Simple RNA Viruses Research Articles

Single-particle studies of the effects of RNA–protein interactions on the self-assembly of RNA virus particles

Understanding the pathways by which simple RNA viruses self-assemble from their coat proteins and RNA is of practical and fundamental interest. Although RNA-protein interactions are thought to play a critical role in the assembly, our understanding of their effects is limited because the assembly process is difficult to observe directly. We address this problem by using interferometric scattering microscopy, a sensitive optical technique with high dynamic range, to follow the invitro assembly kinetics of more than 500 individual particles of brome mosaic virus (BMV)-for which RNA-protein interactions can be controlled by varying the ionic strength of the buffer. We find that when RNA-protein interactions are weak, BMV assembles by a nucleation-and-growth pathway in which a small cluster of RNA-bound proteins must exceed a critical size before additional proteins can bind. As the strength of RNA-protein interactions increases, the nucleation time becomes shorter and more narrowly distributed, but the time to grow a capsid after nucleation is largely unaffected. These results suggest that the nucleation rate is controlled by RNA-protein interactions, while the growth process is driven less by RNA-protein interactions and more by protein-protein interactions and intraprotein forces. The nucleated pathway observed with the plant virus BMV is strikingly similar to that previously observed with bacteriophage MS2, a phylogenetically distinct virus with a different host kingdom. These results raise the possibility that nucleated assembly pathways might be common to other RNA viruses.

Virus Assembly Pathways Inside a Host Cell.

Simple RNA viruses self-assemble spontaneously and encapsulate their genome into a shell called the capsid. This process is mainly driven by the attractive electrostatics interaction between the positive charges on capsid proteins and the negative charges on the genome. Despite its importance and many decades of intense research, how the virus selects and packages its native RNA inside the crowded environment of a host cell cytoplasm in the presence of an abundance of nonviral RNA and other anionic polymers has remained a mystery. In this paper, we perform a series of simulations to monitor the growth of viral shells and find the mechanism by which cargo-coat protein interactions can impact the structure and stability of the viral shells. We show that coat protein subunits can assemble around a globular nucleic acid core by forming nonicosahedral cages, which have been recently observed in assembly experiments involving small pieces of RNA. We find that the resulting cages are strained and can easily be split into fragments along stress lines. This suggests that such metastable nonicosahedral intermediates could be easily reassembled into the stable native icosahedral shells if the larger wild-type genome becomes available, despite the presence of a myriad of nonviral RNAs.

Mitovirus and Mitochondrial Coding Sequences from Basal Fungus Entomophthora muscae.

Fungi constituting the Entomophthora muscae species complex (members of subphylum Entomophthoromycotina, phylum Zoopagamycota) commonly kill their insect hosts and manipulate host behaviors in the process. In this study, we made use of public transcriptome data to identify and characterize eight new species of mitoviruses associated with several different E. muscae isolates. Mitoviruses are simple RNA viruses that replicate in host mitochondria and are frequently found in more phylogenetically apical fungi (members of subphylum Glomeromyoctina, phylum Mucoromycota, phylum Basidiomycota and phylum Ascomycota) as well as in plants. E. muscae is the first fungus from phylum Zoopagomycota, and thereby the most phylogenetically basal fungus, found to harbor mitoviruses to date. Multiple UGA (Trp) codons are found not only in each of the new mitovirus sequences from E. muscae but also in mitochondrial core-gene coding sequences newly assembled from E. muscae transcriptome data, suggesting that UGA (Trp) is not a rarely used codon in the mitochondria of this fungus. The presence of mitoviruses in these basal fungi has possible implications for the evolution of these viruses.

RNA Base Pairing Determines the Conformations of RNA Inside Spherical Viruses.

Many simple RNA viruses enclose their genetic material by a protein shell called the capsid. While the capsid structures are well characterized for most viruses, the structure of RNA inside the shells and the factors contributing to it remain poorly understood. We study the impact of base pairing on the conformations of RNA and find that it undergoes a swollen coil to globule continuous transition as a function of the strength of the pairing interaction. We also observe a first order transition and kink profile as a function of RNA length. All these transitions could explain the different RNA profiles observed inside viral shells.

Transcriptional Profiling of the Immune Response to Marburg Virus Infection.

Marburg virus is a genetically simple RNA virus that causes a severe hemorrhagic fever in humans and nonhuman primates. The mechanism of pathogenesis of the infection is not well understood, but it is well accepted that pathogenesis is appreciably driven by a hyperactive immune response. To better understand the overall response to Marburg virus challenge, we undertook a transcriptomic analysis of immune cells circulating in the blood following aerosol exposure of rhesus macaques to a lethal dose of Marburg virus. Using two-color microarrays, we analyzed the transcriptomes of peripheral blood mononuclear cells that were collected throughout the course of infection from 1 to 9 days postexposure, representing the full course of the infection. The response followed a 3-stage induction (early infection, 1 to 3 days postexposure; midinfection, 5 days postexposure; late infection, 7 to 9 days postexposure) that was led by a robust innate immune response. The host response to aerosolized Marburg virus was evident at 1 day postexposure. Analysis of cytokine transcripts that were overexpressed during infection indicated that previously unanalyzed cytokines are likely induced in response to exposure to Marburg virus and further suggested that the early immune response is skewed toward a Th2 response that would hamper the development of an effective antiviral immune response early in disease. Late infection events included the upregulation of coagulation-associated factors. These findings demonstrate very early host responses to Marburg virus infection and provide a rich data set for identification of factors expressed throughout the course of infection that can be investigated as markers of infection and targets for therapy. Marburg virus causes a severe infection that is associated with high mortality and hemorrhage. The disease is associated with an immune response that contributes to the lethality of the disease. In this study, we investigated how the immune cells circulating in the blood of infected primates respond following exposure to Marburg virus. Our results show that there are three discernible stages of response to infection that correlate with presymptomatic, early, and late symptomatic stages of infection, a response format similar to that seen following challenge with other hemorrhagic fever viruses. In contrast to the ability of the virus to block innate immune signaling in vitro, the earliest and most sustained response is an interferon-like response. Our analysis also identifies a number of cytokines that are transcriptionally upregulated during late stages of infection and suggest that there is a Th2-skewed response to infection. When correlated with companion data describing the animal model from which our samples were collected, our results suggest that the innate immune response may contribute to overall pathogenesis.

RNA topology remolds electrostatic stabilization of viruses.

Simple RNA viruses efficiently encapsulate their genome into a nano-sized protein shell: the capsid. Spontaneous coassembly of the genome and the capsid proteins is driven predominantly by electrostatic interactions between the negatively charged RNA and the positively charged inner capsid wall. Using field theoretic formulation we show that the inherently branched RNA secondary structure allows viruses to maximize the amount of encapsulated genome and make assembly more efficient, allowing viral RNAs to out-compete cellular RNAs during replication in infected host cells.

New Insights into Viral Architecture via Affine Extended Symmetry Groups

Since the seminal work of Caspar and Klug on the structure of the protein containers that encapsulate and hence protect the viral genome, it has been recognized that icosahedral symmetry is crucial for the structural organization of viruses. In particular, icosahedral symmetry has been invoked in order to predict the surface structures of viral capsids in terms of tessellations or tilings that schematically encode the locations of the protein subunits in the capsids. Whilst this approach is capable of predicting the relative locations of the proteins in the capsids, a prediction on the relative sizes of different virus particles in a family cannot be made. Moreover, information on the full 3D structure of viral particles, including the tertiary structures of the capsid proteins and the organization of the viral genome within the capsid are inaccessible with their approach. We develop here a mathematical framework based on affine extensions of the icosahedral group that allows us to address these issues. In particular, we show that the relative radii of viruses in the family of Polyomaviridae and the material boundaries in simple RNA viruses can be determined with our approach. The results complement Caspar and Klug's theory of quasi-equivalence and provide details on virus structure that have not been accessible with previous methods, implying that icosahedral symmetry is more important for virus architecture than previously appreciated.

An animal virus-derived peptide switches membrane morphology: possible relevance to nodaviral transfection processes.

The N-terminal domain of the capsid protein cleavage product of the flock house virus (FHV) consists of 21 residues and forms an amphipathic alpha-helix, which is thought to play a crucial role in permeabilizing biological membranes for RNA translocation in the host cell. We have found that the Met --> Nle variant of this domain (denoted here as gamma1) efficiently induces the formation of the interdigitated gel phase (LbetaI) of 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) bilayers. In situ scanning force microscopy of solid supported bilayers and fluorescence spectroscopy of peptide-treated DPPC vesicles provide evidence for the formation of acyl chain interdigitated lipid domains. It could be shown by fluorescence spectroscopy that the peptide inserts in the DPPC matrix above the main transition temperature of the lipid, while the formation of domains with decreased thickness occurs after the sample is cooled to 25 degrees C. The orientation and secondary structure of the peptide in lipid bilayers were investigated using attenuated total reflectance infrared (ATR-IR) and circular dichroism (CD) spectroscopy. These results enabled us to formulate a mechanistic model for the peptide-mediated induction of interdigitation in DPPC bilayers. Moreover, the membrane activity of gamma1 with gel phase lipids established in this study may have further implications for the infection strategy adopted by simple RNA viruses.

"Rattlesnake" structure of a filamentous plant RNA virus built of two capsid proteins.

Elongated particles of simple RNA viruses of plants are composed of an RNA molecule coated with numerous identical capsid protein subunits to form a regular helical structure, of which tobacco mosaic virus is the archetype. Filamentous particles of the closterovirus beet yellow virus (BYV) reportedly contain approximately 4000 identical 22-kDa (p22) capsid protein subunits. The BYV genome encodes a 24-kDa protein (p24) that is structurally related to the p22. We searched for the p24 in BYV particles by using immunoelectron microscopy with specific antibodies against the recombinant p24 protein and its N-terminal peptide. A 75-nm segment at one end of the 1370-nm filamentous viral particle was found to be consistently labeled with both types of antibodies, thus indicating that p24 is indeed the second capsid protein and that the closterovirus particle, unlike those of other plant viruses with helical symmetry, has a "rattlesnake" rather than uniform structure.

The evolution of RNA viruses.

AbstractThe structures of spherical RNA plant tiruses, containing 180 copies of one protein subunit, are closely related to those of the animal RNA picornaviruses, which are built of 60 copies of each of three larger capsid proteins. Other spherical RNA and DNA viruses utilize the same structural motif as building units of the viral capsid. It is therefore probable that many of the simple RNA viruses, whether found in animals or plants, have had a common genetic origin. The original gene may have coded for a protein that bound to specific molecules on the cell surface.

Molecular organization and stabilizing forces of simple RNA viruses: V. The role of lysyl residues in the stabilization of cucumber mosaic virus strain S

Experiments have been carried out to identify the chemical groups responsible for the stabilization of CMV (strain S). At pH 10.3 CMV-protein and CMV-RNA reassociated into nucleoproteins that possess no specific viruslike structure. In rate zonal centrifugation the nucleoproteins sedimented as three zones trailing the virus marker. The nucleoproteins were isolated and analyzed for RNA component contents with polyacrylamide electrophoresis. In order of increasing sedimentation rates, they contained RNA components 4, 3, and 1 + 2. Separate treatment of CMV-protein and CMV-RNA at pH 10.3, followed by neutralization, had no effect on their capability to reassemble normally at pH 7. The nucleoproteins formed at pH 10.3 could be dissociated by 2.0 M LiCl. The protein reisolated in this manner could also be reassembled at pH 7 with CMV-RNA to form normal virions. These experiments suggest that the aborted reassembly at alkaline pH is caused mainly by a deprotonation of protein amino-acid residues and RNA bases with a pK around 10, and that reprotonation can reverse any pH-induced conformational perturbation of CMV-protein. Reaction of CMV with the lysyl-specific reagent trinitrobenzene-sulfonic acid (TNBS) led to structural transformation of the virions into slower sedimenting trinitrophenylated (TNP)-nucleoproteins with undefined structures. These nucleoproteins also contained CMV-RNA components 4, 3, and 1 + 2 in order of increasing sedimentation rates. The time-course of the TNBS reaction and the structural transformation suggested that approximately 3 of 15 lysyl residues in each subunit are essential to the stability of the virion. Protein could easily be prepared from 3TNP-CMV by means of 2.0 M LiCl dissociation. The yellow color and the spectral properties provided direct identification of CMV-protein as the site of trinitrophenylation. CMV with nine TNP substituents per subunit failed to dissociate under the influence of 2.0 M LiCl. Reassembly at pH 7.2 failed when 3TNP-CMV-protein was used as protein constituent. Reaction of the D-strain of CMV with TNBS was much slower. The structural transformation occurred at a higher TNP substitution level, and was more gradual than with CMV-S. This observation is in agreement with the generally greater stability of CMV-D. Of several bromoviruses tested, the reactivity with TNBS decreased in the following order: BBMV > CCMV > BMV. All but BMV degraded in their entirety under the conditions investigated.

Studies on the stabilizing forces of simple RNA viruses: I. Selective interference with protein—RNA interactions in turnip yellow mosaic virus

To allow an experimental study of the disengagement of the RNA from its linkage with the protein in turnip yellow mosaic virus, several methods were developed which affected the capsid structure to such an extent that upon breakage of the protein-RNA linkage, the RNA was allowed enough mobility to undergo either internal rearrangements or to be released in the reaction mixture. These “capsid-expanding” methods encompassed alkali treatment, moderate heating, and the treatment with 12 m-formamide, all in the presence of high ionic strength (1.0) m-KCl). Moderate alkali treatment of turnip yellow mosaic virus gave rise to an in situ fragmentation of the phosphodiester chain of the RNA followed by a complexing of the RNA fragments inside the intact capsid. In alkali treatment with pH above 11, a large proportion of the virus particles released their fragmented RNA into the reaction mixture. The release of RNA was an all-or-nothing process, and followed single-hit kinetics for the majority of particles in a population. The rate of RNA release increased as the pH was raised, but appeared to be independent of the degree of fragmentation and/or complexing that preceded it. Hence, the release of RNA is primarily controlled by the degree of capsid expansion. Moderate heating or treatment with 12 m-formamide provided a means of capsid perturbation which allowed a direct analysis of the breakage of protein-RNA linkages as a function of pH. With both methods it was found that at pH values under neutrality turnip yellow mosaic virus retained its stability, but that a pH of 7 or higher induced dissociation into capsids and free RNA. The same pH dependence of dissociation of turnip yellow mosaic virus was also found after high doses of ultraviolet light irradiation. Whereas the viral capsid degraded into slowly sedimenting fragments upon irradiation under slightly acid as well as under neutral conditions, the majority of the virus particles remained stable at pH 6, but not at pH 7. It was concluded that in turnip yellow mosaic virus the integrity of the protein-RNA linkages is dependent upon the pH, and that these linkages are broken between pH 6 and 7. The type of interaction compatible with such behavior would be an ionic linkage between protonated histidinyl residues and negatively charged nucleic acid phosphates, or could consist of juxtaposed protein carboxylate and nucleotide amino groups, sharing a hydrogen-bonded proton. The latter model would provide a structural role for the extraordinarily large number of cytidilic acid residues in turnip yellow mosaic virus RNA.

Studies on the stabilizing forces of simple RNA viruses: II. Stability, dissociation and reassembly of cucumber mosaic virus

The stability properties of cucumber mosaic virus were investigated in relation to those of two other, well-described, icosahedral RNA viruses of similar geometry; the cowpea chlorotic mottle virus and the turnip yellow mosaic virus. High concentrations of neutral salts caused the dissociation of cucumber mosaic virus into its constituent RNA and protein subunits irrespective of the pH of the solution. At low ionic strength the effect of pH on the infectivity and the sedimentation behavior of the virus was tested between pH 4.0 and 8.5. No effect was noticed in this range, but significant change became evident at pH 9.8 and was complete at pH 10.45. The products of this alkaline treatment were a mixture of slower sedimenting nucleoproteins. The RNA inside cucumber mosaic virus was accessible to pancreatic ribonuclease. There was little or no pH-dependence of the ribonuclease susceptibility. Under no circumstances were protein capsids of cucumber mosaic virus ever obtained, neither by degradation of the virion, reassembly of the protein subunits, nor directly from the infected plant. These stability properties of cucumber mosaic virus are strikingly different from those of cowpea chlorotic mottle virus and turnip yellow mosaic virus, as reported in the literature, and indicate the possession of only weak inter-protein subunit linkages, or their total absence. Cucumber mosaic virus could be reversibly dissociated by varying the concentration of lithium chloride in its solutions. The reassembled virus was shown to be identical to cucumber mosaic virus in its physical properties, in its reaction with cucumber mosaic virus antiserum, and in the degree of its sensitivity to ribonuclease. The infectivity of the reassembled virus was almost as high as the infectivity of the untreated virus. Cucumber mosaic virus protein combined with great efficiency with turnip yellow mosaic virus RNA of different molecular conformations and size. Fragmented turnip yellow mosaic virus RNA (molecular weight 60,000) combined to yield particles with the sedimentation properties of cucumber mosaic virus ( S 20, w about 93 s); full size turnip yellow mosaic virus RNA gave particles with sedimentation rates of about 130 s. The presence of RNA was mandatory for the reassembly of cucumber mosaic virus protein subunits. No reaggregation of alkali-converted cucumber mosaic virus could be achieved. Salt-dissociated cucumber mosaic virus failed to reassemble in a normal fashion below pH 5, where insoluble nucleoprotein precipitates were formed, or above pH 10, where slower sedimenting nucleoproteins were obtained. These nucleoproteins also could not be assembled further into virus-like particles, and might represent minimum free-energy structures resulting from a less specific interaction of the different sized RNA species of cucumber mosaic virus with protein subunits. Cucumber mosaic virus probably derives its stability from relatively strong ionic linkages between nucleic acid-phosphate residues functioning as a polyanion, and basic amino-acid residues of the protein which function as a polycation.