Molecular characterization and intermolecular interaction of coat protein of Prunus necrotic ringspot virus: implications for virus assembly (original) (raw)
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Virology, 2003
Binding of coat protein (CP) to the 3Ј nontranslated region (3Ј-NTR) of viral RNAs is a crucial requirement to establish the infection of Alfamo-and Ilarviruses. In vitro binding properties of the Prunus necrotic ringspot ilarvirus (PNRSV) CP to the 3Ј-NTR of its genomic RNA using purified E. coli-expressed CP and different synthetic peptides corresponding to a 26-residue sequence near the N-terminus were investigated by electrophoretic mobility shift assays. PNRSV CP bound to, at least, three different sites existing on the 3Ј-NTR. Moreover, the N-terminal region between amino acid residues 25 to 50 of the protein could function as an independent RNA-binding domain. Single exchange of some arginine residues by alanine eliminated the RNA-interaction capacity of the synthetic peptides, consistent with a crucial role for Arg residues common to many RNA-binding proteins possessing Arg-rich domains. Circular dichroism spectroscopy revealed that the RNA conformation is altered when amino-terminal CP peptides bind to the viral RNA. Finally, mutational analysis of the 3Ј-NTR suggested the presence of a pseudoknotted structure at this region on the PNRSV RNA that, when stabilized by the presence of Mg 2ϩ , lost its capability to bind the coat protein. The existence of two mutually exclusive conformations for the 3Ј-NTR of PNRSV strongly suggests a similar regulatory mechanism at the 3Ј-NTR level in Alfamo-and Ilarvirus genera.
Adaptive Covariation between the Coat and Movement Proteins of Prunus Necrotic Ringspot Virus
Journal of Virology, 2006
The relative functional and/or structural importance of different amino acid sites in a protein can be assessed by evaluating the selective constraints to which they have been subjected during the course of evolution. Here we explore such constraints at the linear and three-dimensional levels for the movement protein (MP) and coat protein (CP) encoded by RNA 3 of prunus necrotic ringspot ilarvirus (PNRSV). By a maximum-parsimony approach, the nucleotide sequences from 46 isolates of PNRSV varying in symptomatology, host tree, and geographic origin have been analyzed and sites under different selective pressures have been identified in both proteins. We have also performed covariation analyses to explore whether changes in certain amino acid sites condition subsequent variation in other sites of the same protein or the other protein. These covariation analyses shed light on which particular amino acids should be involved in the physical and functional interaction between MP and CP. Finally, we discuss these findings in the light of what is already known about the implication of certain sites and domains in structure and protein-protein and RNA-protein interactions.
Journal of General Virology, 2006
Interactions between viral proteins are critical for virus viability. Bimolecular fluorescent complementation (BiFC) technique determines protein interactions in real-time under almost normal physiological conditions. The coat protein (CP) of Prunus necrotic ringspot virus is required for multiple functions in its replication cycle. In this study, the region involved in CP dimerization has been mapped by BiFC in both bacteria and plant tissue. Full-length and C-terminal deleted forms of the CP gene were fused in-frame to the N-and C-terminal fragments of the yellow fluorescent protein. The BiFC analysis showed that a domain located between residues 9 and 27 from the C-end plays a critical role in dimerization. The importance of this C-terminal region in dimer formation and the applicability of the BiFC technique to analyse viral protein interactions are discussed.
Archives of Virology, 2008
Prunus necrotic ringspot virus (PNRSV) is distributed worldwide, but no molecular data have been previously reported from South American isolates. The nucleotide sequences corresponding to the movement (MP) and coat (CP) proteins of 23 isolates of PNRSV from Chile, Brazil, and Uruguay, and from different Prunus species, have been obtained. Phylogenetic analysis performed with full-length MP and CP sequences from all the PNRSV isolates confirmed the clustering of the isolates into the previously reported PV32-I, PV96-II and PE5-III phylogroups. No association was found between specific sequences and host, geographic origin or symptomatology. Comparative analysis showed that both MP and CP have phylogroup-specific amino acids and all of the motifs previously characterized for both proteins. The study of the distribution of synonymous and nonsynonymous changes along both open reading frames revealed that most amino acid sites are under the effect of negative purifying selection.
Molecular Variability Among Isolates of Prunus Necrotic Ringspot Virus from Different Prunus spp
Phytopathology®, 1999
Viral sequences amplified by polymerase chain reaction from 25 isolates of Prunus necrotic ringspot virus (PNRSV), varying in the symptomatology they cause in six different Prunus spp., were analyzed for restriction fragment polymorphisms. Most of the isolates could be discriminated by using a combination of three different restriction enzymes. The nucleotide sequences of the RNA 4 of 15 of these isolates were determined. Sequence comparisons and phylogenetic analyses of the RNA 4 and coat proteins (CPs) revealed that all of the isolates clustered into three different groups, represented by three previously sequenced PNRSV isolates: PV32, PE5, and PV96. The PE5-type group was characterized by a 5′ untranslated region that was clearly different from that of the other two groups. The PV32-type group was characterized by an extra hexanucleotide consisting of a duplication of the six immediately preceding nucleotides. Although most of the variability was observed in the first third of t...
Journal of General Virology, 2010
The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is required for viral transport. Previous analysis with MPs of other members of the family Bromoviridae has shown that the C-terminal part of these MPs plays a critical role in the interaction with the cognate coat protein (CP) and in cell-to-cell transport. Bimolecular fluorescence complementation and overlay analysis confirm an interaction between the C-terminal 38 aa of PNRSV MP and its cognate CP. Mutational analysis of the C-terminal region of the PNRSV MP revealed that its C-terminal 38 aa are dispensable for virus transport, however, the 4 aa preceding the dispensable C terminus are necessary to target the MP to the plasmodesmata and for the functionality of the protein. The capacity of the PNRSV MP to use either a CP-dependent or a CP-independent cell-to-cell transport is discussed.
Annals of Applied Biology, 2002
Seven isolates of Prunus necrotic ringspot virus (PNRSV) originating from Slovakia were subjected to biological tests under glasshouse conditions. Mainly mild symptoms were observed on chip-budded test cherry rootstocks. The complete sequence for the capsid protein (CP) gene of four isolates was determined. All sequences were 675 nucleotides long and clustered in the largest of four groups delineated by phylogenetic analyses of all so far known PNRSV CP sequences. A set of restriction endonucleases was suggested to differentiate four isolate clusters by restriction enzyme digestion of CP sequences.
Expression of Prunus Necrotic Ringspot Virus Coat Protein in E. coli
Iranian Journal of Virology, 2019
Background and AimsSerological assay is considered as one of the best choices for conducting large number of infection tests. Recombinant DNA technology has been used for expression of virus coat protein (CP) gene in prokaryotic bacterial cells such as Escherichia coli and the recombinant CP (rCP) is used as immunogen in antibody production. Heterologous CP protein expression and purification of the full length Prunus necrotic ringspot virus-PNRSV, Ilarvirus genus, from an Iranian isolate as an antigen was the aim of the study.Materials and MethodsA predominant Iranian PNRSV isolate (PK5) was selected and its CP gene was amplified using specific primers and the nucleotide sequence has been determined. The amplicon was cloned into pET28a(+) expression vector. The amplified CP gene and linearized pET-28a(+) were purified from gel, ligated and transformed into BL21 strain of E. coli. Expression of rCP in transformed BL21 competent cells was tested using SDS-PAGE and Western Blot assays...