ABSTRACT
Leptospirosis, a global reemerging zoonosis caused by the spirochete Leptospira, has severe human and veterinary implications. Cell wall hydrolase (LIC_10271) with LytM (peptidase M23) and LysM domains are found to be associated with various pathogenic bacteria. These domains regulate effects on extracellular matrix and biofilm components, which promote cell wall remodeling and pathogen dissemination in the host. In this study, we present the cloning, expression, purification, and characterization of LIC_10271. To determine the localization of LIC_10271 within the inner membrane of Leptospira, Triton X-114 subcellular fractionation and immunoblot studies were performed. Furthermore, r-LIC_10271 binds with peptidoglycan, lipopolysaccharide, and laminin in a dose-dependent manner. Analysis of the signal peptide, M23, and LysM domains revealed conservation primarily within the P1 group of Leptospira, which encompasses the most pathogenic species. Moreover, the presence of native-LIC_10271 in the inner membrane and the distribution of M23 and LysM domains across pathogenic strains indicates their potential involvement in the interaction between the host and Leptospira.
Subject(s)
Leptospira interrogans , Leptospira , Humans , Laminin/metabolism , Lipopolysaccharides/metabolism , Peptidoglycan/metabolism , Leptospira interrogans/genetics , Leptospira interrogans/metabolism , Hydrolases/metabolism , Leptospira/genetics , Cell Wall/metabolism , Protein BindingABSTRACT
Banana bract mosaic virus (BBrMV), belongs to the genus Potyvirus and it is an important viral pathogen of bananas and plantains. The eukaryotic translation initiation factor, eIF4E, and its isoform play key roles during the virus infection in plants, particularly Potyvirus. The present study was undertaken to determine the role of BBrMV-viral protein genome-linked (VPg) in virus infectivity by analyzing the interaction with the eukaryotic translation initiation factor eIF4E through yeast two-hybrid system. The results suggest that plantain cv. Nendran eIF4E plays an essential role in the initiation of the translation of capped mRNAs and its association with VPg would point to a role of the viral protein in the translation of the virus and may potentially contribute to BBrMV resistance.
ABSTRACT
The NIa protease of Potyviridae is the major viral protease that processes potyviral polyproteins. The NIa protease coding region of Cardamom mosaic virus (CdMV) is amplified from the viral cDNA, cloned and expressed in Escherichia coli. NIa protease forms inclusion bodies in E.coli. The inclusion bodies are solubilized with 8 M urea, refolded and purified by Nickel-Nitrilotriacetic acid affinity chromatography. Three-dimensional modeling of the CdMV NIa protease is achieved by threading approach using the homologous X-ray crystallographic structure of Tobacco etch mosaic virus NIa protease. The model gave an insight in to the substrate specificities of the NIa proteases and predicted the complementation of nearby residues in the catalytic triad (H42, D74 and C141) mutants in the cis protease activity of CdMV NIa protease.
Subject(s)
Endopeptidases/chemistry , Endopeptidases/isolation & purification , Mosaic Viruses/enzymology , Viral Proteins/chemistry , Viral Proteins/isolation & purification , Amino Acid Sequence , DNA, Complementary/chemistry , DNA, Complementary/metabolism , DNA, Viral/chemistry , DNA, Viral/metabolism , Elettaria/virology , Endopeptidases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Models, Molecular , Molecular Sequence Data , Mosaic Viruses/metabolism , Substrate Specificity , Viral Proteins/metabolismABSTRACT
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)
Elettaria/virology , Genome, Viral/genetics , Inclusion Bodies/genetics , Inclusion Bodies/virology , Models, Molecular , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , Circular Dichroism , Elettaria/metabolism , Inclusion Bodies/metabolism , Mosaic Viruses/genetics , Mosaic Viruses/metabolism , Plant Viruses/genetics , Plant Viruses/metabolism , Potyvirus/genetics , Potyvirus/metabolism , Protein Refolding , Protein Structure, Secondary , RNA-Binding Proteins/isolation & purification , RNA-Binding Proteins/metabolism , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
All RNA viruses encode an RNA-dependent RNA polymerase (RdRP) that is required for replication of the viral genome. Nuclear inclusion b (NIb) gene codes for the RdRp in Potyviridae viruses. In this study, expression, solubilization and purification of NIb protein of Cardamom mosaic virus (CdMV) is reported. The objective of the present study was to express and purify the NIb protein of CdMV on a large scale for structural characterization, as the structure of the RdRp from a plant virus is yet to be determined. However, the expression of NIb protein with hexa-histidine tag in Escherichia coli led to insoluble aggregates. Out of all the approaches [making truncated versions to reduce the size of protein; replacing an amino acid residue likely to be involved in hydrophobic intermolecular interactions with a hydrophilic one; expressing the protein along with chaperones; expression in Origami cells for proper disulphide bond formation, in E. coli as a fusion with maltose-binding protein (MBP) and in Nicotiana tabacum] to obtain the RdRp in a soluble form, only expression in E. coli as a fusion with MBP and its expression in N. tabacum were successful. The NIb expressed in plant or as a fusion with MBP in E. coli can be scaled up for further work.
Subject(s)
DNA-Directed RNA Polymerases/metabolism , Mosaic Viruses/metabolism , Viral Proteins/metabolism , Amino Acid Sequence , Base Sequence , DNA Primers , DNA-Directed RNA Polymerases/chemistry , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/isolation & purification , Electrophoresis, Polyacrylamide Gel , Molecular Sequence Data , Mutagenesis, Site-Directed , Sequence Homology, Amino Acid , Solubility , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/isolation & purificationABSTRACT
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.
