Structure and assembly of turnip crinkle virus: IV. Analysis of the coat protein gene and implications of the subunit primary structure

doi:10.1016/0022-2836(87)90374-3

Journal of Molecular Biology

Volume 194, Issue 2, 20 March 1987, Pages 265-276

https://doi.org/10.1016/0022-2836(87)90374-3 Get rights and content

Abstract

The structure of the turnip crinkle virus (TCV) coat protein and coat protein gene has been examined by cDNA cloning, nucleotide sequencing and high-resolution mRNA mapping. We have cloned a 1450-nucleotide cDNA fragment, representing the 3′ end of the TCV genome, using genomic RNA polyadenylated in vitro as the reverse transcriptional template. Nucleic acid sequence analysis reveals the presence of a 1053 nucleotide open reading frame capable of encoding a protein of 38,131 M_r, identified as the coat protein subunit.

The 1446 base subgenomic mRNA for the coat protein, mapped using high-resolution primer extension techniques, contains a 137 nucleotide leader sequence upstream from the initiation codon. We have characterized a second subgenomic RNA of approximately 1700 bases, roughly 250 nucleotides longer than the 1446 base species in the 5′ direction. No TCV-related RNAs are polyadenylated in vivo.

The derived amino acid sequence of the TCV coat protein has been built into the 3.2 Å resolution electron density map of TCV reported in paper I of this series. We describe here some of the important features of the structure. Alignment of the three-dimensional structures of tomato bushy stunt virus and southern bean mosaic virus shows significant sequence relationships in the arms and S domains, although the conserved residues do not appear to have any special role in stabilizing the β-barrel fold or in mediating subunit interactions. The sequences of TCV and carnation mottle virus can be aligned. Comparisons among the four are discussed in terms of the organization of the S domain.

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The capsid protein (CP) of turnip crinkle virus (TCV) is the elicitor of hypersensitive response (HR) and resistance mediated by the resistance protein HRT in the Di-17 ecotype of Arabidopsis. Here we identified the N-terminal 52-amino-acid R domain of TCV CP as the elicitor of HRT-dependent HR in Nicotiana benthamiana. Mutating this domain at position 6 (R6A), but not at positions 8 (R8A) or 14 (G14A), abolished HR in N. benthamiana. However, on Di-17 Arabidopsis leaves only R8A R domain elicited visible epidermal HR. When incorporated in infectious TCV RNAs, R8A and G14A mutations exerted dramatically different effects in Di-17 plants, as R8A caused systemic cell death whereas G14A led to complete restriction of the mutant virus. This continual spectrum of HR and resistance responses elicited by various R domain mutants suggests that the CP–HRT interaction could be perturbed by conformational changes in the R domain of TCV CP.
Isolation of an asymmetric RNA uncoating intermediate for a single-stranded RNA plant virus
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Citation Excerpt :
Mass spectrometry of these digestion products and N-terminal amino acid sequencing (Fig. 1, and Supplementary Figs. 1 and 2) confirm earlier suggestions that they are proteolysis products of the CP consisting of the S and P domains only.8,10 In this analysis, we observed small differences from the masses expected from the originally published TCV CP sequence.5 We therefore resequenced the CP gene using cDNA derived from the TCV genomic RNA used here (see Materials and Methods).
We have determined the three-dimensional structures of both native and expanded forms of turnip crinkle virus (TCV), using cryo-electron microscopy, which allows direct visualization of the encapsidated single-stranded RNA and coat protein (CP) N-terminal regions not seen in the high-resolution X-ray structure of the virion. The expanded form, which is a putative disassembly intermediate during infection, arises from a separation of the capsid-forming domains of the CP subunits. Capsid expansion leads to the formation of pores that could allow exit of the viral RNA. A subset of the CP N-terminal regions becomes proteolytically accessible in the expanded form, although the RNA remains inaccessible to nuclease. Sedimentation velocity assays suggest that the expanded state is metastable and that expansion is not fully reversible. Proteolytically cleaved CP subunits dissociate from the capsid, presumably leading to increased electrostatic repulsion within the viral RNA. Consistent with this idea, electron microscopy images show that proteolysis introduces asymmetry into the TCV capsid and allows initial extrusion of the genome from a defined site. The apparent formation of polysomes in wheat germ extracts suggests that subsequent uncoating is linked to translation. The implication is that the viral RNA and its capsid play multiple roles during primary infections, consistent with ribosome-mediated genome uncoating to avoid host antiviral activity.
Maize chlorotic mottle machlomovirus expresses its coat protein from a 1.47-kb subgenomic RNA and makes a 0.34-kb subgenomic RNA
2000, Virology
Analysis of double-stranded RNAs produced in maize plants infected with maize chlorotic mottle machlomovirus (MCMV) and Northern blots of total RNA from infected plants or protoplasts showed two subgenomic RNAs (sgRNAs). Primer extension was used to map these sgRNAs, which are 1.47 and 0.34 kb long. The transcription start sites are nucleotide (nt) 2970 or 2971 for sgRNA1 and nt 4101 for sgRNA2. The 5′ ends of the sgRNAs are similar to one another and to the 5′ end of genomic RNA, and 11 nt sequences immediately upstream of their transcription start sites are similar. The location of the sgRNA1 transcription start site indicates that MCMV expresses a 7-kDa open reading frame (ORF) from nt 2995 to 3202 instead of the predicted 9-kDa ORF from nt 2959 to 3202. In protoplast inoculation experiments, a silent mutation at nt 2965 and a 4-nt change at nt 2959–2962 stopped the synthesis of sgRNA1 and expression of the coat protein ORF, which begins more than 400 nt downstream. Replication of MCMV does not require the expression of any of the ORFs encoded on sgRNA1. SgRNA2 has the potential to encode 2.3-, 2.7-, and 4.6-kDa peptides, but the function, if any, of sgRNA2 is unknown.
Symptom attenuation by a satellite RNA in vivo is dependent on reduced levels of virus coat protein
1999, Virology
Many plant RNA viruses provide replication and encapsidation functions for one or more satellite RNAs (sat-RNAs) that can modulate the symptoms of the associated helper virus. Sat-RNA C, a virulent sat-RNA associated with turnip crinkle virus (TCV), normally intensifies symptoms but can attenuate symptoms if the TCV coat protein (CP) is replaced with that of cardamine chlorotic fleck carmovirus [Kong et al. (1995) Plant Cell 7, 1625–1634] or if TCV contains an alteration in the CP initiation codon (TCV-CPm) [Kong et al. (1997b) Plant Cell 9, 2051–2063]. To further elucidate the mechanism of symptom attenuation by sat-RNA C, the composition of the CP produced by TCV-CPm (CP_CPm) was determined. Our results reveal that CP_CPm likely has two additional amino acids at its N-terminus compared with wild-type TCV CP. TCV-CPm produces reduced levels of CP, and this reduction, not the two additional residues at the CP N-terminus, is responsible for symptom attenuation by sat-RNA C.
Mutational analyses of the putative calcium binding site and hinge of the turnip crinkle virus coat protein
1999, Virology
The turnip crinkle carmovirus (TCV) coat protein (CP) is folded into R (RNA-binding), S (shell), and P (protruding) domains. The S domain is an eight-stranded β barrel common to the coat protein subunits of most RNA viruses. A five-amino-acid hinge connects the S and P domains. In assembled particles, each pair of CP subunits is thought to bind a single calcium ion through interactions with three residues of one subunit and two residues of a neighboring subunit. These five residues comprise the putative calcium-binding site (CBS). The putative CBS and hinge are adjacent to one another. Mutations were introduced into the putative CBS or hinge in an effort to further determine the biological functions of TCV CP. One putative CBS mutant, TCV-M32, exhibited wild-type cell-to-cell movement but failed to move systemically in Nicotiana benthamiana, and particles were not detected. Another putative CBS mutant, TCV-M23, exhibited deficient cell-to-cell movement but particles accumulated in isolated protoplasts. Two other putative CBS mutants, TCV-M22 and -M33, showed wild-type cell-to-cell and systemic movement but elicited mild systemic symptoms that were somewhat delayed. All of the hinge mutants exhibited wild-type movement but some elicited non-wild-type symptoms. Point mutations in the putative CBS or hinge appear to alter virus–ion interactions, secondary structure, or particle conformation, thereby affecting interactions between the CP and plant hosts.
Minimal sequence and structural requirements of a subgenomic RNA promoter for turnip crinkle virus
1999, Virology
Infection of plants or protoplasts with turnip crinkle virus (TCV) results in the synthesis of the genomic RNA and two subgenomic (sg) RNAs of 1.7 kb and 1.45 kb, respectively. Both of the sgRNA promoters were characterized previously and their secondary structures predicted by computer analysis [J. Wang and A. E. Simon (1997).Virology232, 174–186]. Secondary structure-sensitive chemical and enzymatic probes have now been used to determine the structure of the promoter directing synthesis of the 1.45-kb sgRNA, namely the 1.45-kb sgRNA promoter, in solution. The newly obtained structure conforms with the previously predicted hairpin structure except for the hairpin base: four CG base pairs and a CA bulge are present instead of an A bulge. Studies of deletions within the 96-nucleotide (nt) 1.45-kb sgRNA promoter defined a minimal 30-nt core sequence as essential for promoter activity: a 21-nt hairpin and a 9-nt flanking single-stranded sequence. Mutational analysis in the stem section of the core promoter supported a role for the primary sequence and secondary structure in promoter activity. Sequence alterations in the flanking single-stranded region further suggest that the sequence CCCAUUA, encompassing the transcription start site, is required for efficient transcription of the 1.45-kb sgRNA by the TCV RNA-dependent RNA polymerasein vivo.

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^☆: Paper III in this series is Stockley et al. (1986).

^☆☆: This work was supported USDA Competitive grant no. 8500068 (to T.J.M.) and NIH grant no. CA-13202 (to S.C.H.).

^‡: Present address: Department of Genetics, University of Leeds, LS2 9JT, England.

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Journal of Molecular Biology

Structure and assembly of turnip crinkle virus: IV. Analysis of the coat protein gene and implications of the subunit primary structure☆,☆☆

Abstract

Virology

Anal. Biochem.

Virology

Virology

Virology

Virology

Biophys. J.

Advan. Virus Res.

TIBS

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

Biochim. Biophys. Acta