ABSTRACT
The viral genome-linked protein (VPg) of Potyviruses is covalently attached to the 5’ end of the genomic RNA. Towards biophysical characterization, the VPg coding region of Cardamom mosaic virus (CdMV) was amplified from the cDNA and expressed in E. coli. Most of the expressed VPg aggregated as inclusion bodies that were solubilized with urea and refolded with L-arginine hydrochloride. The various forms of CdMV VPg (native, denatured and refolded) were purified and the conformational variations between these forms were observed with fluorescence spectroscopy. Native and refolded CdMV VPg showed unordered secondary structure in the circular dichroism (CD) spectrum. The model of CdMV VPg was built based on the crystal structure of phosphotriesterase (from Pseudomonas diminuta), which had the maximum sequence homology with VPg to identify the arrangement of conserved amino acids in the protein to study the functional diversity of VPg. This is the first report on the VPg of CdMV, which is classified as a new member of the Macluravirus genus of the Potyviridae family.
Subject(s)
Circular Dichroism , Elettaria/metabolism , Genome, Viral/genetics , Inclusion Bodies/genetics , Inclusion Bodies/metabolism , Models, Molecular , Mosaic Viruses/genetics , Mosaic Viruses/metabolism , Plant Viruses/genetics , Plant Viruses/metabolism , Potyvirus/genetics , Potyvirus/metabolism , Protein Refolding , Protein Structure, Secondary , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/isolation & purification , RNA-Binding Proteins/metabolism , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
The complete nucleotide sequence of an Indian strain of Cymbidium mosaic virus (CymMV)was determined and compared with other potexviruses. Phylogenetic analyses on the basis of RNA-dependent RNA polymerase (RdRp), triple gene block protein and coat protein (CP) amino acid sequences revealed that CymMV is closely related to the Narcissus mosaic virus (NMV), Scallion virus X (SVX), Pepino mosaic virus (PepMV)and Potato aucuba mosaic virus (PAMV). Different sets of primers were used for the amplification of different regions of the genome through RT-PCR and the amplified genes were cloned in a suitable vector. The full genome of the Indian isolate of CymMV from Phaius tankervilliae shares 96-97% similarity with isolates reported from other countries. It was found that the CP gene of CymMV shares a high similarity with each other and other potexviruses. One of the Indian isolates seems to be a recombinant formed by the intermolecular recombination of two other CymMV isolates. The phylogenetic analyses, Recombination Detection Program (RDP2)analyses and sequence alignment survey provided evidence for the occurrence of a recombination between an Indian isolate (AM055720) as the major parent, and a Korean type-2 isolate (AF016914) as the minor parent. Recombination was also observed between a Singapore isolate (U62963) as the major parent,and a Taiwan CymMV (AY571289) as the minor parent.
Subject(s)
Base Sequence , Cloning, Molecular , DNA Primers , Genome, Viral , Mosaic Viruses/genetics , Open Reading Frames , Potexvirus/genetics , Recombination, Genetic , Sequence Analysis, DNAABSTRACT
A survey was conducted to study the biological and genetic diversity of Cardamom mosaic virus (CdMV) that causes the most widespread disease in the cardamom growing area in the Western Ghats of south India. Six distinct subgroups were derived based on their symptomatology and host range from the sixty isolates collected. The serological variability between the virus isolates was analysed by ELISA and Western blotting. The 3 terminal region consisting of the coat protein (CP) coding sequence and 3 untranslated region (3 UTR) was cloned and sequenced from seven isolates. Sequence comparisons revealed considerable genetic diversity among the isolates in their CP and 3 UTR, making CdMV one of the highly variable members of Potyviridae. The possible occurrence of recombination between the isolates and the movement of the virus in the cardamom tract of south India are discussed.
Subject(s)
3' Untranslated Regions/classification , Amino Acid Sequence , Capsid Proteins/classification , Genetic Variation , India , Molecular Sequence Data , Mosaic Viruses/genetics , Phylogeny , Plant Viruses/genetics , Sequence Alignment , Sequence Analysis, RNAABSTRACT
An antiviral protein (25 kD) isolated from leaves of Celosia cristata (CCP 25) was tested for depurination study on ribosomal RNA from yeast. Ribosomal RNA yielded 360 nucleotide base fragment after treatment with CCP 25 indicating that CCP 25 was a ribosome inactivating protein. CCP 25 also inhibited translation of brome mosaic virus (BMV) and pokeweed mosaic virus (PMV) RNAs in rabbit reticulocyte translation system. The radioactive assay showed that incorporation of [35S]-methionine was less in translation proteins of BMV nucleic acid when CCP 25 was added to translation system. This indicated that antiviral protein from Celosia cristata not only depurinated ribosomal RNA but also inhibited translation of viral RNA in vitro.