Visualization and characterization of tobacco mosaic virus movement protein binding to single-stranded nucleic acids.

Cell-to-cell spread of tobacco mosaic virus (TMV) is presumed to occur through plant intercellular connections, the plasmodesmata. Viral movement is an active process mediated by a specific virus-encoded P30 protein. P30 has at least two functions, to cooperatively bind single-stranded nucleic acids and to increase plasmodesmatal permeability. Here, we visualized P30 complexes with single-stranded DNA and RNA. These complexes are long, unfolded, and very thin (1.5 to 2.0 nm in diameter). Unlike TMV virions (300 x 18 nm), the complexes are compatible in size with the P30-induced increase in plasmodesmatal permeability (2.4 to 3.1 nm), making them likely candidates for the structures involved in the cell-to-cell movement of TMV. Mutational analysis using single and double deletion mutants of P30 revealed three regions potentially important for the protein function. Amino acid residues 65 to 86 possibly are required for correct folding of the active protein, and the regions between amino acid residues 112 to 185 and 185 to 268 potentially contain two independently active single-stranded nucleic acid binding domains designated binding domains A and B, respectively.     

The P30 movement protein of tobacco mosaic virus is a single-strand nucleic acid binding protein

The P30 protein of tobacco mosaic virus (TMV) is required for cell to cell movement of viral RNA, which presumably occurs through plant intercellular connections, the plasmodesmata. The mechanism by which P30 mediates transfer of TMV RNA molecules through plasmodesmata channels is unknown. We have identified P30 as an RNA and single-stranded (ss) DNA binding protein. Binding of purified P30 to ss nucleic acids is strong, highly cooperative, and sequence nonspecific with a minimal binding site of 4-7 nucleotides per P30 monomer. In-frame deletions across P30 were used to localize the ss nucleic acid binding domain to within amino acid residues 65-86 of the protein. We propose that binding of P30 to TMV RNA creates an unfolded protein-RNA complex that functions as an intermediate in virus cell to cell movement through plasmodesmata.     

Distinct functional domains in the cowpea mosaic virus movement protein.

Cell-to-cell movement of cowpea mosaic virus particles in plants takes place with the help of tubules that penetrate presumably modified plasmodesmata. These tubules, which are built up by the virus-encoded 48-kDa movement protein (MP), are also formed on single protoplast cells. To determine whether the MP contains different functional domains, the effect of mutations in its coding region was studied. Mutations between amino acids 1 and 313 led to complete abolishment of the tubule-forming capacity, while a deletion in the C-terminal region resulted in tubules that could not take up virus particles. From these observations, it is concluded that the MP contains at least two distinct domains, one that is involved in tubule formation and that spans amino acids 1 and 313 and a second that is probably involved in the incorporation of virus particles in the tubule and that is located in the C terminus between amino acids 314 and 331.     

Cellular nucleic acid binding protein binds G-rich single-stranded nucleic acids and may function as a nucleic acid chaperone

Cellular nucleic acid binding protein (CNBP) is a small single-stranded nucleic acid binding protein made of seven Zn knuckles and an Arg-Gly rich box. CNBP is strikingly conserved among vertebrates and was reported to play broad-spectrum functions in eukaryotic cells biology. Neither its biological function nor its mechanisms of action were elucidated yet. The main goal of this work was to gain further insights into the CNBP biochemical and molecular features. We studied Bufo arenarum CNBP (bCNBP) binding to single-stranded nucleic acid probes representing the main reported CNBP putative targets. We report that, although bCNBP is able to bind RNA and single-stranded DNA (ssDNA) probes in vitro, it binds RNA as a preformed dimer whereas both monomer and dimer are able to bind to ssDNA. A systematic analysis of variant probes shows that the preferred bCNBP targets contain unpaired guanosine-rich stretches. These data expand the knowledge about CNBP binding stoichiometry and begins to dissect the main features of CNBP nucleic acid targets. Besides, we show that bCNBP presents a highly disordered predicted structure and promotes the annealing and melting of nucleic acids in vitro. These features are typical of proteins that function as nucleic acid chaperones. Based on these data, we propose that CNBP may function as a nucleic acid chaperone through binding, remodeling, and stabilizing nucleic acids secondary structures. This novel CNBP biochemical activity broadens the field of study about its biological function and may be the basis to understand the diverse ways in which CNBP controls gene expression.     

The amino acid sequence of the cowpea strain of tobacco mosaic virus protein.

The amino acid sequence of the coat protein of the cowpea strain of tobacco mosaic virus (cowpea virus) has been determined. The tryptic peptide overlaps were obtained by digesting the protein with chymotrypsin and separating and analysing the lysine-and arginine-containing chymotryptic peptides. The primary structure of cowpea virus protein has been found to differ markedly from that of any other known strain of tobacco mosaic virus, and contains 3 amino acid residues more and 96 amino acid changes from the type strain. The significance of the distribution of those areas of the protein in which the amino acid residues are the same for all naturally occurring strains and chemically induced mutants of tobacco mosaic virus so far studied and the residues that form the important carboxyl-carboxylate pairs are discussed.     

The replication of cowpea mosaic virus

Cowpea mosaic virus (CPMV) is the type member of the comovirus group, which contains 14 different plant viruses that have the same structural organization of genomic RNAs and virions and use the same mechanism for expression of the viral RNAs. The combined structure and organization of the two CPMV genomic RNAs is strikingly similar to that of the single genome of animal picornaviruses. This suggests a common ancestry and similar replication mechanisms. CPMV is by far the best-studied comovirus and we shall limit this review to some recent data on this virus. For additional general information the reader is referred to other recent reviews on CPMV and comoviruses^1,2.     

Murine protein which binds preferentially to oligo-C-rich single-stranded nucleic acids.

Two single-stranded nucleic acid binding proteins mCBP and mCTBP were identified by means of their binding to a potential recombination hotspot in LTRs of mouse retro-transposons. Both are nuclear proteins of 35 and 55 kDa respectively. mCBP binds preferentially to oligo dC, mCTBP to oligo dCdT. mCBP was purified and its cDNA was isolated and sequenced.     

The design of single-stranded nucleic acid knots

A general strategy is described for the synthesis of single-stranded nucleic acid knots. Control of nucleic acid sequence is used to direct the formation of secondary structures that produce the target topology. The key feature of the strategy is the equation of a half-turn of double helical DNA or RNA with a node in a knot. By forming nodes from complementary DNA sequences, it appears possible to direct the assembly of any simple knot. Stabilization of individual nodes may be achieved by constructing them from long regions containing both B-DNA and Z-DNA. Control over the braiding of DNA that acts as a link between node-forming domains can be realized by condensing the nodes into well-defined DNA structures, such as extended domains of linear duplex, branched junctions, antijunctions or mesojunctions. Further topological control may be derived from the pairing of linker regions to complementary single-stranded molecules, thereby preventing them from braiding in an undesirable fashion.     

Rapid detection of cowpea aphid-borne mosaic virus in cowpea seeds

Four cultivars of cowpea (Vigna unguiculata [L]. Walp.) were infected with cowpea aphid-borne mosaic virus (CABMV) by natural infection in field plots. Seeds taken from these plants were tested for the presence of the virus by ELISA and symptom observation on the plantlets grown from the seeds. A biotin/ streptavidin ELISA technique was used and found to be more sensitive than a standard ELISA protocol for detecting CABMV infection in seed. There was a good correlation between the ELISA detection of CABMV in tissue taken from single cowpea seeds and subsequent development of infected plants grown from the same seeds. The ELISA technique is reliable for selecting CABMV-free stocks of cowpea seeds.     

The binding of fd gene-5 protein to single-stranded nucleic acid.

The gene-5 protein of the fd filamentous bacterial virus binds to single-stranded DNA over a pH range of 2-10.3. Binding to fd DNA is several hundred-fold stronger than to bacteriophage R17 RNA or to DNA tetranucleotides.     

Dimerization of recombinant tobacco mosaic virus movement protein

The p30 movement protein (MP) is essential for cell-to-cell spread of tobacco mosaic virus in planta. We used anion-exchange chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to obtain highly purified 30-kDa MP, which migrated as a single band in native PAGE. Analytical ultracentrifugation suggested that the protein was monodisperse and dimeric in the nonionic detergent n-octyl-beta-D-glucopyranoside. Circular dichroism (CD) spectroscopy showed that the detergent-solubilized protein contained significant alpha-helical secondary structure. Proteolysis of the C-tail generated a trypsin-resistant core that was a mixture of primarily monomers and some dimers. We propose that MP dimers are stabilized by electrostatic interactions in the C terminus as well as hydrophobic interactions between putative transmembrane alpha-helical coiled coils.     

Role of the alfalfa mosaic virus movement protein and coat protein in virus transport

The movement protein (MP) and coat protein (CP) encoded by Alfalfa mosaic virus (AMV) RNA 3 are both required for virus transport. RNA 3 vectors that expressed nonfused green fluorescent protein (GFP), MP:GPF fusions, or GFP:CP fusions were used to study the functioning of mutant MP and CP in protoplasts and plants. C-terminal deletions of up to 21 amino acids did not interfere with the function of the CP in cell-to-cell movement, although some of these mutations interfered with virion assembly. Deletion of the N-terminal 11 or C-terminal 45 amino acids did not interfere with the ability of MP to assemble into tubular structures on the protoplast surface. Additionally, N- or C-terminal deletions disrupted tubule formation. A GFP:CP fusion was targeted specifically into tubules consisting of a wild-type MP. All MP deletion mutants that showed cell-to-cell and systemic movement in plants were able to form tubular structures on the surface of protoplasts. Brome mosaic virus (BMV) MP did not support AMV transport. When the C-terminal 48 amino acids were replaced by the C-terminal 44 amino acids of the AMV MP, however, the BMV/AMV chimeric protein permitted wild-type levels of AMV transport. Apparently, the C terminus of the AMV MP, although dispensable for cell-to-cell movement, confers specificity to the transport process.     

The geminivirus BR1 movement protein binds single-stranded DNA and localizes to the cell nucleus.

Plant viruses encode movement proteins that are essential for infection of the host but are not required for viral replication or encapsidation. Squash leaf curl virus (SqLCV), a bipartite geminivirus with a single-stranded DNA genome, encodes two movement proteins, BR1 and BL1, that have been implicated in separate functions in viral movement. To further elucidate these functions, we have investigated the nucleic acid binding properties and cellular localization of BR1 and BL1. In this study, we showed that BR1 binds strongly to single-stranded nucleic acids, with a higher affinity for single-stranded DNA than RNA, and is localized to the nucleus of SqLCV-infected plant cells. In contrast, BL1 binds only weakly to single-stranded nucleic acids and not at all to double-stranded DNA. The nuclear localization of BR1 and the previously demonstrated plasma membrane localization of BL1 were also observed when these proteins were expressed from baculovirus vectors in Spodoptera frugiperda insect cells. The biochemical properties and cellular locations of BR1 and BL1 suggest a model for SqLCV movement whereby BR1 is involved in the shuttling of the genome in and/or out of the nucleus and BL1 acts at the plasma membrane/cell wall to facilitate viral movement across cell boundaries.