Search

Capsid disassembly and genome release are critical steps in the lifecycle of a virus. However, their mechanisms are poorly understood, both in vivo and in vitro. Here, we have identified two in vitro disassembly pathways of the brome mosaic virus (BMV) by charge detection mass spectrometry and transmission electron microscopy. When subjected to a pH jump to a basic environment at low ionic strength, protein-RNA interactions are disrupted. Under these conditions, BMV appears to disassemble mainly through a global cleavage event into two main fragments: a near complete capsid that has released the RNA and the released RNA complexed to a small number of the capsid proteins. Upon slow buffer exchange to remove divalent cations at neutral pH, capsid protein interactions are disrupted. The BMV virions swell but there is no measurable loss of the RNA. Some of the virions break into small fragments, leading to an increase in the abundance of species with masses less than 1 MDa. The peak attributed to the BMV virion shifts to a higher mass with time. The mass increase is attributed to additional capsid proteins associating with the disrupted capsid protein-RNA complex, where the RNA is presumably partially exposed. It is likely that this pathway is more closely related to how the capsid disassembles in vivo, as it offers the advantage of protecting the RNA with the capsid protein until translation begins.

A hallmark feature of (+)-strand RNA viruses of eukaryotic cells is that progeny (+)-strands are accumulated 100-fold over (−)-strands. Previous experimental evidence suggests that, in Brome mosaic virus (BMV), a plant-infecting member of the alphavirus-like superfamily, the addition of RNA3 and, specifically, translation of the wild-type (WT) coat protein (CP) gene contributes to increased accumulation of (+)-strands. It is unclear whether this stimulation of (+)-strand accumulation by CP is due to direct regulation of viral RNA replication or RNA stabilization via encapsidation. Analysis of BMV progeny RNA in Nicotiana benthamiana plants revealed that expression of RNA3 variants that did not express WT CP led to a severe defect in BMV (+)-strand accumulation. The (+)-strand accumulation could be rescued when CP was complemented in trans. To verify whether stimulation of (+)-strand accumulation is coupled with encapsidation, two independent mutations were engineered into CP open reading frames. An N-terminal deletion that prevented CP binding to the viral RNAs resulted in a severe reduction of BMV (+)-strand accumulation but stimulated (−)-strand accumulation over the WT. On the other hand, a C-terminal mutation affecting CP dimerization caused a significant decrease in (+)-strand accumulation but had no detectable effect on (−)-strand accumulation. Nucleotide sequences in the movement protein-coding region were also found to contribute to (+)-strand accumulation, in part by providing packaging signals for efficient RNA3 encapsidation. Overall, these results show that RNA encapsidation is a significant determinant of BMV RNA intracellular accumulation.

Phloem-mobile mRNAs are assumed to contain sequence elements directing RNA to the phloem translocation pathway. One of such elements is represented by tRNA sequences embedded in untranslated regions of many mRNAs, including those proved to be mobile. Genomic RNAs of a number of plant viruses possess a 3'-terminal tRNA-like structures (TLSs) only distantly related to genuine tRNAs, but nevertheless aminoacylated and capable of interaction with some tRNA-binding proteins. Here, we elaborated an experimental system for analysis of RNA phloem transport based on an engineered RNA of Potato virus X capable of replication, but not encapsidation and movement in plants. The TLSs of Brome mosaic virus, Tobacco mosaic virus and Turnip yellow mosaic virus were demonstrated to enable the phloem transport of foreign RNA. A miRNA precursor, pre-miR390b, was also found to render RNA competent for the phloem transport. In line with this, sequences of miRNA precursors were identified in a Cucurbita maxima phloem transcriptome, supporting the hypothesis that, at least in some cases, miRNA phloem signaling can involve miRNA precursors. Collectively, the data presented here suggest that RNA molecules can be directed into the phloem translocation pathway by structured RNA elements such as those of viral TLSs and miRNA precursors.

Viruses avoid exposure of the viral genome to harmful agents with the help of a protective protein shell known as the capsid. A secondary effect of this protective barrier is that macromolecules that may be in high concentration on the outside cannot freely diffuse across it. Therefore, inside the cell and possibly even outside, the intact virus is generally under a state of osmotic stress. Viruses deal with this type of stress in various ways. In some cases, they might harness it for infection. However, the magnitude and influence of osmotic stress on virus physical properties remains virtually unexplored for single-stranded RNA viruses - the most abundant class of viruses. Here, we report on how a model system for the positive-sense RNA icosahedral viruses, brome mosaic virus (BMV), responds to osmotic pressure. Specifically, we study the mechanical properties and structural stability of BMV under controlled molecular crowding conditions. We show that BMV is mechanically reinforced under a small external osmotic pressure but starts to yield after a threshold pressure is reached. We explain this mechanochemical behavior as an effect of the molecular crowding on the entropy of the "breathing"fluctuation modes of the virus shell. The experimental results are consistent with the viral RNA imposing a small negative internal osmotic pressure that prestresses the capsid. Our findings add a new line of inquiry to be considered when addressing the mechanisms of viral disassembly inside the crowded environment of the cell.

Viruses require multifunctional structured RNAs to hijack their host’s biochemistry, but their mechanisms can be obscured by the difficulty of solving conformationally dynamic RNA structures. Using cryo-EM, we visualized the structure of the mysterious viral tRNA-like structure (TLS) from brome mosaic virus (BMV), which affects replication, translation, and genome encapsidation. Structures in isolation and bound to tyrosyl-tRNA synthetase (TyrRS) show that this ∼55 kDa purported tRNA mimic undergoes large conformational rearrangements to bind TyrRS in a form that differs dramatically from tRNA. Our studies reveal how viral RNAs can use a combination of static and dynamic RNA structures to bind host machinery through highly noncanonical interactions and highlights the utility of cryo-EM for visualizing small conformationally dynamic structured RNAs.

Non-enveloped RNA viruses pervade all domains of life. In a cell, they co-assemble from viral RNA and capsid proteins. Virus-like particles can form in vitro where virtually any non-cognate polyanionic cargo can be packaged. How only viral RNA gets selected for packaging in vivo, in presence of myriad other polyanionic species, has been a puzzle. Through a combination of charge detection mass spectrometry and cryo-electron microscopy, it is determined that co-assembling brome mosaic virus (BMV) coat proteins and nucleic acid oligomers results in capsid structures and stoichiometries that differ from the icosahedral virion. These previously unknown shell structures are strained and less stable than the native one. However, they contain large native structure fragments that can be recycled to form BMV virions, should a viral genome become available. The existence of such structures suggest the possibility of a previously unknown regulatory pathway for the packaging process inside cells.

We report the asymmetric reconstruction of the single-stranded RNA (ssRNA) content in one of the three otherwise identical virions of a multipartite RNA virus, brome mosaic virus (BMV). We exploit a sample consisting exclusively of particles with the same RNA content—specifically, RNAs 3 and 4—assembled in planta by agrobacterium-mediated transient expression. We find that the interior of the particle is nearly empty, with most of the RNA genome situated at the capsid shell. However, this density is disordered in the sense that the RNA is not associated with any particular structure but rather, with an ensemble of secondary/tertiary structures that interact with the capsid protein. Our results illustrate a fundamental difference between the ssRNA organization in the multipartite BMV viral capsid and the monopartite bacteriophages MS2 and Qβ for which a dominant RNA conformation is found inside the assembled viral capsids, with RNA density conserved even at the center of the particle. This can be understood in the context of the differing demands on their respective lifecycles: BMV must package separately each of several different RNA molecules and has been shown to replicate and package them in isolated, membrane-bound, cytoplasmic complexes, whereas the bacteriophages exploit sequence-specific “packaging signals” throughout the viral RNA to package their monopartite genomes.

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 (B1(V)) and type 2 (B2(V)) virions package genomic RNA1 and RNA2, respectively, while type 3 (B3+4(V)) virions copackage 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 stability and the conformational dynamics of the individual virions, respectively. The crystallographic structure of the BMV capsid shows four trypsin cleavage sites (K(65), R(103), K(111), and K(165) on the CP subunits) exposed on the exterior of the capsid. Irrespective of the digestion time, while retaining their capsid structural integrity, B1(V) and B2(V) released a single peptide encompassing amino acids 2 to 8 of the N-proximal arginine-rich RNA binding motif. In contrast, B3+4(V) 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. IMPORTANCE The majority of viruses contain RNA genomes protected by a shell of capsid proteins. Although crystallographic studies show that viral capsids are static structures, accumulating evidence suggests that, in solution, virions are highly dynamic assemblies. The three genomic RNAs (RNA1, -2, and -3) and a single subgenomic RNA (RNA4) of Brome mosaic virus (BMV), an RNA virus pathogenic to plants, are distributed among three physically homogeneous virions. This study examines the thermal stability by differential scanning fluorimetry (DSF) and capsid dynamics by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analyses following trypsin digestion of the three virions assembled separately in vivo using the Agrobacterium-mediated transient expression approach. The results provide compelling evidence that virions packaging genomic RNA1 and -2 are distinct from those copackaging RNA3 and -4 in their stability and dynamics, suggesting that RNA-dependent capsid dynamics play an important biological role in the viral life cycle.

Human parechoviruses (HPeV) are picornaviruses with a highly-ordered RNA genome contained within icosahedrally-symmetric capsids. Ordered RNA structures have recently been shown to interact with capsid proteins VP1 and VP3 and facilitate virus assembly in HPeV1. Using an assay that combines reversible cross-linking, RNA affinity purification and peptide mass fingerprinting (RCAP), we mapped the RNA-interacting regions of the capsid proteins from the whole HPeV1 virion in solution. The intrinsically-disordered N-termini of capsid proteins VP1 and VP3, and unexpectedly, VP0, were identified to interact with RNA. Comparing these results to those obtained using recombinantly-expressed VP0 and VP1 confirmed the virion binding regions, and revealed unique RNA binding regions in the isolated VP0 not previously observed in the crystal structure of HPeV1. We used RNA fluorescence anisotropy to confirm the RNA-binding competency of each of the capsid proteins’ N-termini. These findings suggests that dynamic interactions between the viral RNA and the capsid proteins modulate virus assembly, and suggest a novel role for VP0.

In the brome mosaic virus (BMV) virion, the coat protein (CP) selectively contacts the RNA motifs that regulate translation and RNA replication (Hoover et al., 2016. J. Virol. 90, 7748). We hypothesize that the unstructured N-terminal arm (NTA) of the BMV CP can specifically recognize RNA motifs. Using ion mobility spectrometry-mass spectrometry, we demonstrate that peptides containing the NTA of the CP were found to preferentially bind to an RNA hairpin motif that directs the initiation of BMV RNA synthesis. RNA binding causes the peptide to change from heterogeneous structures to a single family of structures. Fluorescence anisotropy, fluorescence quenching and size exclusion chromatography experiments all confirm that the NTA can specific recognize the RNA motif. The peptide introduced into plants along with BMV virion increased accumulation of the BMV CP and accelerated the rate of minus-strand RNA synthesis. The intrinsically disordered BMV NTA could thus specifically recognize BMV RNAs to affect viral infection.

Viral capsids are dynamic assemblies that undergo controlled conformational transitions to perform various biological functions. The replicated three-molecule RNA progeny of Brome mosaic virus (BMV) are packaged by a single capsid protein (CP) into three types of morphologically indistinguishable icosahedral virions with T=3 quasi-symmetry. Type 1 (B1v) and type 2 (B2v) virions respectively package genomic RNA1 or RNA2, while type 3 (B3+4v) co-packages genomic RNA3 (B3) and its sub-genomic RNA4 (B4). In this study, the application of a robust Agrobacterium-mediated transient expression system allowed us to assemble each virion type separately in planta. Physical and biochemical approaches analyzing the morphology, size, and electrophoretic mobility failed to distinguish between the virion types, so protease-based mapping experiments were used to analyze the conformational dynamics of the individual virions. The crystallographic structure of the BMV capsid shows four trypsin-cleavage sites (K65, R103, K111 and K165 on the A, B and C subunits) exposed on the exterior of the capsid. Irrespective of the digestion time, while retaining their capsid structural integrity, B1v and B2v released only two peptides involving amino acids 2-8 and 16-22 from the N-proximal arginine-rich RNA binding motif. In contrast, B3+4v capsids are unstable to trypsin, releasing several peptides in addition to the four sites predicted to be 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.IMPORTANCEThe majority of viruses contain RNA genomes protected by a shell of capsid proteins. Although crystallographic studies show that viral capsids are static structures, accumulating evidence suggests that in solution virions are highly dynamic assemblies. The three genomic RNAs (RNAs 1, 2 and 3) and a single subgenomic RNA (RNA4) of Brome mosaic virus (BMV), an RNA virus pathogenic to plants, are distributed among three physically homogeneous virions. This study examines the capsid dynamics by MALDI-TOF analyses following trypsin digestion of the three virions assembled separately in vivo using the Agrobacterium-mediated transient expression approach. The results provide compelling evidence that virions packaging genomic RNAs1 and 2 are more stable and dynamically distinct from those co-packaging RNA3 and 4, suggesting that RNA-dependent capsid dynamics play an important biological role in the viral life cycle.

The four brome mosaic virus (BMV) RNAs (RNA1 to RNA4) are encapsidated in three distinct virions that have different disassembly rates in infection. The mechanism for the differential release of BMV RNAs from virions is unknown, since 180 copies of the same coat protein (CP) encapsidate each of the BMV genomic RNAs. Using mass spectrometry, we found that the BMV CP contains a complex pattern of posttranslational modifications. Treatment with phosphatase was found to not significantly affect the stability of the virions containing RNA1 but significantly impacted the stability of the virions that encapsidated BMV RNA2 and RNA3/4. Cryo-electron microscopy reconstruction revealed dramatic structural changes in the capsid and the encapsidated RNA. A phosphomimetic mutation in the flexible N-terminal arm of the CP increased BMV RNA replication and virion production. The degree of phosphorylation modulated the interaction of CP with the encapsidated RNA and the release of three of the BMV RNAs. UV cross-linking and immunoprecipitation methods coupled to high-throughput sequencing experiments showed that phosphorylation of the BMV CP can impact binding to RNAs in the virions, including sequences that contain regulatory motifs for BMV RNA gene expression and replication. Phosphatase-treated virions affected the timing of CP expression and viral RNA replication in plants. The degree of phosphorylation decreased when the plant hosts were grown at an elevated temperature. These results show that phosphorylation of the capsid modulates BMV infection. IMPORTANCE How icosahedral viruses regulate the release of viral RNA into the host is not well understood. The selective release of viral RNA can regulate the timing of replication and gene expression. Brome mosaic virus (BMV) is an RNA virus, and its three genomic RNAs are encapsidated in separate virions. Through proteomic, structural, and biochemical analyses, this work shows that posttranslational modifications, specifically, phosphorylation, on the capsid protein regulate the capsid-RNA interaction and the stability of the virions and affect viral gene expression. Mutational analysis confirmed that changes in modification affected virion stability and the timing of viral infection. The mechanism for modification of the virion has striking parallels to the mechanism of regulation of chromatin packaging by nucleosomes.

Metabolite-dependent conformational switching in RNA riboswitches is now widely accepted as a critical regulatory mechanism for gene expression in bacterial systems. More recently, similar gene regulation mechanisms have been found to be important for viral systems as well. One of the most abundant and best-studied systems is the tRNA-like structure (TLS) domain, which has been found to occur in many plant viruses spread across numerous genera. In this work, folding dynamics for the TLS domain of Brome Mosaic Virus have been investigated using single-molecule fluorescence resonance energy transfer techniques. In particular, burst fluorescence methods are exploited to observe metal-ion ([Mn+])-induced folding in freely diffusing RNA constructs resembling the minimal TLS element of brome mosaic virus RNA3. The results of these experiments reveal a complex equilibrium of at least three distinct populations. A stepwise, or consecutive, thermodynamic model for TLS folding is developed, which is in good agreement with the [Mn+]-dependent evolution of conformational populations and existing structural information in the literature. Specifically, this folding pathway explains the metal-ion dependent formation of a functional TLS domain from unfolded RNAs via two consecutive steps: 1) hybridization of a long-range stem interaction, followed by 2) formation of a 3′-terminal pseudoknot. These two conformational transitions are well described by stepwise dissociation constants for [Mg2+] (K1 = 328 ± 30 μM and K2 = 1092 ± 183 μM) and [Na+] (K1 = 74 ± 6 mM and K2 = 243 ± 52 mM)-induced folding. The proposed thermodynamic model is further supported by inhibition studies of the long-range stem interaction using a complementary DNA oligomer, which effectively shifts the dynamic equilibrium toward the unfolded conformation. Implications of this multistep conformational folding mechanism are discussed with regard to regulation of virus replication.

Plant pathogenic single strand positive-sense RNA viruses with the tripartite genome are classified into two families: Bromoviridae and Virgaviridae. Family Bromoviridae contains four genera Bromo, Cucumo, Alfamo, and Ilarviruses characterized by icosahedral particles. By contrast family Virgaviridae contains only one genus, Hordeivirus, with tripartite genome and characterized by helical particles. Unlike in monopartite plant viruses, packaging in tripartite RNA viruses requires a well-orchestrated process to ensure that viral progeny is selectively encapsidated and distributed optimally into three or four different viral capsids. Among the tripartite RNA viruses mentioned above, brome mosaic virus (BMV), the type member of the genus bromovirus, has been extensively used as a model system to unravel the mechanism of genome packaging. Using the available research data on BMV, this review is focused in updating the readers on how various macromolecular interactions (e.g. packaging signals) and biological factors (i.e. type of host plant) modulate genome packaging. The review also offers new directions of research to further our knowledge on the genome packaging in tripartite viruses.

Brome mosaic virus (BMV) is a well-known plant virus representing single-stranded RNA (ssRNA) positive-sense viruses. It has been widely used as a model in multiple studies concerning plant virus biology, epidemiology and the application of viral capsids in nanotechnology. Herein, we describe a method for BMV purification based on ion-exchange and size-exclusion chromatography. The presented method is of similar efficiency to previously described protocols relying on differential centrifugation and can easily be scaled up. The resulting BMV capsids are stable and monodisperse and can be used for further applications.

Structural RNA domains are widely involved in the regulation of biological functions, such as gene expression, gene modification, and gene repair. Activity of these dynamic regions depends sensitively on the global fold of the RNA, in particular, on the binding affinity of individual conformations to effector molecules in solution. Consequently, both the 1) structure and 2) conformational dynamics of noncoding RNAs prove to be essential in understanding the coupling that results in biological function. Toward this end, we recently reported observation of three conformational states in the metal-induced folding pathway of the tRNA-like structure domain of Brome Mosaic Virus, via single-molecule fluorescence resonance energy transfer studies. We report herein selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE)-directed structure predictions as a function of metal ion concentrations ([Mn+]) to confirm the three-state folding model, as well as test 2° structure models from the literature. Specifically, SHAPE reactivity data mapped onto literature models agrees well with the secondary structures observed at 0–10 mM [Mg2+], with only minor discrepancies in the E hairpin domain at low [Mg2+]. SHAPE probing and SHAPE-directed structure predictions further confirm the stepwise unfolding pathway previously observed in our single-molecule studies. Of special relevance, this means that reduction in the metal-ion concentration unfolds the 3′ pseudoknot interaction before unfolding the long-range stem interaction. This work highlights the synergistic power of combining 1) single-molecule Forster resonance energy transfer and 2) SHAPE-directed structure-probing studies for detailed analysis of multiple RNA conformational states. In particular, single-molecule guided deconvolution of the SHAPE reactivities permits 2° structure predictions of isolated RNA conformations, thereby substantially improving on traditional limitations associated with current structure prediction algorithms.

Although RNA viruses evolved the mechanisms of specific encapsidation, miss-packaging of cellular RNAs has been reported in such RNA virus systems as flock house virus or cucumber necrosis virus. To find out if brome mosaic virus (BMV), a tripartite RNA virus, can package cellular RNAs, BMV was propagated in barley and in Nicotiana benthamiana hosts, purified by cesium chloride (CsCl) gradient ultracentrifugation followed by nuclease treatment to remove any contaminating cellular (host) RNAs. The extracted virion RNA was then sequenced by using next-generation sequencing (NGS RNA-Seq) with the Illumina protocol. Bioinformatic analysis revealed the content of host RNAs ranging from 0.07% for BMV extracted from barley to 0.10% for the virus extracted from N. benthamiana. The viruses from two sources appeared to co-encapsidate different patterns of host-RNAs, including ribosomal RNAs (rRNAs), messenger RNAs (mRNAs) but also mitochondrial and plastid RNAs and, interestingly, transposable elements, both transposons and retrotransposons. Our data reveal that BMV virions can carry host RNAs, having a potential to mediate horizontal gene transfer (HGT) in plants.

Positive-strand RNA viruses replicate their genomes in membrane-bound replication compartments. Brome mosaic virus (BMV) replicates in vesicular invaginations of the endoplasmic reticulum membrane. BMV has served as a productive model system to study processes like virus-host interactions, RNA replication and recombination. Here we present multiple lines of evidence showing that the structure of the viral RNA replication compartments plays a fundamental role and that recruitment of parental RNAs to a common replication compartment is a limiting step in intermolecular RNA recombination. We show that a previously defined requirement for an RNA recruitment element on both parental RNAs is not to function as a preferred crossover site, but in order for individual RNAs to be recruited into the replication compartments. Moreover, modulating the form of the replication compartments from spherular vesicles (spherules) to more expansive membrane layers increased intermolecular RNA recombination frequency by 200- to 1000-fold. We propose that intermolecular RNA recombination requires parental RNAs to be recruited into replication compartments as monomers, and that recruitment of multiple RNAs into a contiguous space is much more common for layers than for spherules. These results could explain differences in recombination frequencies between viruses that replicate in association with smaller spherules versus larger double-membrane vesicles and convoluted membranes.

Genomes of single-stranded RNA viruses have evolved to optimize several concurrent properties. One of them is the architecture of their genomic folds, which must not only feature precise structural elements at specific positions, but also allow for overall spatial compactness. The latter was shown to be disrupted by random synonymous mutations, a disruption which can consequently negatively affect genome encapsidation. In this study, we use three mutation schemes with different degrees of locality to mutate the genomes of phage MS2 and Brome Mosaic virus in order to understand the observed sensitivity of the global compactness of their folds. We find that mutating local stretches of their genomes’ sequence or structure is less disruptive to their compactness compared to inducing randomly-distributed mutations. Our findings are indicative of a mechanism for the conservation of compactness acting on a global scale of the genomes, and have several implications for understanding the interplay between local and global architecture of viral RNA genomes.

Positive-strand RNA viruses generally assemble RNA replication complexes on rearranged host membranes. Alphaviruses, other members of the alpha-like virus superfamily, and many other positive-strand RNA viruses invaginate host membrane into vesicular RNA replication compartments, known as spherules, whose interior is connected to the cytoplasm. Brome mosaic virus (BMV) and its close relative, cowpea chlorotic mottle virus (CCMV), form spherules along the endoplasmic reticulum. BMV spherule formation and RNA replication can be fully reconstituted in S. cerevisiae, enabling many studies identifying host factors and viral interactions essential for these processes. To better define and understand the conserved, core pathways of bromovirus RNA replication, we tested the ability of CCMV to similarly support spherule formation and RNA replication in yeast. Paralleling BMV, we found that CCMV RNA replication protein 1a was the only viral factor necessary to induce spherule membrane rearrangements and to recruit the viral 2a polymerase (2apol) to the endoplasmic reticulum. CCMV 1a and 2apol also replicated CCMV and BMV genomic RNA2, demonstrating core functionality of CCMV 1a and 2apol in yeast. However, while BMV and CCMV 1a/2apol strongly replicate each others' genomic RNA3 in plants, neither supported detectable CCMV RNA3 replication in yeast. Moreover, in contrast to plant cells, in yeast CCMV 1a/2apol supported only limited replication of BMV RNA3 (

Brome mosaic virus (BMV) (the Russian strain) infects monocot plants and has been studied extensively in barley and wheat. Here, we report BMV can systemically infect rice (Oryza sativa var. japonica), including cultivars in which the genomes have been determined. The BMV capsid protein can be found throughout the inoculated plants. However, infection in rice exhibits delayed symptom expression or no symptoms when compared with wheat (Triticum aestivum). The sequences of BMV RNAs isolated from rice did not reveal any nucleotide changes in RNA1 or RNA2, while RNA3 had only one synonymous nucleotide change from the inoculum sequence. Preparations of purified BMV virions contained RNA1 at a significantly reduced level relative to the other two RNAs. Analysis of BMV RNA replication in rice revealed that minus-strand RNA1 was replicated at a reduced rate when compared with RNA2. Thus, rice appears to either inhibit RNA1 replication or lacks a sufficient amount of a factor needed to support efficient RNA1 replication.

Reactive oxygen species (ROS), including superoxide anion (O2-), hydrogen peroxide (H2O2), and hydroxyl radical, act as signaling molecules to transduce biotic and abiotic stimuli into stress adaptations in plants. A respiratory burst oxidase homolog B of Nicotiana benthamiana (NbRBOHB) is responsible for O2- production to inhibit pathogen infection during plant innate immunity. RBOH-derived O2- can be immediately converted into H2O2 by the action of superoxide dismutase. Interestingly, we recently showed that red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA [(+)RNA] virus, hijacks the host's ROS-generating machinery during infection. An RCNMV replication protein associates with NbRBOHB and triggers intracellular ROS bursts. These bursts are required for robust viral RNA replication. However, what types of ROS are required for viral replication is currently unknown. Here, we found that RCNMV replication was sensitive to an O2- scavenger but insensitive to an H2O2 scavenger. Interestingly, replication of another plant (+)RNA virus, brome mosaic virus, was sensitive to both types of scavengers. These results indicate a virus-specific pattern requirement of O2- and H2O2 for (+)RNA virus replication and suggest a conserved nature of the roles of ROS in (+)RNA virus replication.

As sessile organisms, plants have to accommodate to rapid changes in their surrounding environment. Reactive oxygen species (ROS) act as signaling molecules to transduce biotic and abiotic stimuli into plant stress adaptations. It is established that a respiratory burst oxidase homolog B of Nicotiana benthamiana (NbRBOHB) produces ROS in response to microbe-associated molecular patterns to inhibit pathogen infection. Plant viruses are also known as causative agents of ROS induction in infected plants; however, the function of ROS in plant-virus interactions remains obscure. Here, we show that the replication of red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA [(+)RNA] virus, requires NbRBOHB-mediated ROS production. The RCNMV replication protein p27 plays a pivotal role in this process, redirecting the subcellular localization of NbRBOHB and a subgroup II calcium-dependent protein kinase of N. benthamiana (NbCDPKiso2) from the plasma membrane to the p27-containing intracellular aggregate structures. p27 also induces an intracellular ROS burst in an RBOH-dependent manner. NbCDPKiso2 was shown to be an activator of the p27-triggered ROS accumulations and to be required for RCNMV replication. Importantly, this RBOH-derived ROS is essential for robust viral RNA replication. The need for RBOH-derived ROS was demonstrated for the replication of another (+)RNA virus, brome mosaic virus, suggesting that this characteristic is true for plant (+)RNA viruses. Collectively, our findings revealed a hitherto unknown viral strategy whereby the host ROS-generating machinery is diverted for robust viral RNA replication.

Melandrium yellow fleck virus belongs to the genus Bromovirus , which is a group of tripartite plant RNA viruses. This virus has an approximately 200-nucleotide direct repeat sequence in the 5′ untranslated region (UTR) of RNA3 that encodes the 3a movement protein. In the present study, protoplast assays suggested that the duplicated region contains amplification-enhancing elements. Deletion analyses of the 5′ UTR of RNA3 showed that mutations in the short base-paired region, which is located dozens of bases upstream of the initiation codon of the 3a gene, greatly reduced the accumulation of RNA3. Disruption and restoration of the base-paired structure caused the accumulation of RNA3 to be decreased and restored, respectively. In vitro translation/replication assays demonstrated that the base-paired structure is important for the efficient amplification of negative-stand RNA3. A similar base-paired structure in RNA3 of another bromovirus, brome mosaic virus (BMV), also facilitated the efficient amplification of BMV RNA3, but only in combination with melandrium yellow fleck virus (MYFV) replicase and not with BMV replicase, thereby suggesting specific interactions between base-paired structures and MYFV replicase.

The three subsets of virions that comprise the Brome mosaic virus (BMV) were previously thought to be indistinguishable. This work tested the hypothesis that distinct capsid-RNA interactions in the BMV virions allow different rates of viral RNA release. Several results support distinct interactions between the capsid and the BMV genomic RNAs. First, the deletion of the first eight residues of the BMV coat protein (CP) resulted in the RNA1-containing particles having altered morphologies, while those containing RNA2 were unaffected. Second, subsets of the BMV particles separated by density gradients into a pool enriched for RNA1 (B1) and for RNA2 and RNA3/4 (B2.3/4) were found to have different physiochemical properties. Compared to the B2.3/4 particles, the B1 particles were more sensitive to protease digestion and had greater resistivity to nanoindentation by atomic force microscopy and increased susceptibility to nuclease digestion. Mapping studies showed that portions of the arginine-rich N-terminal tail of the CP could interact with RNA1. Mutational analysis in the putative RNA1-contacting residues severely reduced encapsidation of BMV RNA1 without affecting the encapsidation of RNA2. Finally, during infection of plants, the more easily released RNA1 accumulated to higher levels early in the infection. IMPORTANCE Viruses with genomes packaged in distinct virions could theoretically release the genomes at different times to regulate the timing of gene expression. Using an RNA virus composed of three particles, we demonstrated that the RNA in one of the virions is released more easily than the other two in vitro . The differential RNA release is due to distinct interactions between the viral capsid protein and the RNAs. The ease of RNA release is also correlated with the more rapid accumulation of that RNA in infected plants. Our study identified a novel role for capsid-RNA interactions in the regulation of a viral infection.

Brome mosaic virus (BMV) packages its genomic and subgenomic RNAs into three separate viral particles. BMV purified from barley, wheat, and tobacco have distinct relative abundances of the encapsidated RNAs. We seek to identify the basis for the host-dependent differences in viral RNA encapsidation. Sequencing of the viral RNAs revealed recombination events in the 3' untranslated region of RNA1 of BMV purified from barley and wheat, but not from tobacco. However, the relative amounts of the BMV RNAs that accumulated in barley and wheat are similar and RNA accumulation is not sufficient to account for the difference in RNA encapsidation. Virions purified from barley and wheat were found to differ in their isoelectric points, resistance to proteolysis, and contacts between the capsid residues and the RNA. Mass spectrometric analyses revealed that virions from the three hosts had different post-translational modifications that should impact the physiochemical properties of the virions. Another major source of variation in RNA encapsidation was due to the purification of BMV particles to homogeneity. Highly enriched BMV present in lysates had a surprising range of sizes, buoyant densities, and distinct relative amounts of encapsidated RNAs. These results show that the encapsidated BMV RNAs reflect a combination of host effects on the physiochemical properties of the viral capsids and the enrichment of a subset of virions. The previously unexpected heterogeneity in BMV should influence the timing of the infection and also the host innate immune responses.

All positive-strand RNA viruses reorganize host intracellular membranes to assemble their viral replication complexes (VRCs); however, how these viruses modulate host lipid metabolism to accommodate such membrane proliferation and rearrangements is not well defined. We show that a significantly increased phosphatidylcholine (PC) content is associated with brome mosaic virus (BMV) replication in both natural host barley and alternate host yeast based on a lipidomic analysis. Enhanced PC levels are primarily associated with the perinuclear ER membrane, where BMV replication takes place. More specifically, BMV replication protein 1a interacts with and recruits Cho2p (choline requiring 2), a host enzyme involved in PC synthesis, to the site of viral replication. These results suggest that PC synthesized at the site of VRC assembly, not the transport of existing PC, is responsible for the enhanced accumulation. Blocking PC synthesis by deleting the CHO2 gene resulted in VRCs with wider diameters than those in wild-type cells; however, BMV replication was significantly inhibited, highlighting the critical role of PC in VRC formation and viral replication. We further show that enhanced PC levels also accumulate at the replication sites of hepatitis C virus and poliovirus, revealing a conserved feature among a group of positive-strand RNA viruses. Our work also highlights a potential broad-spectrum antiviral strategy that would disrupt PC synthesis at the sites of viral replication but would not alter cellular processes.

Highlights ► We model the role of coat protein (CP) during RNA recombination between RNA3 and sgRNA3a in Brome mosaic virus (BMV). ► Mutations in cis-acting RNA motifs responsible for CP binding debilitate recombination. ► Mutations in CP domains responsible for RNA binding debilitate recombination. ► Both, the recombination frequency and distribution of crossover sites are affected by mutations. ► Recombination mechanisms are suggested where CP molecules bring recombining RNAs together during (+) or (−) strand syntheses., We have previously described the efficient homologous recombination system between 5′ subgenomic RNA3a (sgRNA3a) and genomic RNA3 of Brome mosaic virus (BMV) in barley protoplasts (Sztuba-Solińska et al., 2011a). Here, we demonstrated that sequence alterations in the coat protein (CP)-binding cis-acting RNA motifs, the Bbox region (in the intercistronic RNA3 sequence) and the RNA3 packaging element (PE, in the movement protein ORF), reduced crossover frequencies in protoplasts. Additionally, the modification of Bbox-like element in the 5′ UTR region strongly debilitated crossovers. Along the lines of these observations, RNA3 mutants not expressing CP or expressing mutated CPs also reduced recombination. A series of reciprocal transfections demonstrated a functional crosstalk between the Bbox and PE elements. Altogether, our data imply the role of CP in sgRNA3a-directed recombination by either facilitating the interaction of the RNA substrates and/or by creating roadblocks for the viral replicase.

The coat protein of positive-stranded RNA viruses often contains a positively-charged tail that extends toward the center of the capsid and interacts with the viral genome. Electrostatic interaction between the tail and the RNA has been postulated as a major force in virus assembly and stabilization. The goal of this work is to examine the correlation between electrostatic interaction and the amount of RNA packaged in the tripartite Brome Mosaic Virus (BMV). Nanoindentation experiment using atomic force microscopy showed that the stiffness of BMV virions with different RNAs varied by a ten-fold higher range than would be predicted by electrostatics. BMV mutants with decreased positive charges encapsidated lower amounts of RNA while mutants with increased positive charges packaged additional RNAs up to ~900 nucleotides. However, the extra RNAs included truncated BMV RNAs, an additional copy of RNA4, potential cellular RNAs, or a combination of the three, indicating that change in the charge of the capsid could result in several different outcomes in RNA encapsidation. In addition, mutant with specific arginines changed to lysines in the capsid also exhibited defects in the specific encapsidation of one or more of the four BMV positive-strand RNAs. The experimental results indicate that electrostatics is a major component in RNA encapsidation, but was unable to account for all of the observed effects on RNA encapsidation. Thermodynamic modeling incorporating the electrostatics was able to predict the approximate length of the RNA to be encapsidated for the majority of mutant virions, but not for a mutant with extreme clustered positive charges. Cryo-electron microscopy of virions that encapsidated an additional copy of RNA4 revealed that, despite the increase in RNA encapsidated, the capsid structure was minimally changed. These results experimentally demonstrated the impact of electrostatics and additional restraints in the encapsidation of BMV RNAs, which could be applicable to other viruses.

All positive-strand RNA viruses reorganize host intracellular membranes to assemble their replication complexes. Similarly, brome mosaic virus (BMV) induces two alternate forms of membrane-bound RNA replication complexes: vesicular spherules and stacks of appressed double-membrane layers. The mechanisms by which these membrane rearrangements are induced, however, remain unclear. We report here that host ACB1-encoded acyl coenzyme A (acyl-CoA) binding protein (ACBP) is required for the assembly and activity of both BMV RNA replication complexes. ACBP is highly conserved among eukaryotes, specifically binds to long-chain fatty acyl-CoA, and promotes general lipid synthesis. Deleting ACB1 inhibited BMV RNA replication up to 30-fold and resulted in formation of spherules that were ∼50% smaller but ∼4-fold more abundant than those in wild-type (wt) cells, consistent with the idea that BMV 1a invaginates and maintains viral spherules by coating the inner spherule membrane. Furthermore, smaller and more frequent spherules were preferentially formed under conditions that induce layer formation in wt cells. Conversely, cellular karmella structures, which are arrays of endoplasmic reticulum (ER) membranes formed upon overexpression of certain cellular ER membrane proteins, were formed normally, indicating a selective inhibition of 1a-induced membrane rearrangements. Restoring altered lipid composition largely complemented the BMV RNA replication defect, suggesting that ACBP was required for maintaining lipid homeostasis. Smaller and more frequent spherules are also induced by 1a mutants with specific substitutions in a membrane-anchoring amphipathic α-helix, implying that the 1a-lipid interactions play critical roles in viral replication complex assembly.

The first 45 amino acid region of brome mosaic virus (BMV) capsid protein (CP) contains RNA binding and structural domains that are implicated in the assembly of infectious virions. One such important structural domain encompassing amino acids 28QPVIV32, highly conserved between BMV and cowpea chlorotic mottle virus (CCMV), exhibits a β-hexamer structure. In this study we report that alteration of the β-hexamer structure by mutating 28QPVIV32 to 28AAAAA32 had no effect either on symptom phenotype, local and systemic movement in Chenopodium quinoa and RNA profile of in vivo assembled virions. However, sensitivity to RNase and assembly phenotypes distinguished virions assembled with CP subunits having β-hexamer from those of wild type. A comparison of 3-D models obtained by cryo electron microscopy revealed overall similar structural features for wild type and mutant virions, with small but significant differences near the 3-fold axes of symmetry.

Packaging of the segmented RNA genome of Brome mosaic virus (BMV) into discrete particles is an essential step in the virus life cycle; however, questions remain regarding the mechanism of RNA packaging and the degree to which the viral coat protein controls the process. In this study, we used a plant-derived glycosidase, Pokeweed antiviral protein, to remove 14 specific bases from BMV RNA3 to examine the effect of depurination on virus assembly. Depurination of A771 within ORF3 and A1006 in the intergenic region inhibited coat protein binding and prevented RNA3 incorporation into particles. The disruption of interaction was not based on sequence identity, as mutation of these two purines to pyrimidines did not decrease coat protein-binding affinity. Rather, we suggest that base removal results in decreased thermodynamic stability of local RNA structures required for packaging, and that this instability is detected by coat protein. These results describe a new level of discrimination by coat protein, whereby it recognizes damage to specific viral RNA elements in the form of base removal and selects against incorporating the RNA into particles.

Cell-penetrating peptides (CPP) can translocate across the cell membrane and have been extensively studied for the delivery of proteins, nucleic acids, and therapeutics in mammalian cells. However, characterizations of CPP in plants have only recently been initiated. We showed that the intact virion and a recombinant capsid protein (CaP) from a plant-infecting nonenveloped icosahedral RNA virus, Brome mosaic virus (BMV), can penetrate the membranes of plant protoplasts but are trapped by the extracellular matrix. Furthermore, a 22-residue peptide derived from the N-terminal region of the CaP (CPNT) can enter barley protoplasts and cells of intact barley and Arabidopsis roots. An inhibitor of the macropinocytosis reduced CPNT entry, while treatment with NiCl2 changed the cellular localization of CPNT. CPNT increased uptake of the green flourescent protein (GFP) into the cell when covalently fused to GFP or when present in trans of GFP. The BMV CPNT overlaps with the sequence known to bind BMV RNA, and it can deliver BMV RNAs into cells, resulting in viral replication, as well as deliver double-stranded RNAs that can induce gene silencing.

The Brome mosaic virus (BMV) coat protein (CP) accompanies the three BMV genomic RNAs and the subgenomic RNA into and out of cells in an infection cycle. In addition to serving as a protective shell for all of the BMV RNAs, CP plays regulatory roles during the infection process that are mediated through specific binding of RNA elements in the BMV genome. One regulatory RNA element is the B box present in the 5' untranslated region (UTR) of BMV RNA1 and RNA2 that play important roles in the formation of the BMV replication factory, as well as the regulation of translation. A second element is within the tRNA-like 3' UTR of all BMV RNAs that is required for efficient RNA replication. The BMV CP can also encapsidate ligand-coated metal nanoparticles to form virus-like particles (VLPs). This update summarizes the interaction between the BMV CP and RNAs that can regulate RNA synthesis, translation and RNA encapsidation, as well as the formation of VLPs.

Positive-strand RNA viruses replicate their genomes on membranes with virus-induced rearrangements such as single- or double-membrane vesicles, but the mechanisms of such rearrangements, including the role of host proteins, are poorly understood. Brome mosaic virus (BMV) RNA synthesis occurs in ≈70 nm, negatively curved endoplasmic reticulum (ER) membrane invaginations induced by multifunctional BMV protein 1a. We show that BMV RNA replication is inhibited 80–90% by deleting the reticulon homology proteins (RHPs), a family of membrane-shaping proteins that normally induce and stabilize positively curved peripheral ER membrane tubules. In RHP-depleted cells, 1a localized normally to perinuclear ER membranes and recruited the BMV 2a pol polymerase. However, 1a failed to induce ER replication compartments or to recruit viral RNA templates. Partial RHP depletion allowed formation of functional replication vesicles but reduced their diameter by 30–50%. RHPs coimmunoprecipitated with 1a and 1a expression redirected >50% of RHPs from peripheral ER tubules to the interior of BMV-induced RNA replication compartments on perinuclear ER. Moreover, RHP-GFP fusions retained 1a interaction but shifted 1a-induced membrane rearrangements from normal vesicles to double membrane layers, a phenotype also induced by excess 1a-interacting 2a pol . Thus, RHPs interact with 1a, are incorporated into RNA replication compartments, and are required for multiple 1a functions in replication compartment formation and function. The results suggest possible RHP roles in the bodies and necks of replication vesicles.

Defining the shape, conformation, or assembly state of an RNA in solution often requires multiple investigative tools ranging from nucleotide analog interference mapping to X-ray crystallography. A key addition to this toolbox is small-angle X-ray scattering (SAXS). SAXS provides direct structural information regarding the size, shape, and flexibility of the particle in solution and has proven powerful for analyses of RNA structures with minimal requirements for sample concentration and volumes. In principle, SAXS can provide reliable data on small and large RNA molecules. In practice, SAXS investigations of RNA samples can show inconsistencies that suggest limitations in the SAXS experimental analyses or problems with the samples. Here, we show through investigations on the SAM-I riboswitch, the Group I intron P4-P6 domain, 30S ribosomal subunit from Sulfolobus solfataricus (30S), brome mosaic virus tRNA-like structure (BMV TLS), Thermotoga maritima asd lysine riboswitch, the recombinant tRNAval, and yeast tRNAphe that many problems with SAXS experiments on RNA samples derive from heterogeneity of the folded RNA. Furthermore, we propose and test a general approach to reducing these sample limitations for accurate SAXS analyses of RNA. Together our method and results show that SAXS with synchrotron radiation has great potential to provide accurate RNA shapes, conformations, and assembly states in solution that inform RNA biological functions in fundamental ways.

Melandrium yellow fleck bromovirus (MYFV) systemically infected Arabidopsis thaliana, although the susceptibility of several A. thaliana accessions to MYFV differed from their susceptibility to the other two bromoviruses infecting A. thaliana. We constructed full-length cDNA clones of MYFV genomic RNAs 1, 2, and 3 and determined their complete nucleotide sequences. Similar to Broad bean mottle bromovirus, (1) the 5'-terminal nucleotide of the MYFV genomic RNAs was adenine, and (2) the "D-arm" was absent from the tRNA-like structure in the 3' untranslated regions (UTRs) of MYFV RNAs. As unique characteristics, MYFV RNA3 lacked the poly(A) tract in the intercistronic region and contained a directly repeated sequence of about 200 nucleotides and polypyrimidine tracts of heterogeneous lengths in the 5' UTR. Co-infection experiments using RNA3 clones with or without the duplicated sequence demonstrated that the duplication contributed to the competitive fitness of the virus in Nicotiana benthamiana.

Cucumber Mosaic Virus (CMV) is a plant infecting tripartite positive-strand RNA virus. In addition to three genomic and two known subgenomic RNAs, CMV strains of subgroup II (e.g. Q-CMV), but not subgroup I (e.g. Fny-CMV), produce and package a redundant RNA5 encompassing the 3' 304-307 nucleotides of RNAs 2 and 3. The mechanism regulating RNA5 production and its role in CMV life cycle is unknown. In this study, transient expression of Q2 or Q3 by agroinfiltration into Nicotiana benthamiana plants resulted in efficient accumulation of RNA5 suggesting that its production is independent of CMV replication. Deletion and point mutations engineered into a highly conserved region (Box1) adjacent to the 5' end of RNA5 identified sequences required for its efficient production. An experimental system, involving a chimera of Q3 (Q3B3) characterized by having a 3' tRNA-like structure (3'TLS) from Brome mosaic virus (BMV) and RNA5 defective variants of Q1 (Q1Delta), Q2 (Q2Delta) and Q3B3 (Q3DeltaB3), was used to evaluate in vivo the contribution of RNA5 in promoting RNA recombination. Generation of precise homologous recombinants was strictly dependent on sequence identity. When both parental RNAs carried the Box1, recombination occurred preferentially within the Box1. In contrast, generation of non-homologous recombinants occurred only when Q1 and Q2 were competent to produce RNA5. A mechanistic model explaining the functional role played by the RNA5 in generating CMV recombinants was presented.

Pokeweed antiviral protein (PAP) is a glycosidase of plant origin that has been shown to depurinate some viral RNAs in vitro. We have demonstrated previously that treatment of Brome mosaic virus (BMV) RNAs with PAP inhibited their translation in a cell-free system and decreased their accumulation in barley protoplasts. In the current study, we map the depurination sites on BMV RNA3 and describe the mechanism by which replication of the viral RNA is inhibited by depurination. Specifically, we demonstrate that the viral replicase exhibited reduced affinity for depurinated positive-strand RNA3 compared with intact RNA3, resulting in less negative-strand product. This decrease was due to depurination within the intergenic region of RNA3, between ORF3 and 4, and distant from the 3' terminal core promoter required for initiation of negative-strand RNA synthesis. Depurination within the intergenic region alone inhibited the binding of the replicase to full-length RNA3, whereas depurination outside the intergenic region permitted the replicase to initiate negative-strand synthesis; however, elongation of the RNA product was stalled at the abasic nucleotide. These results support a role of the intergenic region in controlling negative-strand RNA synthesis and contribute new insight into the effect of depurination by PAP on BMV replication.

Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein isolated from the pokeweed plant (Phytolacca americana) that exhibits antiviral activity against several plant and animal viruses. We have shown previously that PAP depurinates Brome mosaic virus (BMV) RNAs in vitro and that prior incubation of these RNAs with PAP reduced their synthesis in barley protoplasts. To investigate the post-transcriptional effect of PAP on viral RNA in vivo, we transcribed BMV RNA3 and expressed PAP in the yeast, Saccharomyces cerevisiae, which is a surrogate host for BMV. With an inducible transcription system, we show that the half-life of RNA3 in PAP-expressing cells was significantly less than in cells expressing PAPx, its enzymatically inactive form. PAP bound to RNA3 and depurinated the RNA within open reading frames 3 and 4 and within untranslated regions of the RNA. The depurinated RNA was associated with polysomes, caused ribosomes to stall at the point of depurination, and was targeted for accelerated degradation by components of the No-go decay pathway. As a consequence of translation elongation arrest and increased RNA degradation, expression of PAP in yeast also decreased the level of protein 3a, encoded by the 5'-proximal open reading frame 3 of BMV RNA3. These data provide the first evidence of viral RNA depurination in vivo by any ribosome-inactivating protein and support our hypothesis that depurination contributes to the antiviral activity of PAP, by enhancing viral RNA degradation and reducing translation of viral protein product.

Flock house virus (FHV), a bipartite RNA virus of insects and a member of the Nodaviridae family, shares viral replication features with the tripartite brome mosaic virus (BMV), an RNA virus that infects plants and is a member of the Bromoviridae family. In BMV and FHV, genome packaging is coupled to replication, a widely conserved mechanism among positive-strand RNA viruses of diverse origin. To unravel the events that modulate the mechanism of replication-coupled packaging, in this study, we have extended the transfer DNA (T-DNA)-based agroinfiltration system to express functional genome components of FHV in plant cells ( Nicotiana benthamiana ). Replication, intracellular membrane localization, and packaging characteristics in agroinfiltrated plant cells revealed that T-DNA plasmids of FHV were biologically active and faithfully mimicked complete replication and packaging behavior similar to that observed for insect cells. Synchronized coexpression of wild-type BMV and FHV genome components in plant cells resulted in the assembly of virions packaging the respective viral progeny RNA. To further elucidate the link between replication and packaging, coat protein (CP) open reading frames were precisely exchanged between BMV RNA 3 (B3) and FHV RNA 2 (F2), creating chimeric RNAs expressing heterologous CP genes (B3/FCP and F2/BCP). Coinfiltration of each chimera with its corresponding genome counterpart to provide viral replicase (B1+B2+B3/FCP and F1+F2/BCP) resulted in the expected progeny profiles, but virions exhibited a nonspecific packaging phenotype. Complementation with homologous replicase (with respect to CP) failed to enhance packaging specificity. Taken together, we propose that the transcription of CP mRNA from homologous replication and its translation must be synchronized to confer packaging specificity.