Molecular Studies on Bromovirus Capsid Protein

AbstractTo determine whether the role of coat protein (CP) in cell-to-cell movement of dicot-adapted cowpea chlorotic mottle bromovirus (CCMV) is distinct from that of monocot-adapted brome mosaic bromovirus (BMV), two reporter genes, β-glucuronidase (GUS) and enhanced green fluorescent protein (EGFP), were substituted for the CP in a biologically active clone of CCMV RNA3 (C3). Primary leaves ofNicotiana benthamiana, Chenopodium quinoa,and cowpea were co-inoculated with wild-type (wt) CCMV RNA 1 and -2 and either C3/ΔCP-GUS or C3/ΔCP-EGFP and analyzed for GUS activity or the presence of green fluorescence. The visual appearance of infections caused by GUS or EGFP variants indicated that, in CCMV, epidermal cell-to-cell movement can occur without a functional CP. By contrast, inoculation of MP defective variants of C3/ΔCP-GUS or C3/ΔCP-EGFP resulted in subliminal infections. Additional experiments examining the infectivity of wt BMV RNA 1 and -2 and a BMV RNA3 variant bearing the EGFP in the place of CP (B3/ΔCP-EGFP) confirmed previous observations that, unlike CCMV, epidermal cell-to-cell movement of BMV is dependent on the expression of a functional CP. Taken together, the results demonstrate that BMV and CCMV use different mechanisms for initial epidermal cell-to-cell spread, and the individual role played by the respective CP genes in this active process is discussed

Packaging of Tobacco Mosaic Virus Subgenomic RNAs by Brome Mosaic Virus Coat Protein Exhibits RNA Controlled Polymorphism

AbstractThe coat protein (CP) of icosahedral Brome mosaic virus (BMV) was expressed from a genetically engineered rod-shape Tobacco mosaic virus. Molecular characterization of the progeny recovered from symptomatic plants revealed that BMV CP selectively packaged the three subgenomic RNAs of the hybrid virus into two differently sized icosahedral virus-like particles (VLPs). The smaller VLPs packaged only the two smaller subgenomic RNAs. Additional in vitro reassembly assays with BMV CP subunits and transcripts of hybrid subgenomic RNAs further demonstrated that the ability of BMV capsids to display polymorphism is not dependent on the RNA size alone and appears to be controlled by some other feature(s) of the genetically engineered RNA

Molecular Studies on Bromovirus Capsid Protein II. Functional Analysis of the Amino-Terminal Arginine-Rich Motif and Its Role in Encapsidation, Movement, and Pathology

AbstractThe N-terminal region of the brome mosaic bromovirus (BMV) coat protein (CP) contains an arginine-rich motif that is conserved among plant and nonplant viruses and implicated in binding the RNA during encapsidation. To elucidate the functional significance of this conserved motif in the BMV CP, a series of deletions encompassing the arginine-rich motif was introduced into a biologically active clone of BMV RNA3, and their effect on replication, encapsidation, and infection in plants was examined. Analysis of infection phenotypes elicited onChenopodium quinoarevealed the importance of the first 19 N-proximal amino acids of BMV CP in encapsidation and pathogenicity. Inoculation ofC. quinoawith three viable variants of BMV RNA3 lacking the first 11, 14, and 18 N-terminal amino acids of the CP resulted in the development of necrotic local lesions and restricted the spread of infection to inoculated leaves. Progeny analysis from symptomatic leaves revealed that, in each case, virus accumulation was severely affected by the introduced mutations and each truncated CP differed in its ability to package genomic RNA. In contrast to these observations inC. quinoa,none of the CP variants was able to establish either local or systemic infections in barley plants. The intrinsic role played by the N-terminal arginine-rich motif of BMV CP in packaging viral RNAs and the interactions between the host and the truncated CPs in modulating symptom expression and movement are discussed

Model-selection-based approach for calculating cellular multiplicity of infection during virus colonization of multi-cellular hosts

The cellular multiplicity of infection (MOI) is a key parameter for describing the interactions between virions and cells, predicting the dynamics of mixed-genotype infections, and understanding virus evolution. Two recent studies have reported in vivo MOI estimates for Tobacco mosaic virus (TMV) and Cauliflower mosaic virus (CaMV), using sophisticated approaches to measure the distribution of two virus variants over host cells. Although the experimental approaches were similar, the studies employed different definitions of MOI and estimation methods. Here, new model-selection-based methods for calculating MOI were developed. Seven alternative models for predicting MOI were formulated that incorporate an increasing number of parameters. For both datasets the best-supported model included spatial segregation of virus variants over time, and to a lesser extent aggregation of virus-infected cells was also implicated. Three methods for MOI estimation were then compared: the two previously reported methods and the best-supported model. For CaMV data, all three methods gave comparable results. For TMV data, the previously reported methods both predicted low MOI values (range: 1.04-1.23) over time, whereas the best-supported model predicted a wider range of MOI values (range: 1.01-2.10) and an increase in MOI over time. Model selection can therefore identify suitable alternative MOI models and suggest key mechanisms affecting the frequency of coinfected cells. For the TMV data, this leads to appreciable differences in estimated MOI values.This work was supported by grant BFU2012-30805 (SFE) and by 'Juan de la Cierva' postdoctoral contract JCI-2011-10379 (MPZ) from the Spanish Secretaria de Estado de Investigacion, Desarrollo e Innovacion. 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Unravelling the Stability and Capsid Dynamics of the Three Virions of Brome Mosaic Virus Assembled Autonomously In Vivo

Viral capsids are dynamic assemblies that undergo controlled conformational transitions to perform various biological functions. The replication-derived four-molecule RNA progeny of Brome mosaic virus (BMV) is packaged by a single capsid protein (CP) into three types of morphologically indistinguishable icosahedral virions with T?3 quasisymmetry. Type 1 (B1V) and type 2 (B2V) virions package genomic RNA1 and RNA2, respectively, while type 3 (B3?4V) virions co-package genomic RNA3 (B3) and its subgenomic RNA4 (sgB4). In this study, the application of a robust Agrobacterium-mediated transient expression system allowed us to assemble each virion type separately in planta. Experimental approaches analyzing the morphology, size, and electrophoretic mobility failed to distinguish between the virion types. Thermal denaturation analysis and protease-based peptide mass mapping experiments were used to analyze the stability and the conformational dynamics of the individual virions, respectively. The crystallographic structure of the BMV capsid shows four trypsin cleavage sites (K65, R103, K111, and K165 on the CP subunits) exposed on the exterior of the capsid. Irrespective of the digestion time, while retaining their capsid structural integrity, B1V and B2V released a single peptide encompassing amino acids 2 to 8 of the N-proximal arginine-rich RNA binding motif. In contrast, B3?4V capsids were unstable with trypsin, releasing several peptides in addition to the peptides encompassing four predicted sites exposed on the capsid exterior. These results, demonstrating qualitatively different dynamics for the three types of BMV virions, suggest that the different RNA genes they contain may have different translational timing and efficiency and may even impart different structures to their capsids

Unravelling the Stability and Capsid Dynamics of the Three Virions of Brome Mosaic Virus Assembled Autonomously In Vivo

Viral capsids are dynamic assemblies that undergo controlled conformational transitions to perform various biological functions. The replication-derived four-molecule RNA progeny of Brome mosaic virus (BMV) is packaged by a single capsid protein (CP) into three types of morphologically indistinguishable icosahedral virions with T?3 quasisymmetry. Type 1 (B1V) and type 2 (B2V) virions package genomic RNA1 and RNA2, respectively, while type 3 (B3?4V) virions co-package genomic RNA3 (B3) and its subgenomic RNA4 (sgB4). In this study, the application of a robust Agrobacterium-mediated transient expression system allowed us to assemble each virion type separately in planta. Experimental approaches analyzing the morphology, size, and electrophoretic mobility failed to distinguish between the virion types. Thermal denaturation analysis and protease-based peptide mass mapping experiments were used to analyze the stability and the conformational dynamics of the individual virions, respectively. The crystallographic structure of the BMV capsid shows four trypsin cleavage sites (K65, R103, K111, and K165 on the CP subunits) exposed on the exterior of the capsid. Irrespective of the digestion time, while retaining their capsid structural integrity, B1V and B2V released a single peptide encompassing amino acids 2 to 8 of the N-proximal arginine-rich RNA binding motif. In contrast, B3?4V capsids were unstable with trypsin, releasing several peptides in addition to the peptides encompassing four predicted sites exposed on the capsid exterior. These results, demonstrating qualitatively different dynamics for the three types of BMV virions, suggest that the different RNA genes they contain may have different translational timing and efficiency and may even impart different structures to their capsids

Deletions in the Conserved Amino-Terminal Basic Arm of Cucumber Mosaic Virus Coat Protein Disrupt Virion Assembly but Do Not Abolish Infectivity and Cell-to-Cell Movement

AbstractThe N-terminal basic arm of cucumber mosaic cucumovirus (CMV) coat protein (CP) contains a conserved arginine-rich motif, which is characteristic of RNA binding proteins of several plant and nonplant viruses. To identify regions of the CMV CP N-terminus that are essential for interacting with viral genomic RNA, a comprehensive set of mutations was engineered into biologically active clones of CMV RNA3 and the behavior of each variant with respect to infectivity, packaging and movement was examined. Biological assays conducted inChenopodium quinoa(local lesion host) andNicotiana benthamiana(systemic host) revealed that variants lacking either 12 N-proximal amino acids or a region containing four consecutive arginine residues of the CP N-terminus were competent for assembly into virions and remained infectious in plants. Interestingly, two other variants, lacking either 19 N-proximal amino acids or a domain containing a cluster of six arginines in the arginine-rich motif, were incompetent for virion assembly but retained the ability to move cell to cell. Taken together, these results indicate that a major portion of the N-terminal basic arm of CMV CP is dispensable for CP-RNA interactions and also establish that CMV can move cell to cell in a nonvirion form. The distinctive role played by the viral CP in movement and specifically, the extent to which the CP N-terminal basic arm is involved in the infection cycle of CMV are discussed

Deletion of highly conserved arginine-rich RNA binding motif in cowpea chlorotic mottle virus capsid protein results in virion structural alterations and RNA packaging constraints

The N-proximal region of cowpea chlorotic mottle virus (CCMV) capsid protein (CP) contains an arginine-rich RNA binding motif (ARM) that is also found in the CPs of other members of Bromoviridae and in other RNA binding proteins such as the Tat and Rev proteins of human immunodeficiency virus. To assess the critical role played by this motif during encapsidation, a variant of CCMV RNA3 (C3) precisely lacking the ARM region (C3/Delta919) of its CP gene was constructed. The biology and the competence of the matured CP derived in vivo from C3/Delta919 to assemble and package progeny RNA was examined in whole plants. Image analysis and computer-assisted three-dimensional reconstruction of wild-type and mutant virions revealed that the CP subunits bearing the engineered deletion assembled into polymorphic virions with altered surface topology. Northern blot analysis of virion RNA from mutant progeny demonstrated that the engineered mutation down-regulated packaging of all four viral RNAs; however, the packaging effect was more pronounced on genomic RNA1 and RNA2 than genomic RNA3 and its CP mRNA. In vitro assembly assays with mutant CP subunits and RNA transcripts demonstrated that the mutant CP is inherently not defective in packaging genomic RNA1 (53%) and RNA2 (54%), but their incorporation into virions was competitively inhibited by the presence of other viral RNAs. Northern blot analysis of RNA encapsidation in vivo of two distinct bromovirus RNA3 chimeras, constructed by exchanging CPs having the Delta919 deletion, demonstrated that the role of the conserved N-terminal ARM in recognizing and packaging specific RNA is distinct for each virus

Advanced Modelling and Simulation of Intermetallic Reinforced Composites for Structural and Functional Applications

In recent years, intermetallic reinforced composites (IRCs) have garnered significant attention due to their exceptional mechanical properties, corrosion resistance, and high-temperature stability, making them ideal candidates for both structural and functional applications. This research paper presents an advanced modelling and simulation approach to understand the microstructural evolution, mechanical behaviour, and functional properties of IRCs. Utilizing a combination of finite element analysis (FEA), molecular dynamics (MD), and phase-field modelling, the study offers a comprehensive insight into the intricate interplay between the matrix, reinforcement, and the resultant composite behaviour. The developed models accurately predict the stress-strain response, thermal conductivity, and fatigue life of the IRCs under various loading and environmental conditions. Furthermore, the simulations provide a detailed understanding of the mechanisms governing crack initiation and propagation in these composites. The outcomes of this research not only pave the way for optimizing the design and processing parameters of IRCs but also underscore the potential of these materials in aerospace, automotive, and energy sectors. The findings presented herein serve as a foundational reference for researchers and engineers aiming to harness the full potential of intermetallic reinforced composites in advanced engineering applications