-Strand Synthesis In Vitro by the RNA-Dependent RNA Polymerase of Turnip Yellow Mosaic Virus

1998 ◽  
Vol 72 (5) ◽  
pp. 3965-3972 ◽  
Author(s):  
B. A. L. M. Deiman ◽  
A. K. Koenen ◽  
P. W. G. Verlaan ◽  
C. W. A. Pleij
Keyword(s):  
Rna Polymerase ◽  
Mosaic Virus ◽  
Mutational Analysis ◽  
Yellow Mosaic Virus ◽  
Turnip Yellow Mosaic Virus ◽  
Minus Strand ◽  
Rna Dependent Rna Polymerase ◽  
Substitution Mutations ◽  
Internal Initiation

ABSTRACT From mutational analysis of the 3′-terminal hairpin of turnip yellow mosaic virus (TYMV) RNA and use of nonstructured C-rich RNA templates, we conclude that the main determinant in the tRNA-like structure of TYMV RNA for initiation of minus-strand synthesis by the viral RNA-dependent RNA polymerase (RdRp) is the non-base-paired 3′ ACC(A) end. Base pairing of this 3′ end reduces the transcription efficiency drastically, and deletion of only the 3′-terminal A residue results in a fivefold drop in efficiency. The two C residues of the 3′ ACCA end are required for efficient transcription, as shown by substitution mutations. However, the 5′ A residue is not specifically involved in initiation of transcription, as shown by substitution mutations. Furthermore, the hairpin stem and loop upstream of the 3′ ACCA end also do not interact with the RdRp in a base-specific way. However, for efficient transcription, the hairpin stem should be at least five bp in length, while the calculated ΔG value should be less than −10.5 kcal/mol. Unexpectedly, the use of nonstructured C-rich RNA templates showed that the RdRp can start internally on an NCCN or NUCN sequence. Therefore, a possible function of the tRNA-like structure of TYMV RNA may be to prevent internal initiation of minus-strand synthesis.

scholarly journals Initiation of Genomic Plus-Strand RNA Synthesis from DNA and RNA Templates by a Viral RNA-Dependent RNA Polymerase

1999 ◽  
Vol 73 (8) ◽  
pp. 6415-6423 ◽  
Author(s):  
K. Sivakumaran ◽  
C. Cheng Kao
Keyword(s):  
Rna Polymerase ◽  
Mosaic Virus ◽  
Rna Synthesis ◽  
Mutational Analysis ◽  
Initiation Site ◽  
Brome Mosaic Virus ◽  
Minus Strand ◽  
Rna Dependent Rna Polymerase ◽  
Dna And Rna

ABSTRACT In contrast to the synthesis of minus-strand genomic and plus-strand subgenomic RNAs, the requirements for brome mosaic virus (BMV) genomic plus-strand RNA synthesis in vitro have not been previously reported. Therefore, little is known about the biochemical requirements for directing genomic plus-strand synthesis. Using DNA templates to characterize the requirements for RNA-dependent RNA polymerase template recognition, we found that initiation from the 3′ end of a template requires one nucleotide 3′ of the initiation nucleotide. The addition of a nontemplated nucleotide at the 3′ end of minus-strand BMV RNAs led to initiation of genomic plus-strand RNA in vitro. Genomic plus-strand initiation was specific since cucumber mosaic virus minus-strand RNA templates were unable to direct efficient synthesis under the same conditions. In addition, mutational analysis of the minus-strand template revealed that the −1 nontemplated nucleotide, along with the +1 cytidylate and +2 adenylate, is important for RNA-dependent RNA polymerase interaction. Furthermore, genomic plus-strand RNA synthesis is affected by sequences 5′ of the initiation site.

scholarly journals In Vitro Transcription by the Turnip Yellow Mosaic Virus RNA Polymerase: a Comparison with the Alfalfa Mosaic Virus and Brome Mosaic Virus Replicases

2000 ◽  
Vol 74 (1) ◽  
pp. 264-271 ◽  
Author(s):  
Birgit A. L. M. Deiman ◽  
Paul W. G. Verlaan ◽  
Cornelis W. A. Pleij
Keyword(s):  
Rna Polymerase ◽  
Mosaic Virus ◽  
Transcription Initiation ◽  
Alfalfa Mosaic Virus ◽  
Brome Mosaic Virus ◽  
Yellow Mosaic Virus ◽  
Turnip Yellow Mosaic Virus ◽  
Virus Rna ◽  
Internal Initiation

ABSTRACT Recently, we showed that the main determinant in the tRNA-like structure of turnip yellow mosaic virus RNA to initiate minus-strand synthesis in vitro is the 3′ ACCA end. By mutational analysis of the 3′-terminal hairpin, we show here that only a non-base-paired ACCA end is functional and that the stability of the wild-type 3′-proximal hairpin is the most favorable, in that it has the lowest ΔG value and a high transcription efficiency. With a nested set of RNA fragments, we show that the minimum template length is 9 nucleotides and that transcription is improved with increasing the length of the template. The results also suggest that proper base stacking contributes to efficient transcription initiation. Internal initiation is shown to take place on every NPyCPu sequence of a nonstructured template. However, the position of the internal initiation site in the template is important, and competition between the different sites takes place. Internal initiation was also studied with the RNA-dependent RNA polymerase of brome mosaic virus (BMV) and alfalfa mosaic virus (AlMV). The BMV polymerase can start internally on ACCA sequences, though inefficiently. Unexpectedly, the polymerases of both AlMV and BMV can start efficiently on an internal AUGC sequence.

scholarly journals The Ubiquitin-Proteasome System Regulates the Accumulation of Turnip yellow mosaic virus RNA-Dependent RNA Polymerase during Viral Infection

2010 ◽  
Vol 22 (9) ◽  
pp. 3142-3152 ◽  
Author(s):  
Laurent Camborde ◽  
Séverine Planchais ◽  
Vincent Tournier ◽  
Anna Jakubiec ◽  
Gabrièle Drugeon ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Viral Infection ◽  
Mosaic Virus ◽  
Ubiquitin Proteasome System ◽  
Yellow Mosaic Virus ◽  
Turnip Yellow Mosaic Virus ◽  
Rna Dependent Rna Polymerase ◽  
Virus Rna ◽  
Ubiquitin Proteasome

Evidence for phosphorylation and ubiquitinylation of the turnip yellow mosaic virus RNA-dependent RNA polymerase domain expressed in a baculovirus–insect cell system

2000 ◽  
Vol 349 (2) ◽  
pp. 417-425 ◽  
Author(s):  
François HÉRICOURT ◽  
Stéphane BLANC ◽  
Virginie REDEKER ◽  
Isabelle JUPIN
Keyword(s):  
Rna Polymerase ◽  
Mosaic Virus ◽  
Insect Cells ◽  
Recombinant Baculovirus ◽  
Yellow Mosaic Virus ◽  
Turnip Yellow Mosaic Virus ◽  
Rna Dependent Rna Polymerase ◽  
Native Form ◽  
Pest Sequence ◽  
Peptide Mass

All RNA viruses known to date encode an RNA-dependent RNA polymerase (RdRp) that is required for replication of the viral genome. We have expressed and purified the turnip yellow mosaic virus (TYMV) RdRp in insect cells using a recombinant baculovirus, either in its native form, or fused to an hexa-histidine tag. Phosphorylation of the protein was demonstrated by labelling experiments in vivo, as well as phosphatase treatment of the purified protein in vitro. Phospho amino acid analysis and immunoblotting experiments identified serine and threonine residues as being the subject of phosphorylation. Peptide mass mapping using MS analysis of a protein digest revealed that phosphorylation sites are localized within a putative PEST sequence [a sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues] in the N-terminal region of the protein. Using monoclonal antibodies specific for ubiquitin conjugates, we were able to demonstrate that the TYMV RdRp is conjugated to ubiquitin molecules when expressed in insect cells. These observations suggest that the TYMV RdRp may be processed selectively by the ubiquitin/proteasome degradation system upon phosphorylation of the PEST sequence.

scholarly journals Role of the 3′ tRNA-Like Structure in Tobacco Mosaic Virus Minus-Strand RNA Synthesis by the Viral RNA-Dependent RNA Polymerase In Vitro

2000 ◽  
Vol 74 (24) ◽  
pp. 11671-11680 ◽  
Author(s):  
T. A. M. Osman ◽  
C. L. Hemenway ◽  
K. W. Buck
Keyword(s):  
Rna Polymerase ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Untranslated Region ◽  
Rna Synthesis ◽  
Central Core ◽  
Minus Strand ◽  
Stem Loop ◽  
Polymerase Binding

ABSTRACT A template-dependent RNA polymerase has been used to determine the sequence elements in the 3′ untranslated region of tobacco mosaic virus RNA that are required for promotion of minus-strand RNA synthesis and binding to the RNA polymerase in vitro. Regions which were important for minus-strand synthesis were domain D1, which is equivalent to a tRNA acceptor arm; domain D2, which is similar to a tRNA anticodon arm; an upstream domain, D3; and a central core, C, which connects domains D1, D2, and D3 and determines their relative orientations. Mutational analysis of the 3′-terminal 4 nucleotides of domain D1 indicated the importance of the 3′-terminal CA sequence for minus-strand synthesis, with the sequence CCCA or GGCA giving the highest transcriptional efficiency. Several double-helical regions, but not their sequences, which are essential for forming pseudoknot and/or stem-loop structures in domains D1, D2, and D3 and the central core, C, were shown to be required for high template efficiency. Also important were a bulge sequence in the D2 stem-loop and, to a lesser extent, a loop sequence in a hairpin structure in domain D1. The sequence of the 3′ untranslated region upstream of domain D3 was not required for minus-strand synthesis. Template-RNA polymerase binding competition experiments showed that the highest-affinity RNA polymerase binding element region lay within a region comprising domain D2 and the central core, C, but domains D1 and D3 also bound to the RNA polymerase with lower affinity.

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