A viral RNA hijacks host machinery using dynamic conformational changes of a tRNA-like structure.

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 cryo­electron microscopy (cryo-EM), we visualized the structure of the mysterious viral transfer RNA (tRNA)­like structure (TLS) from the brome mosaic virus, which affects replication, translation, and genome encapsidation. Structures in isolation and those bound to tyrosyl-tRNA synthetase (TyrRS) show that this ~55-kilodalton purported tRNA mimic undergoes large conformational rearrangements to bind TyrRS in a form that differs substantially from that of tRNA. Our study reveals how viral RNAs can use a combination of static and dynamic RNA structures to bind host machinery through highly noncanonical interactions, and we highlight the utility of cryo-EM for visualizing small, conformationally dynamic structured RNAs.

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.

Elucidating the Thermodynamic Driving Forces of Polyanion-Templated Virus-like Particle Assembly.

A virus in its most simple form is comprised of a protein capsid that surrounds and protects the viral genome. The self-assembly of such structures, however, is a highly complex, multiprotein, multiinteraction process and has been a topic of study for a number of years. This self-assembly process is driven by the (mainly electrostatic) interaction between the capsid proteins (CPs) and the genome as well as by the protein-protein interactions, which primarily rely on hydrophobic interactions. Insight in the thermodynamics that is involved in virus and virus-like particle (VLP) formation is crucial in the detailed understanding of this complex assembly process. Therefore, we studied the assembly of CPs of the cowpea chlorotic mottle virus (CCMV) templated by polyanionic species (cargo), that is, single-stranded DNA (ssDNA), and polystyrene sulfonate (PSS) using isothermal titration calorimetry. By separating the electrostatic CP-cargo interaction from the full assembly interaction, we conclude that CP-CP interactions cause an enthalpy change of -3 to -4 kcal mol-1 CP. Furthermore, we quantify that upon reducing the CP-CP interaction, in the case of CCMV by increasing the pH to 7, the CP-cargo starts to dominate VLP formation. This is highlighted by the three times higher affinity between CP and PSS compared to CP and ssDNA, resulting in the disassembly of CCMV at neutral pH in the presence of PSS to yield PSS-filled VLPs.

Characterization of an Ohio Isolate of Brome Mosaic Virus and Its Impact on the Development and Yield of Soft Red Winter Wheat.

Brome mosaic virus (BMV) is generally thought to be of little economic importance to crops; consequently, there is little information about its impact on wheat production under field conditions. After repeated detection of BMV in Ohio wheat fields at incidences up to 25%, the virus was isolated, sequenced, characterized, and tested for its impact on soft red winter wheat (SRWW). The Ohio isolate of brome mosaic virus (BMV-OH) was found to be >99% identical to a BMV-Fescue isolate (accession no. DQ530423-25) and capable of systemically infecting multiple monocot and dicot species, including cowpea and soybean, in experimental inoculations. BMV-OH was used in field experiments during the 2016 and 2017 growing seasons to quantify its effect on SRWW grain yield and development when inoculated at Feekes 1, 5, 8, and 10 in two to four cultivars. Cultivar and timing of inoculation had statistically significant (P < 0.05) main and interaction effects on grain yield, wheat growth, and multiple components of yield. Compared with noninoculated controls, BMV-OH reduced grain yield by up to 61% when inoculated at Feekes 1 and by as much as 25, 36, and 31% for inoculations at Feekes 5, 8, and 10, respectively. The magnitude of the yield reduction varied among cultivars and was associated with reductions in grain size and weight or plant population. These findings suggest that BMV could impact wheat productivity in Ohio and will serve as the basis for more large-scale investigations of the effects of this virus in commercial fields.

Nonequilibrium self-assembly dynamics of icosahedral viral capsids packaging genome or polyelectrolyte.

The survival of viruses partly relies on their ability to self-assemble inside host cells. Although coarse-grained simulations have identified different pathways leading to assembled virions from their components, experimental evidence is severely lacking. Here, we use time-resolved small-angle X-ray scattering to uncover the nonequilibrium self-assembly dynamics of icosahedral viral capsids packaging their full RNA genome. We reveal the formation of amorphous complexes via an en masse pathway and their relaxation into virions via a synchronous pathway. The binding energy of capsid subunits on the genome is moderate (~7kBT0, with kB the Boltzmann constant and T0 = 298 K, the room temperature), while the energy barrier separating the complexes and the virions is high (~ 20kBT0). A synthetic polyelectrolyte can lower this barrier so that filled capsids are formed in conditions where virions cannot build up. We propose a representation of the dynamics on a free energy landscape.

Metal Ion-Induced Self-Assembly and Packaging of CCMV Nanocapsules.

The controlled self-assembly of protein cages is vital for the use of these nanocompartments in biomedical and nanotechnological applications. Recently, we showed that by combining different structural peptide elements, it is possible to assemble viral capsid proteins in distinct well-defined morphologies. In this chapter, a triblock copolypeptide is discussed, consisting of a metal ion-coordinating hexahistidine tag, a stimulus-responsive elastin-like polypeptide and a pH-responsive self-assembling viral capsid protein. This protein is able to form two different types of capsids, depending on the assembly pathway that is followed. Here, we focus on the metal ion-induced assembly process and describe the relevant experimental procedures to induce and utilize this assembly behavior.

A method for efficient Bayesian optimization of self-assembly systems from scattering data.

BACKGROUND: The ability of collections of molecules to spontaneously assemble into large functional complexes is central to all cellular processes. Using the viral capsid as a model system for complicated macro-molecular assembly, we develop methods for probing fine details of the process by learning kinetic rate parameters consistent with experimental measures of assembly. We have previously shown that local rule based stochastic simulation methods in conjunction with bulk indirect experimental data can meaningfully constrain the space of possible assembly trajectories and allow inference of experimentally unobservable features of the real system. RESULTS: In the present work, we introduce a new Bayesian optimization framework using multi-Gaussian process model regression. We also extend our prior work to encompass small-angle X-ray/neutron scattering (SAXS/SANS) as a possibly richer experimental data source than the previously used static light scattering (SLS). Method validation is based on synthetic experiments generated using protein data bank (PDB) structures of cowpea chlorotic mottle virus. We also apply the same approach to computationally cheaper differential equation based simulation models. CONCLUSIONS: We present a flexible approach for the global optimization of computationally costly objective functions associated with dynamic, multidimensional models. When applied to the stochastic viral capsid system, our method outperforms a current state of the art black box solver tailored for use with noisy objectives. Our approach also has wide applicability to general stochastic optimization problems.

Host Pah1p phosphatidate phosphatase limits viral replication by regulating phospholipid synthesis.

Replication of positive-strand RNA viruses [(+)RNA viruses] takes place in membrane-bound viral replication complexes (VRCs). Formation of VRCs requires virus-mediated manipulation of cellular lipid synthesis. Here, we report significantly enhanced brome mosaic virus (BMV) replication and much improved cell growth in yeast cells lacking PAH1 (pah1Δ), the sole yeast ortholog of human LIPIN genes. PAH1 encodes Pah1p (phosphatidic acid phosphohydrolase), which converts phosphatidate (PA) to diacylglycerol that is subsequently used for the synthesis of the storage lipid triacylglycerol. Inactivation of Pah1p leads to altered lipid composition, including high levels of PA, total phospholipids, ergosterol ester, and free fatty acids, as well as expansion of the nuclear membrane. In pah1Δ cells, BMV replication protein 1a and double-stranded RNA localized to the extended nuclear membrane, there was a significant increase in the number of VRCs formed, and BMV genomic replication increased by 2-fold compared to wild-type cells. In another yeast mutant that lacks both PAH1 and DGK1 (encodes diacylglycerol kinase converting diacylglycerol to PA), which has a normal nuclear membrane but maintains similar lipid compositional changes as in pah1Δ cells, BMV replicated as efficiently as in pah1Δ cells, suggesting that the altered lipid composition was responsible for the enhanced BMV replication. We further showed that increased levels of total phospholipids play an important role because the enhanced BMV replication required active synthesis of phosphatidylcholine, the major membrane phospholipid. Moreover, overexpression of a phosphatidylcholine synthesis gene (CHO2) promoted BMV replication. Conversely, overexpression of PAH1 or plant PAH1 orthologs inhibited BMV replication in yeast or Nicotiana benthamiana plants. Competing with its host for limited resources, BMV inhibited host growth, which was markedly alleviated in pah1Δ cells. Our work suggests that Pah1p promotes storage lipid synthesis and thus represses phospholipid synthesis, which in turn restricts both viral replication and cell growth during viral infection.

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.

Orientational phase transitions and the assembly of viral capsids.

We present a Landau theory for large-l orientational phase transitions and apply it to the assembly of icosahedral viral capsids. The theory predicts two distinct types of ordering transitions. Transitions dominated by the l=6,10,12, and 18 icosahedral spherical harmonics resemble robust first-order phase transitions that are not significantly affected by chirality. The remaining transitions depend essentially on including mixed l states denoted as l=15+16 corresponding to a mixture of l=15 and l=16 spherical harmonics. The l=15+16 transition is either continuous or weakly first-order and it is strongly influenced by chirality, which suppresses spontaneous chiral symmetry breaking. The icosahedral state is in close competition with states that have tetrahedral, D_{5}, and octahedral symmetries. We present a group-theoretic method to analyze the competition between the different symmetries. The theory is applied to a variety of viral shells.

The Effect of RNA Secondary Structure on the Self-Assembly of Viral Capsids.

Previous work has shown that purified capsid protein (CP) of cowpea chlorotic mottle virus (CCMV) is capable of packaging both purified single-stranded RNA molecules of normal composition (comparable numbers of A, U, G, and C nucleobases) and of varying length and sequence, and anionic synthetic polymers such as polystyrene sulfonate. We find that CCMV CP is also capable of packaging polyU RNAs, which-unlike normal-composition RNAs-do not form secondary structures and which act as essentially structureless linear polymers. Following our canonical two-step assembly protocol, polyU RNAs ranging in length from 1000 to 9000 nucleotides (nt) are completely packaged. Surprisingly, negative-stain electron microscopy shows that all lengths of polyU are packaged into 22-nm-diameter particles despite the fact that CCMV CP prefers to form 28-nm-diameter (T = 3) particles when packaging normal-composition RNAs. PolyU RNAs >5000 nt in length are packaged into multiplet capsids, in which a single RNA molecule is shared between two or more 22-nm-diameter capsids, in analogy with the multiplets of 28-nm-diameter particles formed with normal-composition RNAs >5000 nt long. Experiments in which viral RNA competes for viral CP with polyUs of equal length show that polyU, despite its lack of secondary structure, is packaged more efficiently than viral RNA. These findings illustrate that the secondary structure of the RNA molecule-and its absence-plays an essential role in determining capsid structure during the self-assembly of CCMV-like particles.

Use of Cellular Decapping Activators by Positive-Strand RNA Viruses.

Positive-strand RNA viruses have evolved multiple strategies to not only circumvent the hostile decay machinery but to trick it into being a priceless collaborator supporting viral RNA translation and replication. In this review, we describe the versatile interaction of positive-strand RNA viruses and the 5'-3' mRNA decay machinery with a focus on the viral subversion of decapping activators. This highly conserved viral trickery is exemplified with the plant Brome mosaic virus, the animal Flock house virus and the human hepatitis C virus.

An unrecognized function for COPII components in recruiting the viral replication protein BMV 1a to the perinuclear ER.

Positive-strand RNA viruses invariably assemble their viral replication complexes (VRCs) by remodeling host intracellular membranes. How viral replication proteins are targeted to specific organelle membranes to initiate VRC assembly remains elusive. Brome mosaic virus (BMV), whose replication can be recapitulated in Saccharomyces cerevisiae, assembles its VRCs by invaginating the outer perinuclear endoplasmic reticulum (ER) membrane. Remarkably, BMV replication protein 1a (BMV 1a) is the only viral protein required for such membrane remodeling. We show that ER-vesicle protein of 14 kD (Erv14), a cargo receptor of coat protein complex II (COPII), interacts with BMV 1a. Moreover, the perinuclear ER localization of BMV 1a is disrupted in cells lacking ERV14 or expressing dysfunctional COPII coat components (Sec13, Sec24 or Sec31). The requirement of Erv14 for the localization of BMV 1a is bypassed by addition of a Sec24-recognizable sorting signal to BMV 1a or by overexpressing Sec24, suggesting a coordinated effort by both Erv14 and Sec24 for the proper localization of BMV 1a. The COPII pathway is well known for being involved in protein secretion; our data suggest that a subset of COPII coat proteins have an unrecognized role in targeting proteins to the perinuclear ER membrane.

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

UNLABELLED: 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.

Positive-strand RNA viruses stimulate host phosphatidylcholine synthesis at viral replication sites.

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.

Virus-resembling nano-structures for near infrared fluorescence imaging of ovarian cancer HER2 receptors.

Ovarian cancer remains the dominant cause of death due to malignancies of the female reproductive system. The capability to identify and remove all tumors during intraoperative procedures may ultimately reduce cancer recurrence, and lead to increased patient survival. The objective of this study is to investigate the effectiveness of an optical nano-structured system for targeted near infrared (NIR) imaging of ovarian cancer cells that over-express the human epidermal growth factor receptor 2 (HER2), an important biomarker associated with ovarian cancer. The nano-structured system is comprised of genome-depleted plant-infecting brome mosaic virus doped with NIR chromophore, indocyanine green, and functionalized at the surface by covalent attachment of monoclonal antibodies against the HER2 receptor. We use absorption and fluorescence spectroscopy, and dynamic light scattering to characterize the physical properties of the constructs. Using fluorescence imaging and flow cytometry, we demonstrate the effectiveness of these nano-structures for targeted NIR imaging of HER2 receptors in vitro. These functionalized nano-materials may provide a platform for NIR imaging of ovarian cancer.

Segmented GFP-like aptamer probes for functional imaging of viral genome trafficking.

Recently developed GFP-like RNA aptamers harbor a few unique potential benefits for in vivo RNA imaging applications, including co-packaging of viral genomes. Here we examine them in the context of co-packaging of RNA strands during virion assembly and trafficking. The approach is applicable both in vitro and in vivo, thus bridging an existing methodological gap. We have found that splitting the aptamer sequence in the loop region into two separate parts allows for subsequent self-assembly into a functional unit, which preserves the dye-binding pocket. In presence of the dye, virus-like particles encapsulating segmented GFP-like aptamers provided bright fluorescence emission and showed negligible bleaching due to continuous chromophore exchange: two desirable characteristics for real-time in vivo single particle studies requiring a broader dynamic range than currently available. Proof-of-principle in vivo imaging experiments confirmed detectability of aptamer-loaded virus-like particles in barley root cells even in presence of significant autofluorescence background.

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.

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.