Brome Mosaic Virus RNA Research Articles

Characterization of Variant RNAs Encapsidated during Bromovirus Infection by High-Throughput Sequencing.

Previously, we described the RNA recombinants accumulating in tissues infected with the bromoviruses BMV (Brome mosaic virus) and CCMV (Cowpea chlorotic mottle virus). In this work, we characterize the recombinants encapsidated inside the purified virion particles of BMV and CCMV. By using a tool called the Viral Recombination Mapper (ViReMa) that detects recombination junctions, we analyzed a high number of high-throughput sequencing (HTS) short RNA sequence reads. Over 28% of BMV or CCMV RNA reads did not perfectly map to the viral genomes. ViReMa identified 1.40% and 1.83% of these unmapped reads as the RNA recombinants, respectively, in BMV and CCMV. Intra-segmental crosses were more frequent than the inter-segmental ones. Most intra-segmental junctions carried short insertions/deletions (indels) and caused frameshift mutations. The mutation hotspots clustered mainly within the open reading frames. Substitutions of various lengths were also identified, whereas a small fraction of crosses occurred between viral and their host RNAs. Our data reveal that the virions can package detectable amounts of multivariate recombinant RNAs, contributing to the flexible nature of the viral genomes.

Brome mosaic virus detected in Kansas wheat co-infected with other common wheat viruses.

Wheat breeders are developing new virus-resistant varieties; however, it is assumed that only a few viruses or well-known viruses are present in the field. New sequencing technology is allowing for better determination of natural field virus populations. For three years, 2019-2021, Kansas wheat field surveys were conducted to determine the constituents of natural field virus populations using nanopore sequencing. During analysis, brome mosaic virus (BMV) was identified for the first time in Kansas but was in association with other wheat viruses. Brome mosaic virus was identified from 29 out of 47 different Kansas counties sampled and 44% of the total samples. BMV was found co-infected with wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) in 27.8% of the samples, with WSMV only (13.9%) and co-infected with WSMV + TriMV + High Plains wheat mosaic emaravirus (HPWMoV) (13.9%). RNA genomes of Kansas BMV isolates had 99.4 to 100% nucleotide and amino acid sequence identity, respectively, to each other. RNA2a possessed relatively high divergence (π = 0.01) compared to RNA1a and RNA3a (π = 0.004). Coding regions of all BMV RNAs were considered negative for purifying selection pressure as nonsynonymous and synonymous nucleotide ratio was less than one (dNs/dS >1). The identification of BMV in Kansas virus populations adds another layer of complexity to plant breeding. This work provides information to improve tools to aid in monitoring, detecting, and determining the variation within BMV.

Regulation of Positive-Strand Accumulation by Capsid Protein During Brome mosaic virus Infection In Planta.

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.

Cowpea chlorotic mottle bromovirus replication proteins support template-selective RNA replication in Saccharomyces cerevisiae.

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 (<5% of that by BMV 1a/2apol). In keeping with this, we found that in yeast CCMV 1a was significantly impaired in recruiting BMV or CCMV RNA3 to the replication complex. Overall, we show that many 1a and 2apol functions essential for replication complex assembly, and their ability to be reconstituted in yeast, are conserved between BMV and CCMV. However, restrictions of CCMV RNA replication in yeast reveal previously unknown 1a-linked, RNA-selective host contributions to the essential early process of recruiting viral RNA templates to the replication complex.

Investigating on the binding characteristics of the important RNA‐protein interactions in Brome Mosaic Virus

RNA‐protein interactions are often crucial for viability of many biological systems, including the replication cycle of RNA viruses. Understanding molecular driving forces behind the binding specificity of these interactions in RNA viruses is important in advancing our knowledge not only in the mechanism of viral replication but also in designing therapies against viral infections. Here, we have investigated the binding characteristics of the N‐terminal region of a viral Coat Protein (CP) to promoter sequences of the genomic RNA in Brome Mosaic Virus (BMV), a granary plant‐infecting RNA virus. BMV has three separate RNA molecules as its genomic RNA and coordinating the timely production of these three RNAs is crucial in successful replication of their progeny viruses. Previous studies showed that the coordinating function is conducted by the CP, which involves its specific binding on BMV RNA. The N‐terminal region of CP binds to a couple of promoter sequence of BMV genomic RNA. Interestingly those RNA motifs all have well‐defined loop structures. We have explored on the importance of the three‐dimensional conformations of these RNA motifs for the recognition by the N‐terminal region of CP. We designed peptides mimicking the N‐terminal region of CP, called CPNT, and also prepared RNA sequences representing the promoter motifs of BMV RNA. We have used Fluorescence quenching &amp; anisotropy as well as NMR and FPLC for investigating the binding characteristics of CPNT to these RNA. Our initial results have shown that one of the RNA promoter motifs, found at the 5′end of BMV genomic RNA, showed specific binding affinity in which the conformation of its triloop (5′AUA3′) serves a key recognition motif for the CPNT‐RNA interaction. We are currently investigating the binding characteristic of other RNA motifs with CPNT as well as the effects of pH, salt, and temperature on them. Fluorescence spectroscopy and Fast Protein Liquid Chromatography are used to measure and compare binding efficiencies of peptide‐RNA complexes for pH 7.5, 7.0, 6.5, 6.0, and 5.5 in sodium chloride and ammonium acetate, with native polyacrylamide gel electrophoresis (PAGE) as a way to visualize binding of those complexes. High‐resolution 1D and 2D NMR is used to gain structural insights into complexes. These new results will be presented at the conference.Support or Funding InformationThis project was supported by the Center for Student Research and by the Faculty Support Grant from CSU East Bay. A Varian 500 MHz FT‐NMR instrument was obtained with funds from NSF MRI grant 0420615 and the CSU East Bay College of Science.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Intermolecular RNA Recombination Occurs at Different Frequencies in Alternate Forms of Brome Mosaic Virus RNA Replication Compartments.

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.

Organelle luminal dependence of (+)strand RNA virus replication reveals a hidden druggable target.

Positive-strand RNA viruses replicate their genomes in membrane-bounded cytoplasmic complexes. We show that endoplasmic reticulum (ER)-linked genomic RNA replication by brome mosaic virus (BMV), a well-studied member of the alphavirus superfamily, depends on the ER luminal thiol oxidase ERO1. We further show that BMV RNA replication protein 1a, a key protein for the formation and function of vesicular BMV RNA replication compartments on ER membranes, permeabilizes these membranes to release oxidizing potential from the ER lumen. Conserved amphipathic sequences in 1a are sufficient to permeabilize liposomes, and mutations in these sequences simultaneously block membrane permeabilization, formation of a disulfide-linked, oxidized 1a multimer, 1a's RNA capping function, and productive genome replication. These results reveal new transmembrane complexities in positive-strand RNA virus replication, show that-as previously reported for certain picornaviruses and flaviviruses-some alphavirus superfamily members encode viroporins, identify roles for such viroporins in genome replication, and provide a potential new foundation for broad-spectrum antivirals.

The intrinsically disordered N-terminal arm of the brome mosaic virus coat protein specifically recognizes the RNA motif that directs the initiation of viral RNA replication.

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.

An Improved Brome mosaic virus Silencing Vector: Greater Insert Stability and More Extensive VIGS.

Virus-induced gene silencing (VIGS) is used extensively for gene function studies in plants. VIGS is inexpensive and rapid compared with silencing conducted through stable transformation, but many virus-silencing vectors, especially in grasses, induce only transient silencing phenotypes. A major reason for transient phenotypes is the instability of the foreign gene fragment (insert) in the vector during VIGS. Here, we report the development of a Brome mosaic virus (BMV)-based vector that better maintains inserts through modification of the original BMV vector RNA sequence. Modification of the BMV RNA3 sequence yielded a vector, BMVCP5, that better maintained phytoene desaturase and heat shock protein70-1 (HSP70-1) inserts in Nicotiana benthamiana and maize (Zea mays). Longer maintenance of inserts was correlated with greater target gene silencing and more extensive visible silencing phenotypes displaying greater tissue penetration and involving more leaves. The modified vector accumulated similarly to the original vector in N. benthamiana after agroinfiltration, thus maintaining a high titer of virus in this intermediate host used to produce virus inoculum for grass hosts. For HSP70, silencing one family member led to a large increase in the expression of another family member, an increase likely related to the target gene knockdown and not a general effect of virus infection. The cause of the increased insert stability in the modified vector is discussed in relationship to its recombination and accumulation potential. The modified vector will improve functional genomic studies in grasses, and the conceptual methods used to improve the vector may be applied to other VIGS vectors.

A novel translational control mechanism involving RNA structures within coding sequences.

The impact of RNA structures in coding sequences (CDS) within mRNAs is poorly understood. Here, we identify a novel and highly conserved mechanism of translational control involving RNA structures within coding sequences and the DEAD-box helicase Dhh1. Using yeast genetics and genome-wide ribosome profiling analyses, we show that this mechanism, initially derived from studies of the Brome Mosaic virus RNA genome, extends to yeast and human mRNAs highly enriched in membrane and secreted proteins. All Dhh1-dependent mRNAs, viral and cellular, share key common features. First, they contain long and highly structured CDSs, including a region located around nucleotide 70 after the translation initiation site; second, they are directly bound by Dhh1 with a specific binding distribution; and third, complementary experimental approaches suggest that they are activated by Dhh1 at the translation initiation step. Our results show that ribosome translocation is not the only unwinding force of CDS and uncover a novel layer of translational control that involves RNA helicases and RNA folding within CDS providing novel opportunities for regulation of membrane and secretome proteins.

Phosphorylation of the Brome Mosaic Virus Capsid Regulates the Timing of Viral Infection.

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. 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.

Single-Molecule FRET Reveals Three Conformations for the TLS Domain of Brome Mosaic Virus Genome

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.

The Lsm1-7-Pat1 complex promotes viral RNA translation and replication by differential mechanisms.

The Lsm1-7-Pat1 complex binds to the 3′ end of cellular mRNAs and promotes 3′ end protection and 5′–3′ decay. Interestingly, this complex also specifically binds to cis-acting regulatory sequences of viral positive-strand RNA genomes promoting their translation and subsequent recruitment from translation to replication. Yet, how the Lsm1-7-Pat1 complex regulates these two processes remains elusive. Here, we show that Lsm1-7-Pat1 complex acts differentially in these processes. By using a collection of well-characterized lsm1 mutant alleles and a system that allows the replication of Brome mosaic virus (BMV) in yeast we show that the Lsm1-7-Pat1 complex integrity is essential for both, translation and recruitment. However, the intrinsic RNA-binding ability of the complex is only required for translation. Consistent with an RNA-binding-independent function of the Lsm1-7-Pat1 complex on BMV RNA recruitment, we show that the BMV 1a protein, the sole viral protein required for recruitment, interacts with this complex in an RNA-independent manner. Together, these results support a model wherein Lsm1-7-Pat1 complex binds consecutively to BMV RNA regulatory sequences and the 1a protein to promote viral RNA translation and later recruitment out of the host translation machinery to the viral replication complexes.

Brome mosaic virus Infection of Rice Results in Decreased Accumulation of RNA1.

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.

Characterization of Pokeweed Antiviral Protein (PAP) Isoforms and Comparison of Their Enzymatic Activities toward the Tobacco Etch Virus RNA

Ribosome‐inactivating proteins (RIPs) are found in a wide range of plants. Their antiviral, antibacterial and antifungal biological activities point toward RIPs playing important roles in plant's defense mechanisms. RIPs are RNA N‐glycosidases. They specifically recognize and depurinate universally conserved sarcin/ricin (S/R) loop of the large rRNA. This leads to ribosomal inactivation and termination of protein synthesis. Phytolacca americana, the common pokeweed plant, produces pokeweed antiviral protein (PAP), a type 1 RIP. PAP possesses antiviral properties and inhibits translation of viral RNAs, including HIV‐1, influenza, poliovirus, brome mosaic virus (BMV) RNAs. PAP inhibits translation of capped RNAs by directly binding to the 5'‐cap of the RNA, and depurinating it downstream of the cap structure. The mechanism of uncapped viral RNA translation inhibition is not known. Pokeweed plant produces nine seasonal tissue‐distinct PAP isoforms. Some isoforms are known to exhibit stronger antiviral potential than others: PAP‐S, isolated from seeds. The exact mechanism of how PAP selects its viral RNA substrates for depurination is not well understood, and is of interest research. This study will provide a deeper understanding of the strategies used by plants to battle infectious pathogens, and may lead to the development of efficient plant antiviral agents, and to improvement of the applied solutions for the regulation of plant and animal diseases. Here, we isolate and characterize PAP isoforms and employ fluorescence spectroscopy and HPLC techniques to compare of their enzymatic activities toward tobacco etch virus (TEV) RNA.

Host ESCRT proteins are required for bromovirus RNA replication compartment assembly and function.

Positive-strand RNA viruses genome replication invariably is associated with vesicles or other rearranged cellular membranes. Brome mosaic virus (BMV) RNA replication occurs on perinuclear endoplasmic reticulum (ER) membranes in ~70 nm vesicular invaginations (spherules). BMV RNA replication vesicles show multiple parallels with membrane-enveloped, budding retrovirus virions, whose envelopment and release depend on the host ESCRT (endosomal sorting complexes required for transport) membrane-remodeling machinery. We now find that deleting components of the ESCRT pathway results in at least two distinct BMV phenotypes. One group of genes regulate RNA replication and the frequency of viral replication complex formation, but had no effect on spherule size, while a second group of genes regulate RNA replication in a way or ways independent of spherule formation. In particular, deleting SNF7 inhibits BMV RNA replication > 25-fold and abolishes detectable BMV spherule formation, even though the BMV RNA replication proteins accumulate and localize normally on perinuclear ER membranes. Moreover, BMV ESCRT recruitment and spherule assembly depend on different sets of protein-protein interactions from those used by multivesicular body vesicles, HIV-1 virion budding, or tomato bushy stunt virus (TBSV) spherule formation. These and other data demonstrate that BMV requires cellular ESCRT components for proper formation and function of its vesicular RNA replication compartments. The results highlight growing but diverse interactions of ESCRT factors with many viruses and viral processes, and potential value of the ESCRT pathway as a target for broad-spectrum antiviral resistance.

The brome mosaic virus 3′ untranslated sequence regulates RNA replication, recombination, and virion assembly

The 3′ untranslated region in each of the three genomic RNAs of Brome mosaic virus (BMV) is highly homologous and contains a sequence that folds into a tRNA-like structure (TLS). Experiments performed over the past four decades revealed that the BMV 3′ TLS regulates many important steps in BMV infection. This review summarizes in vitro and in vivo studies of the roles of the BMV 3′ TLS functioning as a minus-strand promoter, in RNA recombination, and to nucleate virion assembly.

Co-infection with two strains of Brome mosaic bromovirus reveals common RNA recombination sites in different hosts.

We have previously reported intra-segmental crossovers in Brome mosaic virus (BMV) RNAs. In this work, we studied the homologous recombination of BMV RNA in three different hosts: barley (Hordeum vulgare), Chenopodium quinoa, and Nicotiana benthamiana that were co-infected with two strains of BMV: Russian (R) and Fescue (F). Our work aimed at (1) establishing the frequency of recombination, (2) mapping the recombination hot spots, and (3) addressing host effects. The F and R nucleotide sequences differ from each other at many translationally silent nucleotide substitutions. We exploited this natural variability to track the crossover sites. Sequencing of a large number of cDNA clones revealed multiple homologous crossovers in each BMV RNA segment, in both the whole plants and protoplasts. Some recombination hot spots mapped at similar locations in different hosts, suggesting a role for viral factors, but other sites depended on the host. Our results demonstrate the chimeric (‘mosaic’) nature of the BMV RNA genome.

Base-paired structure in the 5′ untranslated region is required for the efficient amplification of negative-strand RNA3 in the bromovirus melandrium yellow fleck virus

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.

A MATHEMATICAL MODEL OF RNA3 RECRUITMENT IN THE REPLICATION CYCLE OF BROME MOSAIC VIRUS

Positive-strand RNA viruses, such as the brome mosaic virus (BMV) and hepatitis C virus, utilize a replication cycle which involves the recruitment of RNA genomes from the cellular translation machinery to the viral replication complexes. Here, we coupled mathematical modeling with a statistical inverse problem methodology to better understand this crucial recruitment process. We developed a discrete-delay differential equation model that describes the production of BMV protein 1a and BMV RNA3, and the effect of protein 1a on RNA3 recruitment. We validated our model with experimental data generated in duplicate from a yeast strain that was engineered to express protein 1a and RNA3 under the control of inducible promoters. We used a statistical model comparison technique to test which biological assumptions in our model were correct. Our results suggest that protein 1a expression is governed by a nonlinear phenomenon and that a time delay is important for modeling RNA3 recruitment. We also performed an uncertainty analysis of two experimental designs and found that we could improve our data collection procedure in future experiments to increase the confidence in our parameter estimates.