Efficient transcription obtained by a new procedure of the tRNA-like structure of turnip yellow mosaic virus by a template-dependent and specific viral RNA polymerase

Deiman, Birgit; Séron, Karin; Jaspars, E.M.J.; Pleij, C.W.A.

doi:10.1016/s0166-0934(96)02166-0

Cited by 23 publications

(50 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…1D). This observation demonstrates that the 115K and 85K proteins are derived solely from the 140K ORF and rule out the possibility that the 115K protein corresponds to a fusion of the helicase and the RdRp protein domains, as previously suggested (11). Whether the 115K protein lacks the N-terminal methyltransferase domain or the C-terminal helicase domain remains to be determined by use of other domainspecific antisera.…”

Section: Discussionsupporting

confidence: 71%

“…Granaat) plants were grown and inoculated with TYMV as described previously (47). At 8 days postinoculation, the young developing leaves from the center of the rosette were collected, and the replication complexes were purified as described previously (11), omitting the DEAE ionexchange chromatography step. The glycerol gradient was subdivided into 18 fractions that were assayed for in vitro RdRp activity with TYMV RNA as a template.…”

Section: Methodsmentioning

confidence: 99%

“…The glycerol gradient was subdivided into 18 fractions that were assayed for in vitro RdRp activity with TYMV RNA as a template. The standard assay was performed by mixing 17.5 l of enzyme fraction and 7.5 l of a reaction mixture containing 75 mM Tris-HCl (pH 9), 8 mM MgCl 2 , 5 mM dithiothreitol, 1 g of actinomycin D, 2.5 g of TYMV RNA, 4.16 mM each ATP, GTP, and CTP, 2.5 Ci of [ 32 P]UTP, and 10 U of RNasin (Promega) to give a final volume of 25 l. The mixture was incubated for 40 min at 30°C, and the incorporation of radioactivity into the acid-insoluble fraction was determined as previously described (11).…”

Section: Methodsmentioning

confidence: 99%

“…To investigate whether the 66K and 140K-derived proteins constituted a functional viral RdRp, replication complexes were prepared from TYMV-infected Chinese cabbage plants and from healthy control plants essentially as previously described (11). They were solubilized from membrane structures by using Lubrol W and centrifuged in a glycerol gradient.…”

Section: Generation and Characterization Of Anti-140k Antibodiesmentioning

confidence: 99%

See 3 more Smart Citations

Assembly of Turnip Yellow Mosaic Virus Replication Complexes: Interaction between the Proteinase and Polymerase Domains of the Replication Proteins

Jakubiec

Notaise

Tournier

et al. 2004

J Virol

View full text Add to dashboard Cite

The replication of positive-strand RNA virus genomes is dependent upon the assembly of a replication complex that is an intricate machinery comprising both virus and host components (reviewed in references 1 and 7). Replication complexes are closely associated with intracellular membranes, and many critical interactions among RNA, proteins, and lipids likely take place to allow a successful replication process.Despite the identification and characterization of many viral replication proteins and the purification of a number of positive-strand RNA virus replication complexes that were extensively used in vitro, there is only a limited understanding of the higher-order interactions among these proteins and the ratios in which they are present in viral replication complexes. Understanding how the various proteins interact in these enzyme complexes is essential for unraveling the mechanism of RNA replication and elucidating the three-dimensional structure and function of RNA virus replication complexes. Furthermore, the fine molecular mapping of the interaction sites may constitute an important step toward the identification of the mechanisms that may eventually regulate these interactions or may constitute a way to identify specific targets for new antiviral compounds.To gain insight into the assembly of positive-strand RNA virus replication complexes, we have studied the interactions among the replication proteins of Turnip yellow mosaic virus (TYMV), the type member of the tymovirus group. TYMV is a small spherical plant virus that shares viral replication features with other positive-strand RNA viruses in the alphaviruslike supergroup (17) and has proven useful for investigating fundamental aspects of viral multiplication (3, 60). The TYMV genome is composed of a monopartite, positive-sense RNA of 6,318 nucleotides (Fig. 1A) that directs the expression of two extensively overlapping nonstructural proteins with molecular weights of 69,000 (69K) and 206,000 (206K) (40,61). A third open reading frame (ORF) encodes the 20-kDa coat protein, which is expressed from a subgenomic RNA.The 206K protein is the only viral protein required for TYMV RNA replication (61), and it shows considerable amino acid sequence similarities with nonstructural putative replication proteins of several positive-strand RNA viruses (31). The 206K protein possesses a modular organization, and domains

show abstract

Section: Discussionsupporting

confidence: 71%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Generation and Characterization Of Anti-140k Antibodiesmentioning

confidence: 99%

See 2 more Smart Citations

Assembly of Turnip Yellow Mosaic Virus Replication Complexes: Interaction between the Proteinase and Polymerase Domains of the Replication Proteins

Jakubiec

Notaise

Tournier

et al. 2004

J Virol

View full text Add to dashboard Cite

show abstract

“…These small vesicles are closely associated with TYMV RNA replication, as revealed by previous in vivo RNA labeling observations (35) and immunocytochemical experiments with antibodies raised against the viral replication complex (19). Consistent with these results, membrane-associ-ated extracts that selectively synthesize TYMV negative-strand RNAs have been isolated from TYMV-infected plant cells (11,44).…”

supporting

confidence: 81%

Targeting of the Turnip Yellow Mosaic Virus 66K Replication Protein to the Chloroplast Envelope Is Mediated by the 140K Protein

Prod'homme

Jakubiec

Tournier

et al. 2003

J Virol

112

View full text Add to dashboard Cite

. During viral infection, the 66K protein localizes to virus-induced chloroplastic membrane vesicles, which are closely associated with TYMV RNA replication. To investigate the determinants of its subcellular localization, the 66K protein was expressed in plant protoplasts from separate plasmids. Green fluorescent protein (GFP) fusion and immunofluorescence experiments demonstrated that the 66K protein displayed a cytoplasmic distribution when expressed individually but that it was relocated to the chloroplast periphery under conditions in which viral replication occurred. The 66K protein produced from an expression vector was functional in viral replication since it could transcomplement a defective replication template. Targeting of the 66K protein to the chloroplast envelope in the course of the viral infection appeared to be solely dependent on the expression of the 140K protein. Analysis of the subcellular localization of the 140K protein fused to GFP demonstrated that it is targeted to the chloroplast envelope in the absence of other viral factors and that it induces the clumping of the chloroplasts, one of the typical cytological effects of TYMV infection. These results suggests that the 140K protein is a key organizer of the assembly of the TYMV replication complexes and a major determinant for their chloroplastic localization and retention.A universal feature of eukaryotic positive-strand RNA viruses is that replication of their genomes is closely associated with intracellular membranes (reviewed in reference 8). Most purified viral RNA replication complexes copurify with membrane extracts from infected cells (reviewed in reference 10) and, although in some cases RNA synthesis activity can be solubilized (24, 67), in vivo and in vitro studies suggest that the presence of membranes and/or phospholipids is essential for at least some steps of RNA replication (37,41,67). It was proposed that these membranes can play both a structural and a functional role in the replication complex.Electron microscopy observations of infected cells revealed that many positive-stranded RNA viruses induce proliferation and/or reorganization of the intracellular membranes of their host to create a membrane compartment in which RNA replication takes place. Depending on the virus, a variety of membrane systems can be concerned, including the early and late endomembrane systems (52, 59), the nuclear envelope (13), the vacuole (64), the endosomes and lysosomes (17, 59), the peroxisomes (56), chloroplasts (35), and mitochondria (14, 40). The fact that distinct types of membranes are involved in the replication of different viruses suggests the establishment of specific interactions between such host membranes and virusencoded proteins. A number of viral proteins that target replication complexes to intracellular membranes have been identified (9,48,55,63). Membrane interaction of host-encoded factors that are part of the viral replication complex has also been reported (22,68).Despite this universal association of positive-strand ...

show abstract

Pseudoknots: A Vital Feature in Viral RNA

Deiman

Pleij

1997

Seminars in Virology

View full text Add to dashboard Cite

Efficient transcription obtained by a new procedure of the tRNA-like structure of turnip yellow mosaic virus by a template-dependent and specific viral RNA polymerase

Cited by 23 publications

References 36 publications

Assembly of Turnip Yellow Mosaic Virus Replication Complexes: Interaction between the Proteinase and Polymerase Domains of the Replication Proteins

Assembly of Turnip Yellow Mosaic Virus Replication Complexes: Interaction between the Proteinase and Polymerase Domains of the Replication Proteins

Targeting of the Turnip Yellow Mosaic Virus 66K Replication Protein to the Chloroplast Envelope Is Mediated by the 140K Protein

Pseudoknots: A Vital Feature in Viral RNA

Contact Info

Product

Resources

About