In vitro and in silico assessment of anti-biofilm and anti-quorum sensing properties of 2,4-Di-tert butylphenol against Acinetobacter baumannii.

Introduction. Acinetobacter baumannii is a nosocomial pathogen with a high potential to cause food-borne infections. It is designated as a critical pathogen by the World Health Organization due to its multi-drug resistance and mortalities reported. Biofilm governs major virulence factors, which promotes drug resistance in A. baumannii. Thus, a compound with minimum selection pressure on the pathogen can be helpful to breach biofilm-related virulence.Hypothesis/Gap Statement. To identify anti-biofilm and anti-virulent metabolites from extracts of wild Mangifera indica (mango) brine pickle bacteria that diminishes pathogenesis and resistance of A. baumannii.Aim. This study reports anti-biofilm and anti-quorum sensing (QS) efficacy of secondary metabolites from bacterial isolates of fermented food origin.Method. Cell-free supernatants (CFS) of 13 bacterial isolates from fermented mango brine pickles were screened for their efficiency in inhibiting biofilm formation and GC-MS was used to identify its metabolites. Anti-biofilm metabolite was tested on early and mature biofilms, pellicle formation, extra polymeric substances (EPS), cellular adherence, motility and resistance of A. baumannii. Gene expression and in silico studies were also carried out to validate the compounds efficacy.Results. CFS of TMP6b identified as Bacillus vallismortis, inhibited biofilm production (83.02 %). Of these, major compound was identified as 2,4-Di-tert-butyl phenol (2,4-DBP). At sub-lethal concentrations, 2,4-DBP disrupted both early and mature biofilm formation. Treatment with 2,4-DBP destructed in situ biofilm formed on glass and plastic. In addition, key virulence traits like pellicle (77.5 %), surfactant (95.3 %), EPS production (3-fold) and cell adherence (65.55 %) reduced significantly. A. baumannii cells treated with 2,4-DBP showed enhanced sensitivity towards antibiotics, oxide radicals and blood cells. Expression of biofilm-concomitant virulence genes like csuA/B, pgaC, pgaA, bap, bfmR, katE and ompA along with QS genes abaI, abaR significantly decreased. The in silico studies further validated the higher binding affinity of 2,4-DBP to the AbaR protein than the cognate ligand molecule.Conclusion. To our knowledge, this is the first report to demonstrate 2,4- DBP has anti-pathogenic potential alone and with antibiotics by in vitro, and in silico studies against A. baumannii. It also indicates its potential use in therapeutics and bio-preservatives.

Normal mode analysis and comparative study of intrinsic dynamics of alcohol oxidase enzymes from GMC protein family.

Glucose-Methanol-Choline (GMC) family enzymes are very important in catalyzing the oxidation of a wide range of structurally diverse substrates. Enzymes that constitute the GMC family, share a common tertiary fold but < 25% sequence identity. Cofactor FAD, FAD binding signature motif, and similar structural scaffold of the active site are common features of oxidoreductase enzymes of the GMC family. Protein functionality mainly depends on protein three-dimensional structures and dynamics. In this study, we used the normal mode analysis method to search the intrinsic dynamics of GMC family enzymes. We have explored the dynamical behavior of enzymes with unique substrate catabolism and active site characteristics from different classes of the GMC family. Analysis of individual enzymes and comparative ensemble analysis of enzymes from different classes has shown conserved dynamic motion at FAD binding sites. The present study revealed that GMC enzymes share a strong dynamic similarity (Bhattacharyya coefficient >90% and root mean squared inner product >52%) despite low sequence identity across the GMC family enzymes. The study predicts that local deformation energy between atoms of the enzyme may be responsible for the catalysis of different substrates. This study may help that intrinsic dynamics can be used to make meaningful classifications of proteins or enzymes from different organisms.Communicated by Ramaswamy H. Sarma.

The application of MD simulation to lead identification, vaccine design, and structural studies in combat against leishmaniasis - A review.

Drug discovery, vaccine design, and protein interaction studies are rapidly moving toward the routine use of molecular dynamics simulations (MDS) and related methods. As a result of MDS, it is possible to gain insights into the dynamics and function of identified drug targets, antibody-antigen interactions, potential vaccine candidates, intrinsically disordered proteins, and essential proteins. The MDS appears to be used in all possible ways in combating diseases such as cancer, however, it has not been well documented as to how effectively it is applied to infectious diseases such as Leishmaniasis. As a result, this systematic review aims to survey the application of MDS in combating leishmaniasis. We have systematically collected articles that illustrate the implementation of MDS in drug discovery, vaccine development, and structural studies related to Leishmaniasis. Of all the articles reviewed, we identified that only a limited number of studies focused on the development of vaccines against Leishmaniasis through MDS. Also, the PCA and FEL studies were not carried out in most of the studies. These two were globally accepted utilities to understand the conformational changes and hence it is recommended that this analysis should be taken up in similar approaches in the future.

pH modulates the role of SP6 RNA polymerase in transcription process: an in silico study.

SP6 RNA polymerase (SP6 RNAP) is an essential enzyme for the transcription process in SP6 bacteriophage. SP6 RNAP plays a vital role in mRNA vaccine designing technology and other translational biotechnology research due to the high specificity towards its promoter. The self-replicating performance also put this polymerase to study extensively. Despite of the reports emphasizing the function of this enzyme, a detailed structural and functional understanding of RNA polymerase is not reported so far. Here, we report the first-ever information about SP6RNAP structure and its effect on promoter binding at different pH environments using molecular docking and molecular dynamics simulation (MDS) study. We also report the changes in polymerase conformations in different pH conditions using in-silico approach. The docking study was also performed for SP6 RNAP with SP6 promoter at different pH environments using the in-silico docking tools and conducted the MDS study for complexes. MM/PBSA and per residue energy contribution has been performed at three different pH environments. The structural aspects confirmed that the pH 7.9 state favors the polymerase functional activity in the transcription process which was in the range reported using transcription assay. This polymerase's unique features may play its emerging role as an efficient transcription factor in translational biological research.Communicated by Ramaswamy H. Sarma.

Computational analysis of marine algal compounds for obesity management against pancreatic lipase.

Obesity is considered a global crisis because of its increased risk factors triggered by lifestyle changes. The prevalence of this condition is increasing at an alarming rate, giving rise to development of novel drugs. Pancreatic lipase possesses higher efficacy in inhibiting this condition among the other drug targets. In this study, virtual screening of 126 plant-derived anti-obesity compounds and 1110 marine algal compounds from seaweed metabolite database were screened and targeted against pancreatic lipase and ranked based on their binding affinity values. A total of 530 compounds that possessed best docked scores of less than -6 kcal/mol were checked for Lipinski's properties through Swiss ADME. Furthermore, these compounds were subjected to toxicity prediction using PROTOX II server. As much as 38 compounds were found to be non-toxic and were subjected to molecular docking analysis. Based on the binding energy, the following compounds RG012 (-10.15 kcal/mol), LIG42 (-9.7 kcal/mol), BC010 (-8.47 kcal/mol), RL073 (-8.2 kcal/mol), and LIG46 (-8.03 kcal/mol) were selected exhibiting higher binding affinity when compared to the standard drug (Orlistat) and hence these compounds were subjected to molecular dynamics simulation using GROMACS. BC010 complex revealed a stable interaction within the binding pocket and the binding free energy is -158.208 kJ/mol which is higher when compared to other complexes in 100 ns simulation. BC010 ((7S,11S,12S,14R)-4',14-dimethoxyamentol) from brown algae Cystophora fibrosa could be considered as a potential drug candidate to suppress obesity by inhibiting pancreatic lipase.Communicated by Ramaswamy H. Sarma.

Molecular evaluation of quorum quenching potential of vanillic acid against Yersinia enterocolitica through transcriptomic and in silico analysis.

Introduction. Yersinia enterocolitica is one of the leading food-borne entero-pathogens causing various illnesses ranging from gastroenteritis to systemic infections. Quorum sensing (QS) is one of the prime mechanisms that control the virulence in Y. enterocolitica.Hypothesis/Gap Statement. Vanillic acid inhibits the quorum sensing and other virulence factors related to Y. enterocolitica. It has been evaluated by transcriptomic and Insilico analysis. Therefore, it can be a prospective agent to develop a therapeutic combination against Y. enterocolitica.Aim. The present study is focused on screening natural anti-quorum-sensing agents against Y. enterocolitica. The effect of selected active principle on various virulence factors was evaluated.Methodology. In total, 12 phytochemicals were screened by swarming assay. MATH assay, EPS and surfactant production assay, SEM analysis, antibiotic and blood sensitivity assay were performed to demonstrate the anti-virulence activity. Further, RNA sequencing and molecular docking studies were carried out to substantiate the anti-QS activity.Results. Vanillic acid (VA) has exhibited significant motility inhibition, thus indicating the anti-QS activity with MQIC of 400 µg ml-1 without altering the cell viability. It has also inhibited the violacein production in Chromobacterium violaceum ATCC 12472, which further confirms the anti-QS activity. VA has inhibited 16 % of cell-surface hydrophobicity (CSH), 52 % of EPS production and 60 % of surfactant production. Moreover, it has increased the sensitivity of Y. enterocolitica towards antibiotics. It has also made the cells upto 91 % more vulnerable towards human immune cells. The transcriptomic analysis by RNA sequencing revealed the down regulation of genes related to motility, virulence, chemotaxis, siderophores and drug resistance. VA treatment has also positively regulated the expression of several stress response genes. In furtherance, the anti-QS potential of VA has been validated with QS regulatory protein YenR by in silico molecular simulation and docking study.Conclusion. The present study is possibly the first attempt to demonstrate the anti-QS and anti-pathogenic potential of VA against Y. enterocolitica by transcriptomic and in silico analysis. It also deciphers that VA can be a promising lead to develop biopreservative and therapeutic regimens to treat Y. enterocolitica infections.

2,4-Di-Tert-Butylphenol Isolated From an Endophytic Fungus, Daldinia eschscholtzii, Reduces Virulence and Quorum Sensing in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is among the top three gram-negative bacteria according to the WHO's critical priority list of pathogens against which newer antibiotics are urgently needed and considered a global threat due to multiple drug resistance. This situation demands unconventional antimicrobial strategies such as the inhibition of quorum sensing to alleviate the manifestation of classical resistance mechanisms. Here, we report that 2,4-di-tert-butylphenol (2,4-DBP), isolated from an endophytic fungus, Daldinia eschscholtzii, inhibits the quorum-sensing properties of P. aeruginosa. We have found that treating P. aeruginosa with 2,4-DBP substantially reduced the secretion of virulence factors as well as biofilm, and its associated factors that are controlled by quorum sensing, in a dose-dependent manner. Concomitantly, 2,4-DBP also significantly reduced the expression of quorum sensing-related genes, i.e., lasI, lasR, rhlI, and rhlR significantly. Importantly, 2,4-DBP restricted the adhesion and invasion of P. aeruginosa to the A549 lung alveolar carcinoma cells. In addition, bactericidal assay with 2,4-DBP exhibited synergism with ampicillin to kill P. aeruginosa. Furthermore, our computational studies predicted that 2,4-DBP could bind to the P. aeruginosa quorum-sensing receptors LasR and RhlR. Collectively, these data suggest that 2,4-DBP can be exploited as a standalone drug or in combination with antibiotic(s) as an anti-virulence and anti-biofilm agent to combat the multidrug resistant P. aeruginosa infection.

Anti-quorum sensing and anti-biofilm activity of 5-hydroxymethylfurfural against Pseudomonas aeruginosa PAO1: Insights from in vitro, in vivo and in silico studies.

Pseudomonas aeruginosa is one of the most common pathogens associated with nosocomial infections and a great concern to immunocompromised individuals especially in the cases of cystic fibrosis, AIDS and burn wounds. The pathogenicity of P. aeruginosa is largely directed by the quorum sensing (QS) system. Hence, QS may be considered an important therapeutic target to combat P. aeruginosa infections. The anti-quorum sensing and anti-biofilm efficacy of aromatic aldehyde, 5-hydroxymethylfurfural (5-HMF) against P. aeruginosa PAO1 were assessed. At the sub-inhibitory concentration, 5-HMF suppressed the production of QS-controlled virulence phenotypes and biofilm formation in P. aeruginosa. It was also able to significantly enhance the survival rate of C. elegans infected with P. aeruginosa. The in silico studies revealed that 5-HMF could serve as a competitive inhibitor for the auto-inducer molecules as it exhibited a strong affinity for the regulatory proteins of the QS-circuits i.e. LasR and RhlR. In addition, a significant down-regulation in the expression of QS-related genes was observed suggesting the ability of 5-HMF in mitigating the pathogenicity of P. aeruginosa.

The highly efficient T7 RNA polymerase: A wonder macromolecule in biological realm.

The study of bacteriophage has always been of keen interest for biologists to understand the fundamentals of biology. Bacteriophage T7 was first isolated in 1945 and its first comprehensive genetic map of was published in 1969. Since then, it has gained immense attention of researchers and became a prime model system for experimental biologists. The major gene product of T7 phage, T7 RNA polymerase (T7RNAP), continues to attract researchers since a long time due to its high and specific processivity with a single subunit structure and its capability of transcribing a complete gene without additional proteins. Since the first review article in 1993 there has been around nine reviews on this polymerase till year 2009, most of which focussed on particular aspects of T7RNAP such as structure and function. However, this review encapsulates a broad view on T7RNAP, one of the simplest macromolecule catalyzing RNA synthesis, including recent updates on its applications, structure, activators and inhibitors. Thus this brief review bridges the huge gap on the recent updates on this polymerase and will help the biologists in their endeavours that include the use of T7RNAP.

Cinnamic acid attenuates quorum sensing associated virulence factors and biofilm formation in Pseudomonas aeruginosa PAO1.

OBJECTIVE: Anti-quorum sensing and anti-biofilm efficacy of Cinnamic acid against Pseudomonas aeruginosa was comparatively assessed with respect to potent quorum sensing inhibitor, Baicalein. RESULTS: At sub-lethal concentration, Cinnamic acid effectively inhibited both the production of the QS-dependent virulence factors and biofilm formation in P. aeruginosa without affecting the viability of the bacterium. The phytocompound interfered with the initial attachment of planktonic cells to the substratum thereby causing reduction in biofilm development. In addition, the in vivo study indicated that the test compound protected Caenorhabditis elegans from the virulence factors of P. aeruginosa leading to reduced mortality. The in silico analysis revealed that Cinnamic acid can act as a competitive inhibitor for the natural ligands towards the ligand binding domain of the transcriptional activators of the quorum sensing circuit in P. aeruginosa, LasR and RhlR. CONCLUSIONS: The findings suggest that Cinnamic acid may serve as a novel quorum sensing based anti-infective in controlling P. aeruginosa infections.

Attenuation of quorum sensing controlled virulence factors and biofilm formation in Pseudomonas aeruginosa by pentacyclic triterpenes, betulin and betulinic acid.

The production of virulence determinants and biofilm formation in numerous pathogens is regulated by the cell-density-dependent phenomenon, Quorum sensing (QS). The QS system in multidrug resistant opportunistic pathogen, P. aeruginosa constitutes of three main regulatory circuits namely Las, Rhl, and Pqs which are closely linked to its pathogenicity and establishment of chronic infections. In spite intensive antibiotic therapy, P. aeruginosa continue to be an important cause of nosocomial infections and also the major cause of mortality in Cystic Fibrosis patients with 80% of the adults suffering from chronic P. aeruginosa infection. Hence, targeting QS circuit offers an effective intervention to the ever increasing problem of drug resistant pathogens. In the present study, the pentacyclic triterpenes i.e. Betulin (BT) and Betulinic acid (BA) exhibited significant attenuation in production of QS-regulated virulence factors and biofilm formation in P. aeruginosa, at the sub-lethal concentration. The test compound remarkably interfered in initial stages of biofilm development by decreasing the exopolysaccharide production and cell surface hydrophobicity. Based on the in vivo studies, the test compounds notably enhanced the survival of Caenorhabditis elegans infected with P. aeruginosa. Furthermore, molecular docking analysis revealed that BT and BA can act as a strong competitive inhibitor for QS receptors, LasR and RhlR. The findings suggest that BT and BA can serve as potential anti-infectives in the controlling chronic infection of P. aeruginosa.

An in-silico glimpse into the pH dependent structural changes of T7 RNA polymerase: a protein with simplicity.

The capability of performing an array of functions with its single subunit structure makes T7 RNA polymerase (T7RNAP) as one of the simplest yet attractive target for various investigations ranging from structure determinations to several biological tests. In this study, with the help of molecular dynamics (MD) calculations and molecular docking, we investigated the effect of varying pH conditions on conformational flexibility of T7RNAP. We also studied its effect on the interactions with a well established inhibitor (heparin), substrate GTP and T7 promoter of T7RNAP. The simulation studies were validated with the help of three dimensional reconstructions of the polymerase at different pH environments using transmission electron microscopy and single particle analysis. On comparing the simulated structures, it was observed that the structure of T7RNAP changes considerably and interactions with its binding partners also changes as the pH shifts from basic to acidic. Further, it was observed that the C-terminal end plays a vital role in the inefficiency of the polymerase at low pH. Thus, this in-silico study may provide a significant insight into the structural investigations on T7RNAP as well as in designing potent inhibitors against it in varying pH environments.

Modeling of alcohol oxidase enzyme of Candida boidinii and in silico analysis of competitive binding of proton ionophores and FAD with enzyme.

Alcohol oxidase (AOX) is an important flavin adenine dinucleotide (FAD) dependent oxidoreductase, which is responsible for converting methanol into formaldehyde and hydrogen peroxide for the growth of methylotrophic yeast Candida boidinii. Although AOX plays a crucial role in methanol catabolism, the experimental structure of AOX from Candida boidinii has not been elucidated. This study reports the first complete in silico model of AOX from C. boidinii. This paper also reports the AOX structure modeled using the threading approach, followed by structure analysis and molecular dynamics simulation. The modeled structure was compared with the aryl alcohol oxidase structure (a glucose-methanol-choline family member, pdbID: 3fim). A docking study was performed to analyze the interaction between AOX and its cofactor FAD. The AOX modeled structure also exhibited high similarity with respect to the FAD binding sites, which are the substrate binding sites as seen with 3fim. It was observed that the adenosine part of FAD was deeply buried inside AOX while the isoalloxazine ring stuck to the surface. This paper reports the interaction of selective proton ionophores (CCCP and DNP) with AOX and also reports their binding sites. These proton ionophores showed competitive binding with FAD. The occupancy of the FAD binding sites by the proton ionophore may lead to blocking of the entry of FAD and thereby disruption of AOX import into peroxisomes.

A computational assessment of pH-dependent differential interaction of T7 lysozyme with T7 RNA polymerase.

BACKGROUND: T7 lysozyme (T7L), also known as N-acetylmuramoyl-L-alanine amidase, is a T7 bacteriophage gene product. It involves two functions: It can cut amide bonds in the bacterial cell wall and interacts with T7 RNA polymerase (T7RNAP) as a part of transcription inhibition. In this study, with the help of molecular dynamics (MD) calculations and computational interaction studies, we investigated the effect of varying pH conditions on conformational flexibilities of T7L and their influence on T7RNAP -T7L interactions. RESULTS: From the MD studies of the T7L at three different pH strengths viz. 5, neutral and 7.9 it was observed that T7L structure at pH 5 exhibited less stable nature with more residue level fluctuations, decrease of secondary structural elements and less compactness as compared to its counterparts: neutral pH and pH 7.9. The T-pad analysis of the MD trajectories identified local fluctuations in few residues that influenced the conformational differences in three pH strengths. From the docking of the minimum energy representative structures of T7L at different pH strengths (obtained from the free energy landscape analysis) with T7RNAP structures at same pH strengths, we saw strong interaction patterns at pH 7.9 and pH 5. The MD analysis of these complexes also confirmed the observations of docking study. From the combined in silico studies, it was observed that there are conformational changes in N-terminal and near helix 1 of T7L at different pH strengths, which are involved in the T7RNAP interaction, thereby varying the interaction pattern. CONCLUSION: Since T7L has been used for developing novel therapeutics and T7RNAP one of the most biologically useful protein in both in-vitro and in vivo experiments, this in silico study of pH dependent conformational differences in T7L and the differential interaction with T7RNAP at different pH can provide a significant insight into the structural investigations on T7L and T7RNAP in varying pH environments.

Interaction Analysis of T7 RNA Polymerase with Heparin and Its Low Molecular Weight Derivatives - An In Silico Approach.

The single subunit T7 RNA polymerase (T7RNAP) is a model enzyme for studying the transcription process and for various biochemical and biophysical studies. Heparin is a commonly used inhibitor against T7RNAP and other RNA polymerases. However, exact interaction between heparin and T7RNAP is still not completely understood. In this work, we analyzed the binding pattern of heparin by docking heparin and few of its low molecular weight derivatives to T7RNAP, which helps in better understanding of T7RNAP inhibition mechanism. The efficiency of the compounds was calculated by docking the selected compounds and post-docking molecular mechanics/generalized Born surface area analysis. Evaluation of the simulation trajectories and binding free energies of the complexes after simulation showed enoxaparin to be the best among low molecular weight heparins. Binding free energy analysis revealed that van der Waals interactions and polar solvation energy provided the substantial driving force for the binding process. Furthermore, per-residue free energy decomposition analysis revealed that the residues Asp 471, Asp 506, Asp 537, Tyr 571, Met 635, Asp 653, Pro 780, and Asp 812 are important for heparin interaction. Apart from these residues, most favorable contribution in all the three complexes came from Asp 506, Tyr 571, Met 635, Glu 652, and Asp 653, which can be essential for binding of heparin-like structures with T7RNAP. The results obtained from this study will be valuable for the future rational design of novel and potent inhibitors against T7RNAP and related proteins.

Insight into virus encapsulation mechanism through in silico interaction study between coat protein and RNA operator loops of Sesbania mosaic virus.

Viruses are parasite by nature and they are responsible for many diseases. Inhibitor development is very difficult for viruses due to their rapid mutative nature. A common approach for treating virus infection is targeting them at the genomic level and an encapsulation mechanism can be one of the targets. Sesbania mosaic virus (SeMV) is a spherical virus and its capsid is formed by a coat protein, which contains the Arginine Rich Motif (ARM). This ARM interacts with RNA operator loops present in their genome and starts encapsulation. Though the structure of SeMV was already solved by crystallography, it lacks the critical ARM domain. We predicted the full-length three-dimensional structure of this protein by using crystal structure (lacking ARM) as a template along with tertiary structure of RNA operator loops. Docking studies were performed to discover the interacting residues of protein and RNA which are driving protein and RNA to interact with each other. We observed that these interactions lead to conformation changes in the coat protein structure, which starts genome encapsulation process. The ARM region is found to be crucial for these interactions. Molecular dynamics simulation studies were performed to check the conformational changes and free energy landscapes were generated to check the viability of these changes in terms of energy. In this work we proposed one RNA operator loop that is responsible for noticeable conformational changes in the SeMV structure and might be involved in the activation of the viral protein. The results of this in silico study can be tested further through in vitro studies and can be used to stop encapsulation.

The Arabidopsis stress responsive gene database.

Plants in nature may face a wide range of favorable or unfavorable biotic and abiotic factors during their life cycle. Any of these factors may cause stress in plants; therefore, they have to be more adaptable to stressful environments and must acquire greater response to different stresses. The objective of this study is to retrieve and arrange data from the literature in a standardized electronic format for the development of information resources on potential stress responsive genes in Arabidopsis thaliana. This provides a powerful mean for manipulation, comparison, search, and retrieval of records describing the nature of various stress responsive genes in Arabidopsis thaliana. The database is based exclusively on published stress tolerance genes associated with plants.

Three dimensional electron microscopy and in silico tools for macromolecular structure determination.

Recently, structural biology witnessed a major tool - electron microscopy - in solving the structures of macromolecules in addition to the conventional techniques, X-ray crystallography and nuclear magnetic resonance (NMR). Three dimensional transmission electron microscopy (3DTEM) is one of the most sophisticated techniques for structure determination of molecular machines. Known to give the 3-dimensional structures in its native form with literally no upper limit on size of the macromolecule, this tool does not need the crystallization of the protein. Combining the 3DTEM data with in silico tools, one can have better refined structure of a desired complex. In this review we are discussing about the recent advancements in three dimensional electron microscopy and tools associated with it.

Effects of amino-acid substitutions in the Brome mosaic virus capsid protein on RNA encapsidation.

Brome mosaic virus (BMV) packages its genomic RNAs (RNA1, RNA2, and RNA3) and subgenomic RNA4 into three different particles. However, since the RNAs in the virions have distinct lengths and electrostatic charges, we hypothesize that subsets of the virions should have distinct properties. A glutamine to cysteine substitution at position 120 of the capsid protein (CP) was found to result in a mutant virus named QC that exhibited a dramatically altered ratio of the RNAs in virions. RNA2 was far more abundant than the other RNAs, although the ratios could be affected by the host plant species. RNAs with the QC mutation were competent for replication early in the infection, suggesting that they were either selectively packaged or degraded after packaging. In support of the latter idea, low concentrations of truncated RNA1 that co-migrated with RNA2 were found in the QC virions. Spectroscopic analysis and peptide fingerprinting experiments showed that the QC virus capsid interacted with the encapsidated RNAs differently than did the wild type. Furthermore, wild-type BMV RNA1 was found to be more susceptible to nuclease digestion relative to RNA2 as a function of the buffer pH. Other BMV capsid mutants also had altered ratios of packaged RNAs.

Role of surface charge density in nanoparticle-templated assembly of bromovirus protein cages.

Self-assembling icosahedral protein cages have potentially useful physical and chemical characteristics for a variety of nanotechnology applications, ranging from therapeutic or diagnostic vectors to building blocks for hierarchical materials. For application-specific functional control of protein cage assemblies, a deeper understanding of the interaction between the protein cage and its payload is necessary. Protein-cage encapsulated nanoparticles, with their well-defined surface chemistry, allow for systematic control over key parameters of encapsulation such as the surface charge, hydrophobicity, and size. Independent control over these variables allows experimental testing of different assembly mechanism models. Previous studies done with Brome mosaic virus capsids and negatively charged gold nanoparticles indicated that the result of the self-assembly process depends on the diameter of the particle. However, in these experiments, the surface-ligand density was maintained at saturation levels, while the total charge and the radius of curvature remained coupled variables, making the interpretation of the observed dependence on the core size difficult. The current work furnishes evidence of a critical surface charge density for assembly through an analysis aimed at decoupling the surface charge and the core size.