The Potato Leafroll Luteovirus 17K Protein is a Single-stranded Nucleic Acid-binding Protein

Tacke, Eckhard; Prüfer, Dirk; Schmitz, Jürgen; Rohde, Wolfgang

doi:10.1099/0022-1317-72-8-2035

Cited by 70 publications

(35 citation statements)

References 25 publications

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“…Examination of the RNA 2-encoded proteins has not provided any evidence to support this contention. In direct amino acid sequence comparisons using the programs COMPARE, D O T P L O T and GAP, there was no evidence of statistically significant sequence homology, nor was there evidence of the acidic-basic residue charge distribution reported by Tacke et al (1991) in the PLRV 17K protein• A notable structural characteristic of the PEMV 25K protein is a preponderance of basic residues in the N-terminal half of the molecule (38 of 126 residues), although we can ascribe no functional significance to this observation at this time.…”

Section: Analysis O F Pea Enation Mosaic Virus R N a 2 9 (A)mentioning

confidence: 99%

The chimeric nature of the genome of pea enation mosaic virus: the independent replication of RNA 2

Demler

Rucker

Zoeten

1993

Journal of General Virology

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The genome of pea enation mosaic virus (PEMV) consists of two plus-sense RNAs, both of which are required for mechanical transmission. RNA 1 (5706 nucleotides) has strong sequence similarity with members of the luteovirus group, a similarity that is also manifested in the symptomatology, cytopathology and vector transmission of this virus. RNA 2 (4253 nucleotides) is hypothesized to facilitate systemic invasion and mechanical transmission, attributes that distinguish PEMV from the phloem-limited luteoviruses. Sequence analysis of RNA 2 has demonstrated that PEMV is unique among multicomponent viruses in that it lacks Tand 5'-terminal homology between its genomic RNAs. Sequence analysis of RNA 2 has identified an open reading frame encoding a putative product of 65K that contains a series of polymerase-like motifs typical of viral RNA-dependent RNA polymerases. This protein sequence lacks homology with the polymerase encoded on RNA 1 of PEMV, instead being more closely affiliated with the polymerases of viruses related to the carmoand tombusvirus groups. Inoculation of pea protoplasts with RNA transcripts derived from a full-length cDNA clone ofRNA 2 has demonstrated that RNA 2 replicates autonomously in the absence of RNA 1, although comparable inoculation of whole plants failed to establish a systemic infection. There is no evidence that RNA 2 encodes structural proteins, suggesting that encapsidation functions are supplied in trans by RNA 1, comparable to the helper-dependent complexes occurring within the luteovirus group. These data suggest that the PEMV genome can be characterized as a symbiotic association of two taxonomically distinct viral RNAs cooperatively interacting in the establishment of a systemic virus infection.

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Section: Analysis O F Pea Enation Mosaic Virus R N a 2 9 (A)mentioning

confidence: 99%

The chimeric nature of the genome of pea enation mosaic virus: the independent replication of RNA 2

Demler

Rucker

Zoeten

1993

Journal of General Virology

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“…As the basic region of another PLRV protein (ORF4, coding for a 17 kDa protein; Figure IA) has recently been shown to display nucleic acid-binding properties (Tacke et al, 1991), we examined the possibility that the ORF2b basic amino acid cluster would exhibit a similar activity. For this the close to full-length ORF2b protein as well as several deletion mutants ( Figure SA) were expressed in E. coli as fusion proteins with glutathione-S-transferase (GST) in the pGEX expression vector system (Smith and Johnson, 1988).…”

Section: Requirements Of Downstream Sequences For Efficient Frameshifmentioning

confidence: 99%

“…For this the close to full-length ORF2b protein as well as several deletion mutants ( Figure SA) were expressed in E. coli as fusion proteins with glutathione-S-transferase (GST) in the pGEX expression vector system (Smith and Johnson, 1988). Total bacterial extracts were subjected to PAGE (Figure SB), the separated proteins were electroblotted to nitrocellulose membranes and RNA-binding experiments with radiolabelled single-stranded (+) PLRV RNA were performed as described previously (Gramstat et al, 1990;Tacke et al, 1991). Nonspecific binding to E. coli proteins of low molecular weight was observed in all lanes.…”

Section: Requirements Of Downstream Sequences For Efficient Frameshifmentioning

confidence: 99%

Ribosomal frameshifting in plants: a novel signal directs the −1 frameshift in the synthesis of the putative viral replicase of potato leafroll luteovirus.

et al. 1992

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The 5.8 kb RNA genome of potato leafroll luteovirus (PLRV) contains two overlapping open reading frames, ORF2a and ORF2b, which are characterized by helicase and RNA polymerase motifs, respectively, and possibly represent the viral replicase. Within the overlap, ORF2b lacks an AUG translational start codon and is therefore presumably translated by -1 ribosomal frameshifting as a transframe protein with ORF2a. This hypothesis was studied by introducing the putative frameshift region into an internal position of the 3-glucuronidase (GUS) gene and testing for the occurrence of frameshifting in vivo by transient expression of GUS activity in potato protoplasts as well as in vitro by translation in the reticulocyte system. Both experimental approaches demonstrate that a -1 frameshift occurs at a frequency of -1%. Sitedirected mutagenesis identified the frameshift region and the involvement of the novel heptanucleotide motif UUUAAAU in conjunction with an adjacent stem -loop structure. Part of this stem -loop encodes a basic region in the ORF2b moiety of the transframe protein which was shown by binding experiments with PLRV RNA to represent a nucleic acid-binding domain. These data support a possible biological significance of the frameshift to occur at this position of the large overlap by including the putative RNA template-binding site of the PLRV replicase in the ORF2a/ORF2b transframe protein.

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“…Fax j49 241 8888 182. e-mail Rainer.Fischer!post.rwth-aachen.de Osman et al, 1992 ;Schoumacher et al, 1992 a, b ;Tacke et al, 1991 ;Rouleau et al, 1994 ;Ivanov et al, 1994 ;Bleykasten et al, 1996). Distinct domains essential for this binding activity have been identified in several movement proteins, such as the 30 kDa (' 30K ') protein of tobacco mosaic virus (TMV) (Citovsky et al, 1992), the 35 kDa protein of red clover necrotic mosaic virus (RCNMV) (Osman et al, 1993 ;GiesmanCookmayer & Lommel, 1993), the P3 protein of alfalfa mosaic virus (AlMV) (Schoumacher et al, 1994) and the P1 protein of cauliflower mosaic virus (CaMV) (Thomas & Maule, 1995).…”

mentioning

confidence: 99%

Mapping of the RNA-binding domain of the cucumber mosaic virus movement protein.

Vaquero

Liao

Nähring

et al. 1997

Journal of General Virology

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A series of in-frame deletion mutants was used to identify a domain within the 3a protein of cucumber mosaic virus (CMV) that is required for RNA-binding activity. Deletions in the 3a gene were generated by PCR and restriction digestion, and the resulting mutated 3a sequences were cloned either in pT7-7 or in pGEX-5X3 expression vectors. The mutated 3a proteins or fusions with glutathione S-transferase (GST) were expressed in E. coli, purified, and their nucleic acid-binding activities analysed by photochemical UV cross-linking assays using digoxigenin-UTP-labelled RNA probes. Comparative analyses of seven mutated 3a proteins obtained from inclusion bodies and eight GST fusion proteins revealed that there is an RNA-binding domain located between amino acids 174 and 233. This RNA-binding domain is able to bind single-stranded RNA out of the context of the complete 3a movement protein and is highly conserved within both subgroups of CMV.

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The Potato Leafroll Luteovirus 17K Protein is a Single-stranded Nucleic Acid-binding Protein

Cited by 70 publications

References 25 publications

The chimeric nature of the genome of pea enation mosaic virus: the independent replication of RNA 2

The chimeric nature of the genome of pea enation mosaic virus: the independent replication of RNA 2

Ribosomal frameshifting in plants: a novel signal directs the −1 frameshift in the synthesis of the putative viral replicase of potato leafroll luteovirus.

Mapping of the RNA-binding domain of the cucumber mosaic virus movement protein.

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