Natural languages and RNA virus evolution.

Information concepts from physics, mathematics and computer science support many areas of research in biology. Their focus is on objective information, which provides correlations and patterns related to objects, processes, marks and signals. In these approaches only the quantitative aspects of the meaning of the information is relevant. In other areas of biology, 'meaningful information', which is subjective in nature, relies on the physiology of the organism's sensory organs and on the interpretation of the perceived signals, which is then translated into action, even if this is only mental (in brained animals). Information is involved, in terms of both amount and quality. Here we contextualize and review the main theories that deal with 'meaningful-information' at a molecular level from different areas of natural language research, namely biosemiotics, code-biology, biocommunication and biohermeneutics. As this information mediates between the organism and its environment, we emphasize how such theories compare with the neo-Darwinian treatment of genetic information, and how they project onto the rapid evolution of RNA viruses.

Viruses as archaeological tools for uncovering ancient molecular relationships.

The entry of a virus into the host cell always implies the alteration of certain intracellular molecular relationships, some of which may involve the recovery of ancient cellular activities. In this sense, viruses are archaeological tools for identifying unexpressed activities in noninfected cells. Among these, activities that hinder virus propagation may represent cellular defense mechanisms, for example, activities that mutagenize the viral genome such as ADAR-1 or APOBEC activities. Instead, those that facilitate virus propagation can be interpreted as the result of viral adaptation to-or mimicking-cellular structures, enabling the virus to perform anthropomorphic activities, including hijacking, manipulating, and reorganizing cellular factors for their own benefit. The alternative we consider here is that some of these second set of cellular activities were already in the uninfected cell but silenced, under the negative control of the cell or lineage, and that they represent a necessary precondition for viral infection. For example, specifically loading an amino acid at the 3'-end of the mRNA of some plant viruses by aminoacyl-tRNA synthetases has proved essential for virus infection despite this reaction not occurring with cellular mRNAs. Other activities of this type are discussed here, together with the biological context in which they acquire a coherent meaning, that is, genetic latency and molecular conflict.

Archaeological approaches to RNA virus evolution.

Studies with RNA enzymes (ribozymes) and protein enzymes have identified certain structural elements that are present in some cellular mRNAs and viral RNAs. These elements do not share a primary structure and, thus, are not phylogenetically related. However, they have common (secondary/tertiary) structural folds that, according to some lines of evidence, may have an ancient and common origin. The term 'mRNA archaeology' has been coined to refer to the search for such structural/functional relics that may be informative of early evolutionary developments in the cellular and viral worlds and have lasted to the present day. Such identified RNA elements may have developed as biological signals with structural and functional relevance (as if they were buried objects with archaeological value), and coexist with the standard linear information of nucleic acid molecules that is translated into proteins. However, there is a key difference between the methods that extract information from either the primary structure of mRNA or the signals provided by secondary and tertiary structures. The former (sequence comparison and phylogenetic analysis) requires strict continuity of the material vehicle of information during evolution, whereas the archaeological method does not require such continuity. The tools of RNA archaeology (including the use of ribozymes and enzymes to investigate the reactivity of the RNA elements) establish links between the concepts of communication and language theories that have not been incorporated into knowledge of virology, as well as experimental studies on the search for functionally relevant RNA structures.

X-Ray Crystallography to Study Conformational Changes in a TPP Riboswitch.

Conformational rearrangements are key to the function of riboswitches. These regulatory mRNA regions specifically bind to cellular metabolites using evolutionarily conserved sensing domains and modulate gene expression via adjacent downstream expression platforms, which carry gene expression signals. The regulation is achieved through the ligand-dependent formation of two alternative and mutually exclusive conformations involving the same RNA region. While X-ray crystallography cannot visualize dynamics of such dramatic conformational rearrangements, this method is pivotal to understand RNA-ligand interaction that stabilize the sensing domain and drive folding of the expression platform. X-ray crystallography can reveal local changes in RNA necessary for discriminating cognate and noncognate ligands. This chapter describes preparation of thiamine pyrophosphate riboswitch RNAs and its crystallization with different ligands, resulting in structures with local conformational changes in RNA. These structures can help to derive information on the dynamics of the RNA essential for specific binding to small molecules, with potential for using this information for developing designer riboswitch-ligand systems.

Application of archaeological concepts to the interpretation of RNA virus quasi species evolution.

An enduring problem concerning the evolution of RNA viruses stems from the fact that their long-term rates of evolution (substitutions/ site/year) are lower than those calculated by comparing sequences of isolates collected over short time periods or within a single host (shortterm or intra-host evolution). This inconsistency has been attributed to several reasons, including deviations from the assumption of a molecularclock (constancy of mutational inputs as a function of time) and variations in viral multiplication rates, among others. We previously proposed a non-phylogenetic method for extracting information contained in mRNAs, that cannot be identified from examination of primary sequences alone, and that we called «archaeological¼ information. In this new approach, mRNAs are of interest as molecules, not for their primary sequence or encoded proteins but for encrypted information established in a remote past. In the present article, we propose that an archaeological approach may also contribute to explain higher short-term than long-term evolution rates in RNA viruses, in this case, by using the archaeological concept of palimpsest. The palimpsest is a record of historical changes, but it is not a successively ordered or a complete record, rather it is the product of two opposing activities, one of writing and rewriting and the other of erasing. In RNA virus quasispecies, the gain or loss of mutations is reflected in changes in the submolar frequency of myriads of variants in the population. The fact that mutation elimination is not always complete, turns viral quasispecies into complex palimpsests of viral variants or sub-populations thereof. Here we relate two main different temporalities of the quasispecies palimpsest (short- and long-term) to the stability of mutations in response to changes related to three components of the virus: the virions, the infected cell and the host cell lineage. Host cell lineage-related viral memory would be mostly irre versible as they are adaptive products to host cell changes. In contrast, memories related to the environment of the virion or responsive to the environment of the infected cell, which is shortterm mutational input, is less constrained provided the alteration in the ancestral information carried by the RNA is only transient. The two intermixed memory components result in two differently contributing mutation rates whose influence in the final result depends on whether the timescales used to take the sequences for comparison are short or long term.

Riboswitch Mechanisms: New Tricks for an Old Dog.

Discovered almost twenty years ago, riboswitches turned out to be one of the most common regulatory systems in bacteria, with representatives found in eukaryotes and archaea. Unlike many other regulatory elements, riboswitches are entirely composed of RNA and capable of modulating expression of genes by direct binding of small cellular molecules. While bacterial riboswitches had been initially thought to control production of enzymes and transporters associated with small organic molecules via feedback regulatory circuits, later findings identified riboswitches directing expression of a wide range of genes and responding to various classes of molecules, including ions, signaling molecules, and others. The 5'-untranslated mRNA regions host a vast majority of riboswitches, which modulate transcription or translation of downstream genes through conformational rearrangements in the ligand-sensing domains and adjacent expression-controlling platforms. Over years, the repertoire of regulatory mechanisms employed by riboswitches has greatly expanded; most recent studies have highlighted the importance of alternative mechanisms, such as RNA degradation, for the riboswitch-mediated genetic circuits. This review discusses the plethora of bacterial riboswitch mechanisms and illustrates how riboswitches utilize different features and approaches to elicit various regulatory responses.

The archaeology of coding RNA.

Different theories concerning the origin of RNA (and, in particular, mRNA) point to the concatenation and expansion of proto-tRNA-like structures. Different biochemical and biophysical tools have been used to search for ancient-like RNA elements with a specific structure in genomic viral RNAs, including that of the hepatitis C virus, as well as in cellular mRNA populations, in particular those of human hepatocytes. We define this method as "archaeological," and it has been designed to discover evolutionary patterns through a nonphylogenetic and nonrepresentational strategy. tRNA-like elements were found in structurally or functionally relevant positions both in viral RNA and in one of the liver mRNAs examined, the antagonist interferon-alpha subtype 5 (IFNA5) mRNA. Additionally, tRNA-like elements are highly represented within the hepatic mRNA population, which suggests that they could have participated in the formation of coding RNAs in the distant past. Expanding on this finding, we have observed a recurring dsRNA-like motif next to the tRNA-like elements in both viral RNAs and IFNA5 mRNA. This suggested that the concatenation of these RNA motifs was an activity present in the RNA pools that might have been relevant in the RNA world. The extensive alteration of sequences that likely triggered the transition from the predecessors of coding RNAs to the first fully functional mRNAs (which was not the case in the stepwise construction of noncoding rRNAs) hinders the phylogeny-based identification of RNA elements (both sequences and structures) that might have been active before the advent of protein synthesis. Therefore, our RNA archaeological method is presented as a way to better understand the structural/functional versatility of a variety of RNA elements, which might represent "the losers" in the process of RNA evolution as they had to adapt to the selective pressures favoring the coding capacity of the progressively longer mRNAs.

Viral tRNA Mimicry from a Biocommunicative Perspective.

RNA viruses have very small genomes which limits the functions they can encode. One of the strategies employed by these viruses is to mimic key factors of the host cell so they can take advantage of the interactions and activities these factors typically participate in. The viral RNA genome itself was first observed to mimic cellular tRNA over 40 years ago. Since then researchers have confirmed that distinct families of RNA viruses are accessible to a battery of cellular factors involved in tRNA-related activities. Recently, potential tRNA-like structures have been detected within the sequences of a 100 mRNAs taken from human cells, one of these being the host defense interferon-alpha mRNA; these are then additional to the examples found in bacterial and yeast mRNAs. The mimetic relationship between tRNA, cellular mRNA, and viral RNA is the central focus of two considerations described below. These are subsequently used as a preface for a final hypothesis drawing on concepts relating to mimicry from the social sciences and humanities, such as power relations and creativity. Firstly, the presence of tRNA-like structures in mRNAs indicates that the viral tRNA-like signal could be mimicking tRNA-like elements that are contextualized by the specific carrier mRNAs, rather than, or in addition to, the tRNA itself, which would significantly increase the number of potential semiotic relations mediated by the viral signals. Secondly, and in particular, mimicking a host defense mRNA could be considered a potential new viral strategy for survival. Finally, we propose that mRNA's mimicry of tRNA could be indicative of an ancestral intracellular conflict in which species of mRNAs invaded the cell, but from within. As the meaning of the mimetic signal depends on the context, in this case, the conflict that arises when the viral signal enters the cell can change the meaning of the mRNAs' internal tRNA-like signals, from their current significance to that they had in the distant past.

Geneticin Stabilizes the Open Conformation of the 5' Region of Hepatitis C Virus RNA and Inhibits Viral Replication.

The aminoglycoside Geneticin (G418) is known to inhibit cell culture proliferation, via virus-specific mechanisms, of two different virus genera from the family Flaviviridae. Here, we tried to determine whether Geneticin can selectively alter the switching of the nucleotide 1 to 570 RNA region of hepatitis C virus (HCV) and, if so, whether this inhibits viral growth. Two structure-dependent RNases known to specifically cleave HCV RNA were tested in the presence or absence of the drug. One was the Synechocystis sp. RNase P ribozyme, which cleaves the tRNA-like domain around the AUG start codon under high-salt buffer conditions; the second was Escherichia coli RNase III, which recognizes a double-helical RNA switch element that changes the internal ribosome entry site (IRES) from a closed (C) conformation to an open (O) one. While the drug did not affect RNase P activity, it did inhibit RNase III in the micromolar range. Kinetic studies indicated that the drug favors the switch from the C to the O conformation of the IRES by stabilizing the distal double-stranded element and inhibiting further processing of the O form. We demonstrate that, because the RNA in this region is highly conserved and essential for virus survival, Geneticin inhibits HCV Jc1 NS3 expression, the release of the viral genomic RNA, and the propagation of HCV in Huh 7.5 cells. Our study highlights the crucial role of riboswitches in HCV replication and suggests the therapeutic potential of viral-RNA-targeted antivirals.

Virus is a Signal for the Host Cell.

Currently, the concept of the cell as a society or an ecosystem of molecular elements is gaining increasing acceptance. The basic idea arose in the 19th century, from the surmise that there is not just a single unit underlying an individual's appearance, but a plurality of entities with both collaborative and conflicting relationships. The following hypothesis is based around this model. The incompatible activities taking place between different original elements, which were subsumed into the first cell and could not be eliminated, had to be controlled very closely. Similarly, a strong level of control had to be developed over many cellular elements after the cell changed its genome to DNA. We assume that at least some of those original RNA agents and other biomolecules which carry incompatibilities and risks, are retained within current cells, although they are now under strict control. A virus functions as a signal informing these repressed cellular RNAs and other elements of ancient origin how to restore suppressed degrees of molecular freedom, favoring pre-existing molecular affinities and activities, re-establishing ancient molecular webs of interactions, and giving fragments of ancient coded information (mostly in the form of RNA structural motifs) the opportunity to be re-expressed. Collectively, these newly activated mechanisms lead to different possibilities for pathological cell states. All these processes are opposed by cell-control mechanisms. Thus, in this new scenario, the battle is considered intracellular rather than between the virus and the cell. And so the virus is treated as the signal that precipitates the cell's change from a latent to an active pathological state.

A magnesium-induced RNA conformational switch at the internal ribosome entry site of hepatitis C virus genome visualized by atomic force microscopy.

The 5' untranslated region of hepatitis C virus (HCV) genomic RNA contains an internal ribosome entry site (IRES) element, composed of domains II-IV, which is required for cap-independent translation initiation. Little information on the 3D structure of the whole functional HCV IRES is still available. Here, we use atomic force microscopy to visualize the HCV IRES conformation in its natural sequence context, which includes the upstream domain I and the essential, downstream domains V and VI. The 574 nt-long molecule analyzed underwent an unexpected, Mg(2+)-induced switch between two alternative conformations: from 'open', elongated morphologies at 0-2 mM Mg(2+) concentration to a 'closed', comma-shaped conformation at 4-6 mM Mg(2+). This sharp transition, confirmed by gel-shift analysis and partial RNase T1 cleavage, was hindered by the microRNA miR-122. The comma-shaped IRES-574 molecules visualized at 4-6 mM Mg(2+) in the absence of miR-122 showed two arms. Our data support that the first arm would contain domain III, while the second one would be composed of domains (I-II)+(V-VI) thanks to a long-range RNA interaction between the I-II spacer and the basal region of domain VI. This reinforces the previously described structural continuity between the HCV IRES and its flanking domains I, V and VI.

RNA self-cleavage activated by ultraviolet light-induced oxidation.

A novel UV-C-light-induced ribozyme activity was discovered within the highly structured 5'-genomic regions of both Hepatitis C Virus (HCV) and the related Classic Swine Fever Virus (CSFV). Cleavage is mediated by exposure to UV-C light but not by exogenous oxygen radicals. It is also very selective, occurring at base positions HCV C(79) and CSFV A(45) in some molecules and at the immediately adjacent 5'-positions HCV U(78) and CSFV U(44) in others. Among other reaction products, the majority of biochemically active products detected contained 3'-phosphate and 5'-phosphate-end groups at the newly generated termini, along with a much lower amount of 3'-hydroxyl end group. While preservation of an E-loop RNA structure in the vicinity of the cleavage site was a requisite for HCV RNA self-cleavage, this was not the case for CSFV RNA. The short size of the reactive domains (~33 nt), which are compatible with primitive RNA motifs, and the lack of sequence homology, indicate that as-yet unidentified UV-activated ribozymes are likely to be found throughout structured RNAs, thereby providing clues to whether early RNA self-cleavage events were mediated by photosensitive RNA structures.

In vitro characterization of a miR-122-sensitive double-helical switch element in the 5' region of hepatitis C virus RNA.

It has been proposed that the hepatitis C virus (HCV) internal ribosome entry site (IRES) resides within a locked conformation, owing to annealing of its immediate flanking sequences. In this study, structure probing using Escherichia coli dsRNA-specific RNase III and other classical tools showed that this region switches to an open conformation triggered by the liver-specific microRNA, miR-122. This structural transition, observed in vitro, may be the mechanistic basis for the involvement of downstream IRES structural domain VI in translation, as well as providing a role of liver-specific miR-122 in HCV infection. In addition, the induced RNA switching at the 5' untranslated region could ultimately represent a new mechanism of action of micro-RNAs.