Insertion of sequences containing the coat protein subgenomic RNA promoter and leader in front of the tobacco mosaic virus 30K ORF delays its expression and causes defective cell-to-cell movement

doi:10.1016/0042-6822(90)90063-W

Virology

Volume 174, Issue 1, January 1990, Pages 145-157

https://doi.org/10.1016/0042-6822(90)90063-W Get rights and content

Abstract

The regulation of the internal open reading frame (ORF) of tobacco mosaic virus (TMV) that encodes the 30K movement protein was examined by constructing mutants in vitro with the putative coat protein subgenomic RNA promoter and leader sequences inserted upstream of the 30K ORF. A mutant with a 49-nucleotide fragment of the promoter region inserted replicated only transiently before being overtaken by a progeny wild-type virus with the insert deleted. A mutant with a 253-nucleotide promoter region fragment inserted replicated stably, and the inserted promoter was active in its new location. The production of 30K protein was not enhanced by this promoter/leader insertion to a level similar to that of coat protein. However, the accumulation of 30K protein was delayed, suggesting that different promoters/leader sequences determine the time of expression of the genes. This mutant was deficient in movement. A similar mutant, but with increased production of 30K protein, overcame the movement deficiency, suggesting that 30K protein is needed during the early stages of infection for efficient cell-to-cell movement of the virus.

References (44)

R.N. Beachy et al.
Characterization and in vitro translation of the RNAs from less-than-full-length, virus related, nucleoprotein rods present in tobacco mosaic virus preparations
Virology
(1977)
W.O. Dawson et al.
A tobacco mosaic virus-hybrid expresses and loses an added gene
Virology
(1989)
W.O. Dawson et al.
Synthesis of tobacco mosaic virus in intact tobacco leaves inoculated by differential temperature treatment
Virology
(1975)
S.D. Dunn
Effects of modification of transfer buffer, composition, and renaturation in gels on the recognition of proteins on western blots by monoclonal antibodies
Anal. Biochem.
(1986)
S. Joshi et al.
Properties of the tobacco mosaic virus intermediate RNA2 and its translation
Virology
(1983)
T.A. Kunkel et al.
Rapid and efficient site-specific mutagenesis without phenotypic selection
D.A. Leonard et al.
A temperature-sensitive strain of tobacco mosaic virus defective in cell-to-cell movement generates an altered viral-coded protein
Virology
(1982)
J. Logemann et al.
Improved method for isolation of RNA from plant tissue
Anal. Biochem.
(1987)
I. Ooshika et al.
Identification of the 30K protein of TMV by immunoprecipitation with antibodies directed against a synthetic peptide
Virology
(1984)
A.J. Shatkin
Capping of eucaryotic mRNAs
Cell
(1976)

A. Siegel et al.

The effects of tobacco mosaic virus infection on host and virus-specific protein synthesis in protoplasts

Virology

(1978)

A. Siegel et al.

A messenger RNA for coat protein isolated from tobacco mosaic virus infected tissue

Virology

(1976)

Y. Watanabe et al.

Synthesis of TMV specific RNAs and proteins at the early stage of infection in tobacco protoplasts: Transient expression of the 30K protein and its mRNA

Virology

(1984)

Y. Watanabe et al.

The initiation site for transcription of the TMV 30-kDa protein messenger RNA

FEBS Lett.

(1984)

R. Zagursky et al.

Rapid and easy sequencing of large linear double-stranded DNA and supercoiled plasmid DNA

Gene Anal. Techn.

(1985)

P. Ahlquist et al.

cDNA cloning and in vitro transcription of the complete brome mosaic virus genome

Mol. Cell. Biol.

(1984)

M. Ausubel et al.

D.A. Beck

Utilization of Infectious cDNA Clones of Tobacco Mosaic Virus to Better Understand the Virus/Host Interaction

J.N. Culver et al.

Point mutations in the coat protein gene of tobacco mosaic virus induce hypersensitivity in Nicotiana sylvestris

Mol. Plant-Microbe Interact.

(1989)

W.O. Dawson et al.

cDNA cloning of the complete genome of tobacco mosaic virus and production of infectious transcripts

W.O. Dawson et al.

Modifications of the tobacco mosaic virus coat protein gene affecting replication, movement, and symptomatology

Phytopathology

(1988)

R.S.S. Fraser

Biochemistry of Virus-Infected Plants

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Mapping of the Tobacco mosaic virus movement protein and coat protein subgenomic RNA promoters in vivo
2000, Virology
The Tobacco mosaic virus movement protein (MP) and coat protein (CP) are expressed from 3′-coterminal subgenomic RNAs (sgRNAs). The transcription start site of the MP sgRNA, previously mapped to positions 4838 (Y. Watanabe, T. Meshi, and Y. Okada (1984), FEBS Lett. 173, 247–250) and 4828 (K. Lehto, G. L. Grantham, and W. O. Dawson (1990), Virology 174, 145–157) for the TMV OM and U1 strains, respectively, has been reexamined and mapped to position 4838 for strain U1. Sequences of the MP and CP sgRNA promoters were delineated by deletion analysis. The boundaries for minimal and full MP sgRNA promoter activity were localized between −35 and +10 and −95 and +40, respectively, relative to the transcription start site. The minimal CP sgRNA promoter was mapped between −69 and +12, whereas the boundaries of the fully active promoter were between −157 and +54. Computer analysis predicted two stem–loop structures (SL1 and SL2) upstream of the MP sgRNA transcription start site. Deletion analysis and site-directed mutagenesis suggested that SL1 secondary structure, but not its sequence, was required for MP sgRNA promoter activity, whereas a 39-nt deletion removing most of the SL2 region increased MP sgRNA accumulation fourfold. Computer-predicted folding of the fully active CP sgRNA promoter revealed one long stem–loop structure. Deletion analysis suggested that the upper part of this stem–loop, located upstream of the transcription start site, was essential for transcription and that the lower part of the stem had an enhancing role.
Synthesis of subgenomic RNAs by positive-strand RNA viruses
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Internal initiation of translation directed by the 5'-untranslated region of the tobamovirus subgenomic RNA I<inf>2</inf>
1999, Virology
Previously we reported that, unlike RNA of typical tobamoviruses, the translation of the coat protein (CP) gene of a crucifer-infecting tobamovirus (crTMV) in vitro occurred by an internal ribosome entry mechanism mediated by the 148-nt region that contained an internal ribosome entry site (IRES_CP,148^CR). The equivalent 148-nt sequence from TMV U1 RNA (U1_CP,148^SP) was incapable of promoting internal initiation. In the present work, we have found that the 228-nt region upstream of the movement protein (MP) gene of crTMV RNA (IRES_MP,228^CR) contained an IRES element that directed in vitro translation of the 3′-proximal reporter genes from chimeric dicistronic transcripts. Surprisingly, the equivalent 228-nt sequence upstream from the MP gene of TMV U1 directed translation of the downstream gene of a dicistronic transcripts as well. Consequently this sequence was termed IRES_MP,228^U1. It was shown that IRES_MP,228^CR, IRES_MP,228^U1, and IRES_CP,148^CR could mediate expression of the 3′-proximal GUS gene from dicistronic 35S promoter-based constructs in vivo in experiments on transfection of tobacco protoplasts and particle bombardment of Nicotiana benthamiana leaves. The results indicated that an IRES element was located within the 75-nt region upstream of MP gene (IRES_MP,75), which corresponded closely to the length of the 5′UTR of TMV subgenomic RNA (sgRNA) I₂. The RNA transcripts structurally equivalent to I₂ sgRNAs of TMV U1 and crTMV, but containing a hairpin structure (H) immediately upstream of IRES_MP,75 (HIRES_MP,₇₅^CR-MP-CP-3′UTR; HIRES_MP,75^U1-MP-CP-3′UTR), were able to express the MP gene in vitro. The capacity of HIRES_MP,75^CR sequence for mediating internal translation of the 3′-proximal GUS gene in vivo, in tobacco protoplasts, was demonstrated. We suggested that expression of the MP gene from I₂ sgRNAs might proceed via internal ribosome entry pathway mediated by IRES_MP element contained in the 75-nt 5′UTR. Our results admit that a ribosome scanning mechanism of the MP gene expression from I₂ sgRNA operates concurrently.
Heterologous sequences greatly affect foreign gene expression in tobacco mosaic virus-based vectors
1999, Virology
A series of tobacco mosaic virus (TMV)-based hybrid vectors for transient gene expression were constructed with similar designs but differing in the source of heterologous tobamovirus sequence:Odontoglossumringspot virus, tobacco mild green mosaic virus variants U2 and U5, tomato mosaic virus, and sunn-hemp mosaic virus. These vectors contained a heterologous coat protein subgenomic mRNA promoter and coat protein open reading frame (ORF) and either TMV or heterologous 3′ nontranslated region. The foreign ORF, from the jellyfish green fluorescent protein (GFP) gene, was transcribed from the native TMV coat protein subgenomic mRNA promoter, which extended into the coat protein ORF. The presence of an in-frame stop codon within the GFP mRNA leader and the choice of sequence of GFP ORFs substantially affected translational efficiency. However, the major regulatory component of gene expression in these vectors appeared to be transcriptional rather than translational. There was an inverse relationship between expression of GFP and the heterologous coat protein genes that was reflected in accumulation of the respective mRNAs and proteins. The most effective vector in this series (30B) contained sequences encoding the coat protein subgenomic mRNA promoter, coat protein ORF, and 3′ nontranslated region from tobacco mild green mosaic virus U5. Expressed from 30B, GFP accumulated up to 10% of total soluble protein in leaves.
Mutations that alter a conserved element upstream of the potato virus X triple block and coat protein genes affect subgenomic RNA accumulation
1997, Virology
The putative subgenomic RNA (sgRNA) promoter regions upstream of the potato virus X (PVX) triple block and coat protein (CP) genes contain sequences common to other potexviruses. The importance of these sequences to PVX sgRNA accumulation was determined by inoculation ofNicotiana tabacumNT1 cell suspension protoplasts with transcripts derived from wild-type and modified PVX cDNA clones. Analyses of RNA accumulation by S1 nuclease digestion and primer extension indicated that a conserved octanucleotide sequence element and the spacing between this element and the start-site for sgRNA synthesis are critical for accumulation of the two major sgRNA species. The impact of mutations on CP sgRNA levels was also reflected in the accumulation of CP. In contrast, genomic minus- and plus-strand RNA accumulation were not significantly affected by mutations in these regions. Studies involving inoculation of tobacco plants with the modified transcripts suggested that the conserved octanucleotide element functions in sgRNA accumulation and some other aspect of the infection process.
Deletion analysis of the promoter for the cucumber necrosis virus 0.9-kb subgenomic RNA
1995, Virology
Sequences comprising the core promoter for the cucumber necrosis virus (CNV) 0.9-kb subgenomic RNA have been determined using deletion analysis and site-directed mutagenesis. The deletion studies indicate that the promoter lies within a region located 20 nucleotides upstream and 6 nucleotides downstream and including the subgenomic start site. Sequences further upstream or downstream of the core promoter do not appear to strongly affect promoter activity or viral RNA accumulation. Results of site-directed mutagenesis studies indicate that nucleotides immediately surrounding the subgenomic start site regulate promoter activity. Comparison of sequences within the CNV promoter region with the corresponding region of other tombusviruses shows that the tombusvirus promoter shares a region of near complete identity in 14 of the 26 core promoter nucleotides. Little similarity exists between the CNV 0.9-kb subgenomic RNA promoter and the region surrounding the transcription initiation site for the CNV 2.1-kb subgenomic RNA. Likewise, limited similarity occurs with the 5′ region of CNV genomic RNA. Sequences similar to the ICR2-like motifs found in the promoters of several alphavirus-like (supergroup III) plant and animal viruses are not apparent. This study represents the first analysis of a subgenomic promoter from a member of supergroup II of positive-strand RNA viruses.

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Insertion of sequences containing the coat protein subgenomic RNA promoter and leader in front of the tobacco mosaic virus 30K ORF delays its expression and causes defective cell-to-cell movement

Abstract

Virology

Virology

Virology

Anal. Biochem.

Virology

Virology

Anal. Biochem.

Virology

Cell

Virology

Virology

Virology

FEBS Lett.

Gene Anal. Techn.

cDNA cloning and in vitro transcription of the complete brome mosaic virus genome

Mol. Cell. Biol.

Utilization of Infectious cDNA Clones of Tobacco Mosaic Virus to Better Understand the Virus/Host Interaction

Point mutations in the coat protein gene of tobacco mosaic virus induce hypersensitivity in Nicotiana sylvestris

Mol. Plant-Microbe Interact.

cDNA cloning of the complete genome of tobacco mosaic virus and production of infectious transcripts

Modifications of the tobacco mosaic virus coat protein gene affecting replication, movement, and symptomatology

Phytopathology

Biochemistry of Virus-Infected Plants