ABSTRACT
Diverse animal and plant viruses are able to translocate their virions between neighboring cells via intercellular connections. In this work, we analyze the virion assembly and cell-to-cell movement of a plant closterovirus and reveal a strong correlation between these two processes. The filamentous virions of a closterovirus possess a long body formed by the major capsid protein (CP) and a short tail formed by the minor capsid protein (CPm). Genetic and biochemical analyses show that the functions of these virion components are distinct. A virion body is required primarily for genome protection, whereas a tail represents a specialized device for cell-to-cell movement. Furthermore, tail assembly is mediated by the viral Hsp70 homolog (Hsp70h) that becomes an integral part of the virion. Inactivation of the ATPase domain of Hsp70h results in assembly of tailless virions that are incapable of translocation. A dual role for the viral molecular chaperone Hsp70h in virion assembly and transport, combined with the previous finding of this protein in intercellular channels, allowed us to propose a model of closteroviral movement from cell to cell.
Subject(s)
Capsid/physiology , Cell Movement , Closterovirus/physiology , HSP70 Heat-Shock Proteins/physiology , Membrane Fusion , Plants/virology , Virus Assembly , Amino Acid Sequence , Genome, Viral , Molecular Sequence Data , Sequence Homology, Amino AcidABSTRACT
Three site-specific endonucleases were found in thermophilic strain Bacillus species D6. One of them, BspD6II, is an isoschizomer of Eco57I. The second, BspD6III, is present in the strain in very small amount; therefore, it has not been characterized. This paper is devoted to the third, BspD6I, which recognizes pentanucleotide site 5'-GAGTC-3' and cleaves only one DNA strand at a distance of 4 nucleotides from the site in the 3'-direction in the chain with the GAGTC sequence, i.e., it behaves as a site-specific nickase. Nickase N.BspD6I cleaves one DNA strand only in double-stranded DNA and does not cleave single-stranded DNA. Site-specific methylase SscL1I (an isohypectomer of M*HinfI) that methylates adenine in the sequence 5'-GAGTC-3' prevents DNA hydrolysis by nickase BspD6I.
Subject(s)
Bacillus/enzymology , Deoxyribonuclease I/metabolism , Bacteriophage T7/genetics , Base Sequence , DNA, Viral/metabolism , Deoxyribonuclease I/chemistry , Substrate SpecificityABSTRACT
A beet yellows closterovirus (BYV) variant expressing green fluorescent protein and leaves of BYV local lesion host Claytonia perfoliata were used to reveal genetic requirements for BYV cell-to-cell movement in leaf epidermis and mesophyll. A series of mutations targeting genes that are not involved in amplification of the viral positive-strand RNA was analyzed. The products of genes coding for a 6-kDa hydrophobic protein (p6) and a 64-kDa protein (p64), as well as for minor and major capsid proteins, were found to be essential for intercellular translocation of BYV. In a previous work, we have demonstrated that the BYV HSP70-homolog (HSP70h) also plays a critical role in viral movement (V. V. Peremyslov, Y. Hagiwara, and V. V. Dolja, 1999, Proc. Natl. Acad. Sci. USA, 96, 14771-14776). Altogether, a unique protein quintet including three dedicated movement proteins (p6, p64, and HSP70h) and two structural proteins is required to potentiate the cell-to-cell movement of a closterovirus. The corresponding BYV genes are clustered in a block that is conserved among diverse representatives of the family Closteroviridae.
Subject(s)
Closterovirus/genetics , Closterovirus/physiology , Plants/virology , Capsid/metabolism , Closterovirus/metabolism , Green Fluorescent Proteins , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Movement , Plant Leaves/virology , Plant Viral Movement Proteins , RNA, Viral/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Viral Proteins/genetics , Viral Proteins/metabolism , Viral Structural Proteins/genetics , Viral Structural Proteins/metabolismABSTRACT
The preparation of site-specific endonuclease Bsp123 I was isolated and purified from the thermophilic strain Bacillus species 123. Endonuclease Bsp123 I recognizes the sequence CGCG and cleaves it in the middle between nucleotides G and C, producing blunt ends. Thus, Bsp123 I is an isoschizomer of FnuDII. The maximal activity of the enzyme is displayed at 42 degrees C, pH 8.0, 100 mM NaCl, 10 mM MgCl2.
Subject(s)
Bacillus/enzymology , Deoxyribonucleases, Type II Site-Specific/isolation & purification , DNA, Viral/metabolism , Deoxyribonucleases, Type II Site-Specific/metabolism , Substrate SpecificityABSTRACT
Thermophilic strain Bacillus species AA contains several site-specific endonucleases and three of them have been identified. BspAAI recognizes the sequence 5'-C downward arrow TCGAG-3' and cleaves it after the first "C" forming 4-nucleotide 5'-ends and is an isoschizomer of XhoI. BspAAII recognizes the sequence 5'-T downward arrow CTAGA-3' and cleaves it after the first "T" forming 4-nucleotide 5'-ends and is an isoschizomer of XbaI. BspAAIII recognizes the sequence 5'-GGATCC-3' and is an isomer of BamHI. The optimal temperature and pH values are 42-48 degreesC and 7.0-8.0, respectively.
