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Brownian motion of Cowpea mosaic virus (CPMV) in water was measured using small-angle X-ray photon correlation spectroscopy (SA-XPCS) at 19.2 µs time resolution. It was found that the decorrelation time τ(Q) = 1/DQ 2 up to Q = 0.091 nm -1 . The hydrodynamic radius R H determined from XPCS using Stokes-Einstein diffusion D = kT/(6πηR H ) is 43% larger than the geometric radius R 0 determined from SAXS in the 0.007 M K 3 PO 4 buffer solution, whereas it is 80% larger for CPMV in 0.5 M NaCl and 104% larger in 0.5 M (NH 4 ) 2 SO 4 , a possible effect of aggregation as well as slight variation of the structures of the capsid resulting from the salt-protein interactions., (open access.)

Prognosis and treatment of metastatic cancer continues to be one of the most difficult and challenging areas of oncology. Treatment usually consists of chemotherapeutics, which may be ineffective due to drug resistance, adverse effects, and dose-limiting toxicity. Therefore, novel approaches such as immunotherapy have been investigated to improve patient outcomes and minimize side effects. S100A9 is a calcium-binding protein implicated in tumor metastasis, progression, and aggressiveness that modulates the tumor microenvironment into an immunosuppressive state. S100A9 is expressed in and secreted by immune cells in the pre-metastatic niche, as well as, post-tumor development, therefore making it a suitable targeted for prophylaxis and therapy. In previous work, it is demonstrated that cowpea mosaic virus (CPMV) acts as an adjuvant when administered intratumorally. Here, it is demonstrated that systemically administered, S100A9-targeted CPMV homes to the lungs leading to recruitment of innate immune cells. This approach is efficacious both prophylactically and therapeutically against lung metastasis from melanoma and breast cancer. The current research will facilitate and accelerate the development of next-generation targeted immunotherapies administered as prophylaxis, that is, after surgery of a primary breast tumor to prevent outgrowth of metastasis, as well as, therapy to treat established metastatic disease., (© 2021 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Cowpea mosaic virus (CPMV) is a plant virus that has been developed for multiple biomedical and nanotechnology applications, including immunotherapy. Two key platforms are available: virus nanoparticles (VNPs) based on the complete CMPV virion, including the genomic RNA, and virus-like nanoparticles (VLPs) based on the empty CPMV (eCPMV) virion. It is unclear whether these platforms differ in terms of immunotherapeutic potential. We therefore compared their physicochemical properties and immunomodulatory activities following in situ vaccination of an aggressive ovarian tumor mouse model (ID8-Defb29/Vegf-A). In physicochemical terms, CPMV and eCPMV were very similar, and both significantly increased the survival of tumor-bearing mice and showed promising antitumor efficacy. However, they demonstrated distinct yet overlapping immunostimulatory effects due to the presence of virus RNA in wild-type particles, indicating their suitability for different immunotherapeutic strategies. Specifically, we found that the formulations had similar effects on most secreted cytokines and immune cells, but the RNA-containing CPMV particles were uniquely able to boost populations of potent antigen-presenting cells, such as tumor-infiltrating neutrophils and activated dendritic cells. Our results will facilitate the development of CPMV and eCPMV as immunotherapeutic vaccine platforms with tailored responses. IMPORTANCE The engagement of antiviral effector responses caused by viral infection is essential when using viruses or virus-like particles (VLPs) as an immunotherapeutic agent. Here, we compare the chemophysical and immunostimulatory properties of wild-type cowpea mosaic virus (CPMV) (RNA containing) and eCPMV (RNA-free VLPs) produced from two expression systems (agrobacterium-based plant expression system and baculovirus-insect cell expression). CPMV and eCPMV could each be developed as novel adjuvants to overcome immunosuppression and thus promote tumor regression in ovarian cancer (and other tumor types). To our knowledge, this is the first study to define the immunotherapeutic differences between CPMV and eCPMV, which is essential for the further development of biomedical applications for plant viruses and the selection of rational combinations of immunomodulatory reagents., (Copyright © 2019 American Society for Microbiology.)

The icosahedral capsid of cowpea mosaic virus is formed by 60 copies of the large (L) and small (S) coat protein subunits. The 24-amino-acid C-terminal peptide of the S coat protein can undergo proteolytic cleavage without affecting particle stability or infectivity. Mutagenic studies have shown that this sequence is involved in particle assembly, virus movement, RNA encapsidation and suppression of gene silencing. However, it is unclear how these processes are related, and which part(s) of the sequence are involved in each process. Here, we have analysed the effect of mutations in the C-terminal region of the S protein on the assembly of empty virus-like particles and on the systemic movement of infectious virus. The results confirmed the importance of positively charged amino acids adjacent to the cleavage site for particle assembly and revealed that the C-terminal 11 amino acids are important for efficient systemic movement of the virus.

Empty virus-like particles (eVLPs) of Cowpea mosaic virus (CPMV) are currently being utilized as reagents in various biomedical and nanotechnology applications. Here, we report the crystal structure of CPMV eVLPs determined using X-ray crystallography at 2.3 Å resolution and compare it with previously reported cryo-electron microscopy (cryo-EM) of eVLPs and virion crystal structures. Although the X-ray and cryo-EM structures of eVLPs are mostly similar, there exist significant differences at the C terminus of the small (S) subunit. The intact C terminus of the S subunit plays a critical role in enabling the efficient assembly of CPMV virions and eVLPs, but undergoes proteolysis after particle formation. In addition, we report the results of mass spectrometry-based proteomics analysis of coat protein subunits from CPMV eVLPs and virions that identify the C termini of S subunits undergo proteolytic cleavages at multiple sites instead of a single cleavage site as previously observed., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Unlabelled: Bean pod mottle virus (BPMV) is a bipartite, positive-sense (+) RNA plant virus in the Secoviridae family. Its RNA1 encodes proteins required for genome replication, whereas RNA2 primarily encodes proteins needed for virion assembly and cell-to-cell movement. However, the function of a 58-kDa protein (P58) encoded by RNA2 has not been resolved. P58 and the movement protein (MP) of BPMV are two largely identical proteins differing only at their N termini, with P58 extending MP upstream by 102 amino acid residues. In this report, we unveil a unique role for P58. We show that BPMV RNA2 accumulation in infected cells was abolished when the start codon of P58 was eliminated. The role of P58 does not require the region shared by MP, as RNA2 accumulation in individual cells remained robust even when most of the MP coding sequence was removed. Importantly, the function of P58 required the P58 protein, rather than its coding RNA, as compensatory mutants could be isolated that restored RNA2 accumulation by acquiring new start codons upstream of the original one. Most strikingly, loss of P58 function could not be complemented by P58 provided in trans, suggesting that P58 functions in cis to selectively promote the accumulation of RNA2 copies that encode a functional P58 protein. Finally, we found that all RNA1-encoded proteins are cis-acting relative to RNA1. Together, our results suggest that P58 probably functions by recruiting the RNA1-encoded polyprotein to RNA2 to enable RNA2 reproduction., Importance: Bean pod mottle virus (BPMV) is one of the most important pathogens of the crop plant soybean, yet its replication mechanism is not well understood, hindering the development of knowledge-based control measures. The current study examined the replication strategy of BPMV RNA2, one of the two genomic RNA segments of this virus, and established an essential role for P58, one of the RNA2-encoded proteins, in the process of RNA2 replication. Our study demonstrates for the first time that P58 functions preferentially with the very RNA from which it is translated, thus greatly advancing our understanding of the replication mechanisms of this and related viruses. Furthermore, this study is important because it provides a potential target for BPMV-specific control, and hence could help to mitigate soybean production losses caused by this virus.

Due to the nanoscale size and the strictly controlled and consistent morphologies of viruses, there has been a recent interest in utilizing them in nanotechnology. The structure, surface chemistries and physical properties of many viruses have been well elucidated, which have allowed identification of regions of their capsids which can be modified either chemically or genetically for nanotechnological uses. In this review we focus on the use of such modifications for the functionalization and production of viruses and empty viral capsids that can be readily decorated with metals in a highly tuned manner. In particular, we discuss the use of two plant viruses (Cowpea mosaic virus and Tobacco mosaic virus) which have been extensively used for production of novel metal nanoparticles (<100nm), composites and building blocks for 2D and 3D materials, and illustrate their applications., (© 2013 Elsevier Inc. All rights reserved.)

Bean pod mottle virus (BPMV) is a bipartite, positive-sense (+) RNA plant virus of the family Secoviridae. Its RNA1 encodes all proteins needed for genome replication and is capable of autonomous replication. By contrast, BPMV RNA2 must utilize RNA1-encoded proteins for replication. Here, we sought to identify RNA elements in RNA2 required for its replication. The exchange of 5' untranslated regions (UTRs) between genome segments revealed an RNA2-specific element in its 5' UTR. Further mapping localized a 66 nucleotide region that was predicted to fold into an RNA stem-loop structure, designated SLC. Additional functional analysis indicated the importance of the middle portion of the stem and an adjacent two-base mismatch. This is the first report of a cis-acting RNA element in RNA2 of a bipartite secovirus.

Assembling and interconnecting the building blocks of nanoscale devices and being able to electronically address or measure responses at the molecular level remains an important challenge for nanotechnology. Here we show the usefulness of bottom-up self-assembly for building electronic nanosensors from multiple components that have been designed to interact in a controlled manner. Cowpea mosaic virus was used as a scaffold to control the positions of gold nanoparticles. The nanoparticles were then interconnected using thiol-terminated conjugated organic molecules, resulting in a three-dimensional conductive network. Biotin molecules were attached to the virus scaffold using linkers to act as molecular receptors. We demonstrated that binding avidin to the biotin receptors on the self-assembled nanosensors causes a significant change in the network conductance that is dependent on the charge of the avidin protein., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The broadly neutralizing antibody 2G12 recognizes a conserved cluster of high-mannose glycans on the surface envelope spike of HIV, suggesting that the "glycan shield" defense of the virus can be breached and may, under the right circumstances, serve as a vaccine target. In an attempt to recreate features of the glycan shield semisynthetically, oligomannosides were coupled to surface lysines on the icosahedral capsids of bacteriophage Q beta and cowpea mosaic virus (CPMV). The Q beta glycoconjugates, but not CPMV, presented oligomannose clusters that bind the antibody 2G12 with high affinity. However, antibodies against these 2G12 epitopes were not detected in immunized rabbits. Rather, alternative oligomannose epitopes on the conjugates were immunodominant and elicited high titers of anti-mannose antibodies that do not crossreact with the HIV envelope. The results presented reveal important design considerations for a carbohydrate-based vaccine component for HIV., ((c) 2010 Elsevier Ltd. All rights reserved.)

Previous studies have shown that a functionalized viral nanoparticle can be used as a fluorescent signal-generating element and enhance detection sensitivity for immunoassays and low density microarrays. In this study, we further tested this ability in commercial DNA microarrays, including Affymetrix high density resequencing microarray. Optimum conditions for NeutrAvidin and dye coupling to a double-cysteine mutant of cowpea mosaic virus (CPMV) were found to be comparable to the commonly used streptavidin-phycoerythrin (SAPE) for high density resequencing microarray. A 3-fold signal enhancement in comparison to Cy5-dCTP controls was obtained when using nanoparticles on control scorecard expression microarrays. Hybridization results from commercially available 8000 rat expression arrays indicate an increment of 14% on the detected features when the virus complex was used as the staining reagent in comparison to Cy5-dCTP controls. The current work shows the utility of the CPMV-dye nanoparticles as a detection reagent in well-established detection platforms.

A significant impediment to the widespread use of noninvasive in vivo vascular imaging techniques is the current lack of suitable intravital imaging probes. We describe here a new strategy to use viral nanoparticles as a platform for the multivalent display of fluorescent dyes to image tissues deep inside living organisms. The bioavailable cowpea mosaic virus (CPMV) can be fluorescently labeled to high densities with no measurable quenching, resulting in exceptionally bright particles with in vivo dispersion properties that allow high-resolution intravital imaging of vascular endothelium for periods of at least 72 h. We show that CPMV nanoparticles can be used to visualize the vasculature and blood flow in living mouse and chick embryos to a depth of up to 500 microm. Furthermore, we show that the intravital visualization of human fibrosarcoma-mediated tumor angiogenesis using fluorescent CPMV provides a means to identify arterial and venous vessels and to monitor the neovascularization of the tumor microenvironment.

The structure of cubic Cowpea mosaic virus crystals, compressed at 330 MPa in a diamond anvil cell, was refined at 2.8 A from data collected using ultrashort-wavelength (0.331 A) synchrotron radiation. With respect to the structure at atmospheric pressure, order is increased with lower Debye Waller factors and a larger number of ordered water molecules. Hydrogen-bond lengths are on average shorter and the cavity volume is strongly reduced. A tentative mechanistic explanation is given for the coexistence of disordered and ordered cubic crystals in crystallization drops and for the disorder-order transition observed in disordered crystals submitted to high pressure. Based on such explanation, it can be concluded that pressure would in general improve, albeit to a variable extent, the order in macromolecular crystals.

Cowpea mosaic virus (CPMV) is a robust, icosahedrally symmetric platform successfully used for attaching a variety of molecular substrates including proteins, fluorescent labels, and metals. The symmetric distribution and high local concentration of the attached molecules generates novel properties for the 30 nm particles. We report new CPMV reagent particles generated by systematic replacement of surface lysines with arginine residues. The relative reactivity of each lysine on the native particle was determined, and the two most reactive lysine residues were then created as single attachment sites by replacing all other lysines with arginine residues. Structural analysis of gold derivatization not only corroborated the specific reactivity of these unique lysine residues but also demonstrated their dramatically different presentation environment. Combined with site-directed cystine mutations, it is now possible to uniquely double label CPMV, expanding its use as an addressable nanoblock.

Cowpea mosaic virus is composed of 60 identical copies of a two-subunit protein organized in pentameric assemblies around the icosahedral 5-fold symmetry axis. Treatment of the virus with the Ni(II) complex of the tripeptide GGH and a peroxide oxidant, or irradiation in the presence of Ru(bpy)(3)(2+) and persulfate generates covalent crosslinks across the pentameric subunit boundaries, effectively stitching the subunits together. Intersubunit crosslinking was found to occur exclusively at adjacent tyrosine residues (Y52-Y103), as predicted from the X-ray crystal structure of the capsid, and to be more extensive with the photochemical ruthenium system. The Ni/GGH oxidative procedure was also used to make covalent attachments to the virion by trapping with a functionalized disulfide reagent.

Ordered nucleic acid in an icosahedral virus was first visualized in the X-ray structure of the Picorna-like plant virus, Bean pod mottle virus (BPMV). Virus particles containing the 3500 nucleotide segment of the BPMV bipartite RNA genome (middle component) had nearly 20% of the genome ordered. Here we report the refined structures of the middle component, bottom component (particles containing the 5800 nucleotide segment of the genome), and top component (empty particles of BPMV capsid protein). The bottom component particles contain ordered RNA in the same location as middle component. Although the ordered RNA density in both nucleoprotein particles is the average of the contents of 60 icosahedral asymmetric units, both nucleoprotein components show that the base density for the first two nucleotides is predominantly purine, while the next five appear to be predominantly pyrimidine. The empty capsid demonstrates that RNA dictates the order of the N-terminal 19 residues of the large subunit because these residues are invisible in the top component.

Background & Objectives: Foreign peptide sequences can be inserted into the betaB-betaC loop of the cowpea mosaic virus (CPMV) small coat protein (SCP) to yield functional chimaeric viruses. Immunisation with chimaeric CPMV elicits immune responses that protect against human immunodeficiency and mink enteritis viruses. The present study was undertaken to investigate the expression of a B cell epitope from the merozoite surface antigen-1 of the malaria parasite Plasmodium falciparum (PfMSP1) in CPMV for an epitope based vaccine., Methods: DNA encoding a 19 aa sequence (VTHESYQEL VKKLEALEDA, termed P109), the N-terminus of the mature PfMSP1, was cloned into SCP gene yielding a chimaeric virus CPMV-P109. CPMV-P109 was propagated in cowpea plants. The immunogenicity of purified recombinant virus in rabbits was investigated., Results: CPMV-P109 developed a systemically spreading infection in cowpea, with normal viral morphology. The P109 epitope was detected on CPMV-P109 by ELISA with an antiserum produced against homopolymeric P109. Immunisation of rabbits with CPMV-P109 yielded antibodies that, although were predominantly directed against virus-specific epitopes, also recognized the P109 peptide on the recombinant virus and free P109 peptide. These antibodies however, did not react with the native antigen on merozoite by immunofluorescence., Interpretation & Conclusion: The results indicate that selecting immunodominant peptide epitopes and presenting them in a near native conformation are important for generating biologically relevant antibodies in the CPMV expression system. Further, the findings draw attention to the importance of measuring immune responses to the viral vector antigens, a preponderance of which can result in undesirable effects such as autoimmunity and hypersensitivity in immunized hosts.

A vibrational Raman optical activity (ROA) study of a range of different structural types of virus exemplified by filamentous bacteriophage fd, tobacco mosaic virus, satellite tobacco mosaic virus, bacteriophage MS2 and cowpea mosaic virus has revealed that, on account of its sensitivity to chirality, ROA is an incisive probe of their aqueous solution structures at the molecular level. Protein ROA bands are especially prominent from which, as we have shown by comparison with the ROA spectra of proteins with known structures and by using a pattern recognition program, the folds of the major coat protein subunits may be deduced. Information about amino acid side-chain conformations, exemplified here by the determination of the sign and magnitude of the torsion angle chi(2,1) for tryptophan in fd, may also sometimes be obtained. By subtracting the ROA spectrum of the empty protein capsid (top component) of cowpea mosaic virus from those of the intact middle and bottom-upper components separated by means of a caesium chloride density gradient, the ROA spectrum of the viral RNA was obtained, which revealed that the RNA takes up an A-type single-stranded helical conformation and that the RNA conformations in the middle and bottom-upper components are very similar. This information is not available from the X-ray crystal structure of cowpea mosaic virus since no nucleic acid is visible.

Cowpea mosaic virus (CPMV) can be isolated in gram quantities, possesses a structure that is known to atomic resolution, and is quite stable. It is therefore of potential use as a molecular entity in synthesis, particularly as a building block on the nanochemical scale. CPMV was found to possess a lysine residue with enhanced reactivity in each asymmetric unit, and thus 60 such lysines per virus particle. The identity of this residue was established by a combination of acylation, protein digestion, and mass spectrometry. Under forcing conditions, up to four lysine residues per asymmetric unit can be addressed. In combination with engineered cysteine reactivity described in the accompanying paper, this provides a powerful platform for the alteration of the chemical and physical properties of CPMV particles.

Wild-type Cowpea mosaic virus (CPMV) displays no cysteine side chains on the exterior capsid surface and is therefore relatively unreactive with thiol-selective reagents. Four CPMV mutants bearing cysteine residues in one of two exterior positions of the asymmetric unit were created. The mutants were shown to aggregate by virtue of disulfide bond formation in the absence of added reducing agent, bind to metallic gold, and undergo selective reactions at the introduced thiol residues. Controlled aggregation by virtue of biotin-avidin interactions was demonstrated, as was the independent derivatization of reactive lysine and cysteine positions. The ability to introduce such reactivity into a system that can be readily prepared and isolated in gram quantities should open new doors to applications in biochemistry, materials science, and catalysis.

Cowpea mosaic virus (CPMV) replication occurs in close association with small membranous vesicles in the host cell. The CPMV RNA1-encoded 60 kDa nucleotide-binding protein ('60K') plays a role in the formation of these vesicles. In this study, five cellular proteins were identified that interacted with different domains of 60K using a yeast two-hybrid search of an Arabidopsis cDNA library. Two of these host proteins (termed VAP27-1 and VAP27-2), with high homology to the VAP33 family of SNARE-like proteins from animals, interacted specifically with the C-terminal domain of 60K and upon transient expression colocalized with 60K in CPMV-infected cowpea protoplasts. eEF1-beta, picked up using the central domain of 60K, was also found to colocalize with 60K. The possible role of these host proteins in the viral replicative cycle is discussed.

Red clover mottle virus (RCMV) is a member of the comoviruses, a group of picornavirus-like plant viruses. The X-ray structure of RCMV strain S has been determined and refined to 2.4 A. The overall structure of RCMV is similar to that of two other comoviruses, Cowpea mosaic virus (CPMV) and Bean pod mottle virus (BPMV). The sequence of the coat proteins of RCMV strain O were modeled into the capsid structure of strain S without causing any distortion, confirming the close resemblance between the two strains. By comparing the RCMV structure with that of other comoviruses, a structural fingerprint at the N terminus of the small subunit was identified which allowed subgrouping of comoviruses into CPMV-like and BPMV-like viruses.

Tubular structures involved in the cell-to-cell movement of cowpea mosaic virus (CPMV) were partially purified from infected cowpea protoplasts to identify the structural components. A relatively pure fraction could be obtained by differential centrifugation and this was analysed by PAGE and immunoblotting. Besides the movement protein (MP) and capsid proteins (CP) of CPMV, no other major infection-specific proteins could be detected, suggesting that host proteins are not a major structural component of the movement tubule.

Background: We employed a genetically engineered icosahedral plant virus, cowpea mosaic virus (CPMV), as an expression and presentation system to display a 14 amino acid linear antigenic epitope found in a capsid protein of human rhinovirus 14 (HRV14)., Result: Gram quantities of the CPMV/HRV 14 chimera were made in plants and purified particles were crystallized in a form isomorphous with wild-type CPMV. The 2.8 A resolution structure of the chimera shows that the inserted loop is well ordered and that if the loop were intact, a phenylalanine residue of CPMV would be placed in a hydrophilic environment. The resultant strain may make the loop an attractive substrate for endogenous plant proteases, as roughly 80% of the inserted polypeptides are cleaved, allowing the phenylalanine to be partially buried. Altering the phenylalanine to an arginine could relieve the stress, reducing the propensity for cleavage and increasing the likelihood that the peptide will assume a structure closely similar to its structure in HRV14., Conclusions: Successful crystallization of other CPMV chimeras in forms isomorphous with the native virus suggests that this is a viable system for structure-based design of peptide presentation.

The capsid is modeled as a region of constant electron density located between inner and outer envelopes that exhibit icosahedral symmetry. For computational purposes the envelopes are represented as truncated sums of weighted icosahedral harmonics. Methods are described for estimating the weights from x-ray solution scattering patterns based on nonlinear least squares, and two examples of the procedure, for viruses with known atomic-resolution structures, are given.

The sequence of the 3'-terminal 2424 nucleotides of RNA-2 of the flowering cherry strain of strawberry latent ringspot virus (SLRV) was determined from cDNA clones. The sequence contains a reading frame in the virus-sense strand of 2070 nucleotides, a 3' untranslated region of 552 nucleotides and a 3'-terminal poly(A) tract. The positions of the two coat proteins of SLRV within the reading frame were determined from sequence data obtained by N-terminal sequencing using Edman degradation. The larger coat protein with an M(r) of 43K is located 5' of the smaller coat protein of 27K, and the two proteins are apparently cleaved at a Ser-Gly bond. Although there are numerous similarities between SLRV and the nepoviruses and comoviruses, there is no significant homology between the SLRV coat proteins and the coat proteins of either group. Furthermore, the hydropathy profiles of the SLRV coat proteins are unlike those of either group. No comparisons could be made with the fabaviruses owing to lack of sequencing information. This lack of homology suggests that SLRV is more distantly related to the nepoviruses and comoviruses than has been considered previously.

We describe a novel approach to investigating exchange kinetics in biological assemblies. The method makes use of a Raman multichannel analyzer coupled with a dialysis flow cell. We employ this methodology to determine exchange rates of labile hydrogens in both the packaged RNA genome and protein subunits of bean pod mottle virus (BPMV). In the BPMV assembly, which is similar to human picornaviruses, the x-ray structure indicates that about 20% of the ssRNA chain is ordered at the threefold vertices of the icosahedral capsid, although the nucleotide bases in the ordered segments are not known (Chen et al., 1989). Here, we compare exchange profiles of the native virus with those of the empty capsid, model nucleic acids and aqueous solvent to reveal the following exchange characteristics of BPMV RNA and protein: (i) Base-specific retardation of exchange is observed in the packaged RNA. (ii) Retardation is greatest for uracil residues, for which the first-order exchange rate constant (kU = 0.18 +/- 0.02 min-1) is 40% lower than that of either the H2O solvent or adenine or cytosine groups of RNA (ksolv approximately kA approximately kC = 0.30 +/- 0.02 min-1). (iii) Retardation of exchange is also observed for the guanine residues of packaged RNA. (iv) No appreciable exchange of amide NH groups of capsid subunits occurs within the time of complete exchange (t approximately 10 min) of packaged RNA or bulk solvent. Thus, the present results identify sites in both the protein subunits (amide NH) and RNA nucleotides (amino NH2 and imino NH) which are resistant to solvent-catalyzed hydrogen exchange. We propose that retardation of exchange of labile sites of the RNA nucleotides is a consequence of the organization of the RNA chromosome within the virion. Our findings support a model for BPMV in which surface and buried domains of capsid subunits are extensively and rigidly hydrogen-bonded, and in which uracil and guanine exocyclic donor groups of packaged RNA are the principal targets for subunit interaction at the threefold vertices of the capsid.