The motif V of plum pox potyvirus CI RNA helicase is involved in NTP hydrolysis and is essential for virus RNA replication

Fernández, Andrés

doi:10.1093/nar/25.22.4474

Cited by 90 publications

(65 citation statements)

References 29 publications

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“…The importance of these motifs in helicase function is further supported by the inability of the IR-nHEL polypeptide, which lacks these motifs, to hydrolyze NTP or bind RNA. Taken together, these findings are consistent with the functions of similarly characterized viral helicases (9,14,19).…”

Section: Discussionsupporting

confidence: 87%

Oligomerization and Activity of the Helicase Domain of the Tobacco Mosaic Virus 126- and 183-Kilodalton Replicase Proteins

Goregaoker

Culver

2003

J Virol

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A protein-protein interaction within the helicase domain of the Tobacco mosaic virus (TMV) 126-and 183-kDa replicase proteins was previously implicated in virus replication (S. Goregaoker, D. Lewandowski, and J. Culver, Virology 282:320-328, 2001). To further characterize the interaction, polypeptides covering the interacting portions of the TMV helicase domain were expressed and purified. Biochemical characterizations demonstrated that the helicase domain polypeptides hydrolyzed ATP and bound both single-stranded and duplexed RNA in an ATP-controlled fashion. A TMV helicase polypeptide also was capable of unwinding duplexed RNA, confirming the predicted helicase function of the domain. Biochemically active helicase polypeptides were shown by gel filtration to form high-molecular-weight complexes. Electron microscopy studies revealed the presence of ring-like oligomers that displayed six-sided symmetry. Taken together, these data demonstrate that the TMV helicase domain interacts with itself to produce hexamer-like oligomers. Within the context of the full-length 126-and 183-kDa proteins, these findings suggest that the TMV replicase may form a similar oligomer.Positive-stranded RNA viruses are a diverse group of pathogens that cause diseases in humans, plants, and animals. Although this group of pathogens is taxonomically diverse, they all encode replicase proteins involved in the synthesis of viral RNA. Enzymatic motifs within these replicase proteins can include methyltransferase (MT), helicase, and RNA-dependent RNA polymerase (POL) activities. These motifs may be present within a single multidomain protein, as found within the Tobacco mosaic virus (TMV) 183-kDa protein, or separated onto two or more virus-encoded proteins, as found in the 1a MT-HEL and 2a POL proteins of Brome mosaic virus (BMV) (2, 38). In infected cells, viral replicase proteins associate with host proteins as well as cellular membranes to produce replicase complexes that function in viral RNA synthesis. Despite the essential role of these replicase complexes in virus replication, little is known about their structure and the mechanisms that control their assembly.TMV is a positive-stranded RNA virus that has served as a model for the study of RNA replication (3). TMV is the type member of the genus Tobamovirus and a member of the alphavirus supergroup. Its genome encodes at least four proteins (11) (Fig. 1). The 5Ј-proximal open reading frames (ORFs) encode 126-and 183-kDa proteins, the larger produced by the read-through of an amber stop codon (33). Both the 126-and 183-kDa proteins are necessary for efficient virus replication (17,18,25). Homology studies indicate that the 126-kDa-protein-encoding ORF encodes MT and helicase domains divided by an uncharacterized intervening region (IR), while the readthrough portion of the 183-kDa-protein-encoding ORF encodes the POL domain (20,21,22) (Fig. 1). A 30-kDa cell-tocell movement protein and a 17.5-kDa coat protein are produced from 3Ј coterminal subgenomic mRNAs (6,16,28).Biochemical charac...

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Section: Discussionsupporting

confidence: 87%

Oligomerization and Activity of the Helicase Domain of the Tobacco Mosaic Virus 126- and 183-Kilodalton Replicase Proteins

Goregaoker

Culver

2003

J Virol

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“…For preparation of viral genomic RNA, total RNA was isolated from inoculated plants by a hot-phenol method (Fernandez et al, 1997). Other RNA was extracted from plants by Trizol.…”

Section: Nucleic Acid Extraction and Rna Gel Blottingmentioning

confidence: 99%

RNA-Dependent RNA Polymerase 1 from Nicotiana tabacum Suppresses RNA Silencing and Enhances Viral Infection in Nicotiana benthamiana

et al. 2010

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Endogenous eukaryotic RNA-dependent RNA polymerases (RDRs) produce double-stranded RNA intermediates in diverse processes of small RNA synthesis in RNA silencing pathways. RDR6 is required in plants for posttranscriptional gene silencing induced by sense transgenes (S-PTGS) and has an important role in amplification of antiviral silencing. Whereas RDR1 is also involved in antiviral defense in plants, this does not necessarily proceed through triggering silencing. In this study, we show that Nicotiana benthamiana transformed with RDR1 from Nicotiana tabacum (Nt-RDR1 plants) exhibits hypersusceptibility to Plum pox potyvirus and other viruses, resembling RDR6-silenced (RDR6i) N. benthamiana. Analysis of transient induction of RNA silencing in N. benthamiana Nt-RDR1 and RDR6i plants revealed that Nt-RDR1 possesses silencing suppression activity. We found that Nt-RDR1 does not interfere with RDR6-dependent siRNA accumulation but turns out to suppress RDR6-dependent S-PTGS. Our results, together with previously published data, suggest that RDR1 might have a dual role, contributing, on one hand, to salicylic acid-mediated antiviral defense, and suppressing, on the other hand, the RDR6-mediated antiviral RNA silencing. We propose a scenario in which the natural loss-of-function variant of RDR1 in N. benthamiana may be the outcome of selective pressure to maintain a high RDR6-dependent antiviral defense, which would be required to face the hypersensitivity of this plant to a large number of viruses.

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“…Within helicase motif V, insertions at four amino acids were deleterious, whereas those at eight others were tolerated. Two of these deleterious insertions (Thr 1455 , and between Ile 1458 and Glu 1459 ) correspond to conserved residues in which different types of mutations make TVMV and Plum pox potyvirus noninfectious (Klein et al 1994;Fernández et al 1997). Insertions within helicase motif VI between Gly 1501 and Glu 1502 , between Val 1510 and Gly 1511 , and at conserved Arg 1512 were deleterious.…”

Section: Regionmentioning

confidence: 99%

Functional Genomics on Potato Virus A: Virus Genome-Wide Map of Sites Essential for Virus Propagation

Kekarainen¹,

Savilahti²,

Valkonen³

2002

Genome Res.

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Transposition-based in vitro insertional mutagenesis strategies provide promising new approaches for functional characterization of any cloned gene or genome region. We have extended the methodology and scope of such analysis to a complete viral genome. To map genome regions both essential and nonessential for Potato virus A propagation, we generated a genomic 15-bp insertion mutant library utilizing the efficient in vitro DNA transposition reaction of phage Mu. We then determined the proficiency of 1125 mutants to propagate in tobacco protoplasts by using a genetic footprinting strategy that simultaneously mapped the genomic insertion sites. Over 300 sites critical for virus propagation were identified, and many of them were located in positions previously not assigned to any viral functions. Many genome regions tolerated insertions indicating less important sites for virus propagation and thus pinpointed potential locations for further genome manipulation. The methodology described is applicable to a detailed functional analysis of any viral nucleic acid cloned as DNA and can be used to address many different processes during viral infection cycles

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The motif V of plum pox potyvirus CI RNA helicase is involved in NTP hydrolysis and is essential for virus RNA replication

Cited by 90 publications

References 29 publications

Oligomerization and Activity of the Helicase Domain of the Tobacco Mosaic Virus 126- and 183-Kilodalton Replicase Proteins

Oligomerization and Activity of the Helicase Domain of the Tobacco Mosaic Virus 126- and 183-Kilodalton Replicase Proteins

RNA-Dependent RNA Polymerase 1 from Nicotiana tabacum Suppresses RNA Silencing and Enhances Viral Infection in Nicotiana benthamiana

Functional Genomics on Potato Virus A: Virus Genome-Wide Map of Sites Essential for Virus Propagation

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