Electrostatic properties of cowpea chlorotic mottle virus and cucumber mosaic virus capsids

Electrostatic properties of cowpea chlorotic mottle virus (CCMV) and cucumber mosaic virus (CMV) were investigated using numerical solutions to the Poisson-Boltzmann equation. Experimentally, it has been shown that CCMV particles swell in the absence of divalent cations when the pH is raised from 5 to 7. CMV, although structurally homologous, does not undergo this transition. An analysis of the calculated electrostatic potential confirms that a strong electrostatic repulsion at the calcium-binding sites in the CCMV capsid is most likely the driving force for the capsid swelling process during the release of calcium. The binding interaction between the encapsulated genome material (RNA) inside of the capsid and the inner capsid shell is weakened during the swelling transition. This probably aids in the RNA release process, but it is unlikely that the RNA is released through capsid openings due to unfavorable electrostatic interaction between the RNA and capsid inner shell residues at these openings. Calculations of the calcium binding energies show that Ca(2+) can bind both to the native and swollen forms of the CCMV virion. Favorable binding to the swollen form suggests that Ca(2+) ions can induce the capsid contraction and stabilize the native form.     

Diversity and identity of mechanical properties of icosahedral viral capsids studied with elastic network normal mode analysis

We analyze the mechanical properties and putative dynamical fluctuations of a variety of viral capsids comprising different sizes and quasi-equivalent symmetries by performing normal mode analysis using the elastic network model. The expansion of the capsid to a swollen state is studied using normal modes and is compared with the experimentally observed conformational change for three of the viruses for which experimental data exist. We show that a combination of one or two normal modes captures remarkably well the overall translation that dominates the motion between the two conformational states, and reproduces the overall conformational change. We observe for all of the viral capsids that the nature of the modes is different. In particular for the T=7 virus, HK97, for which the shape of the capsid changes from spherical to faceted polyhedra, two modes are necessary to accomplish the conformational transition. In addition, we extend our study to viral capsids with other T numbers, and discuss the similarities and differences in the features of virus capsid conformational dynamics. We note that the pentamers generally have higher flexibility and propensity to move freely from the other capsomers, which facilitates the shape adaptation that may be important in the viral life cycle.     

Interaction with capsid protein alters RNA structure and the pathway for in vitro assembly of cowpea chlorotic mottle virus

Viruses use sophisticated mechanisms to allow the specific packaging of their genome over that of host nucleic acids. We examined the in vitro assembly of the Cowpea chlorotic mottle virus (CCMV) and observed that assembly with viral RNA follows two different mechanisms. Initially, CCMV capsid protein (CP) dimers bind RNA with low cooperativity and form virus-like particles of 90 CP dimers and one copy of RNA. Longer incubation reveals a different assembly path. At a stoichiometry of about ten CP dimers per RNA, the CP slowly folds the RNA into a compact structure that can be bound with high cooperativity by additional CP dimers. This folding process is exclusively a function of CP quaternary structure and is independent of RNA sequence. CP-induced folding is distinct from RNA folding that depends on base-pairing to stabilize tertiary structure. We hypothesize that specific encapsidation of viral RNA is a three-step process: specific binding by a few copies of CP, RNA folding, and then cooperative binding of CP to the "labeled" nucleoprotein complex. This mechanism, observed in a plant virus, may be applicable to other viruses that do not halt synthesis of host nucleic acid, including HIV.     

The structure of cucumber mosaic virus and comparison to cowpea chlorotic mottle virus

The structure of cucumber mosaic virus (CMV; strain Fny) has been determined to a 3.2-A resolution using X-ray crystallography. Despite the fact that CMV has only 19% capsid protein sequence identity (34% similarity) to cowpea chlorotic mottle virus (CCMV), the core structures of these two members of the Bromoviridae family are highly homologous. As suggested by a previous low-resolution structural study, the 305-A diameter (maximum) of CMV is approximately 12 A larger than that of CCMV. In CCMV, the structures of the A, B, and C subunits are nearly identical except in their N termini. In contrast, the structures of two loops in subunit A of CMV differ from those in B and C. These loops are 6 and 7 residues longer than the analogous regions in CCMV. Unlike that of CCMV, the capsid of CMV does not undergo swelling at pH 7.0 and is stable at pH 9.0. This may be partly due to the fact that the N termini of the B and C subunits form a unique bundle of six amphipathic helices oriented down into the virion core at the threefold axes. In addition, while CCMV has a cluster of aspartic acid residues at the quasi-threefold axis that are proposed to bind metal in a pH-dependent manner, this cluster is replaced by complementing acids and bases in CMV. Finally, this structure clearly demonstrates that the residues important for aphid transmission lie at the outermost portion of the betaH-betaI loop and yields details of the portions of the virus that are hypothesized to mediate binding to aphid mouthparts.     

Virion swelling is not required for cotranslational disassembly of cowpea chlorotic mottle virus in vitro.

The mechanism by which virions of cowpea chlorotic mottle virus (CCMV) disassemble and allow for translation of the virion RNA is not well understood. Previous models have suggested that virion swelling is required to expose the virion RNA for translation in a process referred to as cotranslational disassembly (M. Brisco, R. Hull, and T. M. A. Wilson, Virology 148:210-217, 1986; J. W. Roenhorst, J. W. M. van Lent, and B. J. M. Verduin, Virology 164:91-98, 1988; J. W. Roenhorst, J. M. Verduin, and R. W. Goldbach, Virology 168:138-146, 1989). Previous work in our laboratory has identified point mutations in the CCMV coat protein which result in virions with altered swelling characteristics (J. Fox, F. G. Albert, J. Speir, and M. J. Young, Virology 227:229-233, 1997; J. M. Fox, X. Zhao, J. A. Speir, and M. J. Young, Virology 222:115-122, 1996). The wild-type and mutant CCMV virions were used to correlate virion swelling with the ability of virion RNA to be translated in a cell-free wheat germ extract. Mutant virions unable to swell (cpK42R) are as infectious as wild-type virions in vivo, and the levels of translated encapsidated virion RNA are similar to those of wild-type virions in vitro. Mutant virions capable of swelling but not of disassembling in vitro (cpR26C) are noninfectious and have severely reduced levels of translation of the encapsidated virion RNA in vitro. These studies suggest that virion swelling is not required for the cotranslational disassembly of CCMV. Additionally, the results indicate that there is a pH-dependent structural transition in the virion, other than swelling, that results in the RNA's being exposed for translation in vitro. An alternative model suggesting that cotranslational disassembly of CCMV involves presentation of the virion RNA through the virion fivefold axis is proposed.     

Metal binding to cowpea chlorotic mottle virus using terbium(III) fluorescence

Metals are thought to play a role in the structure of many viruses. The crystal structure of the T=3 icosahedral cowpea chlorotic mottle virus (CCMV) suggests the presence of 180 unique metal-binding sites in the assembled protein cage. Each of these sites is thought to involve the coordination of the metal by five amino acids contributed from two adjacent coat protein subunits. We have used fluorescence resonance energy transfer (FRET), from tryptophan residues proximal to the putative metal-binding sites, to probe Tb(III) binding to the virus. Binding of Tb(III) was investigated on the wild-type virus and a mutant where the RNA binding ability of the virus was removed. Tb(III) binding was observed both in the wild-type virus (K_d=19 M) and the mutant (K_d=17 M), as monitored by the increase in Tb(III) fluorescence (545 nm) and concomitant decrease in tryptophan fluorescence (342 nm). Competitive binding experiments showed Ca(II) to have about 100-fold less affinity for the binding sites (K_d=1.97 mM). This is the first direct evidence of metal binding to the putative metal-binding sites, originally suggested from the crystal structure of CCMV.     

The amino acid sequences of the tryptic peptides of cowpea chlorotic mottle virus protein

The amino acid sequences of the major tryptic peptides from the coat protein of wild type cowpea chlorotic mottle virus are presented. The sequences have been determined by a combination of enzyme hydrolysis, mass spectrometry and Edman degradation, and the relative usefulness of mass spectrometry in this peptide sequence determination is discussed.     

Encapsulation and crystallization of Prussian blue nanoparticles by cowpea chlorotic mottle virus capsids

Cowpea chlorotic mottle virus (CCMV) capsids were used to encapsulate Prussian blue (PB) particles based on electrostatic interaction. A negatively-charged metal complex, hexacyanoferrate (III), was entrapped inside the capsids through the disassembly/reassembly process under a pH change from 7.5 to 5.2. The loaded capsids reacted with a second Fe(II) to fabricate PB particles. The synthesis of PB in CCMV capsids was confirmed by a unique colour transition at 710 nm and by size-exclusion FPLC. Transmission electron microscopy images of PB-CCMV biohybrids presented discrete spherical particles with a relatively homogeneous size. Dynamic light scattering of PB-CCMV showed two peaks of 29.2 ± 1.7 nm corresponding to triangulation number T = 3 particles, and 17.5 ± 1.2 nm of pseudo T = 2 particles. The encapsulation and crystallization of PB in CCMV provided an efficient method for the self-organization of bimetallic nanoparticles.     

RNA-protein interactions and secondary structures of cowpea chlorotic mottle virus for in vitro assembly

Laser Raman spectroscopy of the cowpea chlorotic mottle virus (CCMV) in native (pH 5.0) and partially swollen (pH 7.5) states reveals the presence of small percentages of protonated adenine (less than 15%) and cytosine (less than 7%) bases in the encapsidated RNA molecule of the native virion. The protonated bases are titrated with pH-induced swelling of the virus. Titration of putative COOH groups of aspartic and glutamic side chains of the virion subunit cannot be detected over the same pH range, which suggests that carboxyl anions (CO-2) and protonated bases are both available at pH 5 to stabilize the ribonucleoprotein particles by electrostatic interactions. The highly (95%) ordered secondary structure of encapsidated RNA may undergo a small additional increase (less than 3%) in ordered structure with release from the virion, suggesting at most a marginal structure-distorting influence from protein contacts in the native particle. The Raman spectra of the virion are also compared by difference spectroscopy with spectra of capsids (empty shells devoid of RNA), subunit dimers, and protein-free RNA. The results indicate that the subunit structure is altered by the release of RNA from the virion, as well as by the swelling of the virion. Amino acid residues and protein secondary structures that are affected in these in vitro assembly and disassembly processes are identified from their characteristic Raman lines. Two classes of cysteinyl SH groups, solvent exposed and solvent protected, are revealed for the capsid and virion subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Infection of cowpea mesophyll protoplasts with cowpea chlorotic mottle virus (CCMV) RNA encapsulated in large liposomes

It has been demonstrated that cowpea chlorotic mottle virus RNA encapsulated in phosphatidyl serine/cholesterol reverse evaporation vesicles (REV) could infect cowpea mesophyll protoplasts under conditions known to enhance liposome-protoplast interactions. Positively charged phosphatidylcholine/stearylamine multilamellar liposomes did not deliver functional CCMV RNA despite their very high nucleic acid trapping capacity and their high affinity for protoplasts.     

Dipsacus fullonum: an Alternative Host of Sunflower chlorotic mottle virus in Argentina

Dipsacus fullonum L. is a biennial weed that is widely spread in the Pampean region of Argentina. Plants of this species showing leaf mosaic symptoms were collected near sunflower ( Helianthus annuus L.) crops infected with Sunflower chlorotic mottle virus (SuCMoV). Symptomatic plants were tested by biological, serological and molecular assays. Sunflower and Nicotiana occidentalis L. plants mechanically inoculated with extracts from symptomatic D. fullonum leaves developed typical SuCMoV symptoms. Samples were DAS-ELISA positive when probed with a SuCMoV antiserum. RT-PCR was used to amplify the whole coat protein gene of the infecting virus. An 807 bp fragment was amplified, cloned and sequenced. The deduced amino acid sequence (EU606023) shared 96% amino acid identity with the SuCMoV sequence (AF255677). These results indicate that D. fullonum mosaic is caused by an isolate of SuCMoV. Thus, this finding has epidemiological relevance as the weed may act as a natural virus reservoir.     

Expression and self-assembly of cowpea chlorotic mottle virus-like particles in Pseudomonas fluorescens

Coat protein of the cowpea chlorotic mottle virus (CCMV), a plant bromovirus, has been expressed in a soluble form in a prokaryote, Pseudomonas fluorescens, and assembled into virus-like particles (VLPs) in vivo that were structurally similar to the native CCMV particles derived from plants. The CCMV VLPs were purified by PEG precipitation followed by separation on a sucrose density gradient and analyzed by size exclusion chromatography, UV spectrometry, and transmission electron microscopy. DNA microarray experiments revealed that the VLPs encapsulated very large numbers of different host RNAs in a non-specific manner. The development of a P. fluorescens expression system now enables production of CCMV VLPs by bacterial fermentation for use in pharmaceutical or nanotechnology applications.     

Complete nucleotide sequences of the coat protein messenger RNAs of brome mosaic virus and cowpea chlorotic mottle virus

The nucleotide sequences of the subgenomic coat protein messengers (RNA4's) of two related bromoviruses, brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV), have been determined by direct RNA and CDNA sequencing without cloning. BMV RNA4 is 876 b long including a 5' noncoding region of nine nucleotides and a 3' noncoding region of 300 nucleotides. CCMV RNA 4 is 824 b long, including a 5' noncoding region of 10 nucleotides and a 3' noncoding region of 244 nucleotides. The encoded coat proteins are similar in length (188 amino acids for BMV and 189 amino acids for CCMV) and display about 70% homology in their amino acid sequences. Length difference between the two RNAs is due mostly to a single deletion, in CCMV with respect to BMV, of about 57 b immediately following the coding region. Allowing for this deletion the RNAs are indicate that mutations leading to divergence were constrained in the coding region primarily by the requirement of maintaining a favorable coat protein structure and in the 3' noncoding region primarily by the requirement of maintaining a favorable RNA spatial configuration.     

Electrostatic interaction between RNA and protein capsid in cowpea chlorotic mottle virus simulated by a coarse-grain RNA model and a Monte Carlo approach

Although many viruses have been crystallized and the protein capsid structures have been determined by x-ray crystallography, the nucleic acids often cannot be resolved. This is especially true for RNA viruses. The lack of information about the conformation of DNA/RNA greatly hinders our understanding of the assembly mechanism of various viruses. Here we combine a coarse-grain model and a Monte Carlo method to simulate the distribution of viral RNA inside the capsid of cowpea chlorotic mottle virus. Our results show that there is very strong interaction between the N-terminal residues of the capsid proteins, which are highly positive charged, and the viral RNA. Without these residues, the binding energy disfavors the binding of RNA by the capsid. The RNA forms a shell close to the capsid with the highest densities associated with the capsid dimers. These high-density regions are connected to each other in the shape of a continuous net of triangles. The overall icosahedral shape of the net overlaps with the capsid subunit icosahedral organization. Medium density of RNA is found under the pentamers of the capsid. These findings are consistent with experimental observations.     

Normal mode calculations of icosahedral viruses with full dihedral flexibility by use of molecular symmetry

The study of the dynamics and thermodynamics of small icosahedral virus capsids is an active field of research. Normal mode analysis is one of the computational tools that can provide important insights into the conformational changes of the virus associated with cell entry or caused by changing of the physicochemical environment. Normal mode analysis of virus capsids has been limited due to the size of these systems, which often exceed 50,000 residues. Here we present the first normal mode calculation with full dihedral flexibility of several virus capsids, including poliovirus, rhinovirus, and cowpea chlorotic mottle virus. The calculations were made possible by applying group theoretical methods, which greatly simplified the calculations without any approximation beyond the all-atom force field representations in general use for smaller protein systems. Since a full Cartesian basis set was too large to be handled by the available computer memory, we used a basis set that includes all internal dihedral angles of the system with the exception of the peptide bonds, which were assumed rigid. The fluctuations of the normal modes are shown to correlate well with crystallographic temperature factors. The motions of the first several normal modes of each symmetry type are described. A hinge bending motion in poliovirus was found that may be involved in the mechanism by which bound small molecules inhibit conformational changes of the capsid. Fully flexible normal mode calculations of virus capsids are expected to increase our understanding of virus dynamics and thermodynamics, and can be useful in the refinement of cryo-electron microscopy structures of viruses.     

Molecular modeling of the RNA binding N-terminal part of cowpea chlorotic mottle virus coat protein in solution with phosphate ions.

The RNA-binding N-terminal arm of the coat protein of cowpea chlorotic mottle virus has been studied with five molecular dynamics simulations of 2.0 ns each. This 25-residue peptide (pep25) is highly charged: it contains six Arg and three Lys residues. An alpha-helical fraction of the sequence is stabilized in vitro by salts. The interaction of monophosphate (Pi) ions with pep25 was studied, and it was found that only two Pi ions are bound to pep25 on average, but water-mediated interactions between pep25 and Pi, which provide electrostatic screening for intrapeptide interactions, are abundant. Shielding by the Pi ions of repulsive electrostatic interactions between Arg sidechains increases the alpha-helicity of pep25. A hydrogen bond at the N-terminal end of the alpha-helix renders extension of the alpha-helix in the N-terminal direction impossible, in agreement with evidence from nuclear magnetic resonance experiments.     

Agrobacterium-mediated transformation of monocot and dicot plants using the NCR promoter derived from soybean chlorotic mottle virus

The NCR promoter (PNCR) from soybean chlorotic mottle virus (SoyCMV) was used to express the selectable marker, neomycin phosphotransferase (nptII) gene, in Agrobacterium-mediated transformation of both monocot (rice) and dicot (tobacco) plants. A multi-cloning site for insertion of a gene of interest into the binary vector pTN is located proximal to the right border region of T-DNA. When chimeric genes under the control of other strong promoters were located in a head-to-head orientation to the PNCR-nptII gene, kanamycin-resistant tobacco shoots were generated more efficiently than when using the original pTN vectors. This suggests that the enhancer-like sequences in the promoters adjacent to PNCR may promote expression of the PNCR-nptII gene. Received: 20 August 1999 / Revision received: 16 November 1999 / Accepted: 19 November 1999