Characterization of Mutational Tolerance of a Viral RNA-Protein Interaction.

Replication of RNA viruses is generally markedly error-prone. Nevertheless, these viruses usually retain their identity under more or less constant conditions due to different mechanisms of mutation tolerance. However, there exists only limited information on quantitative aspects of the mutational tolerance of distinct viral functions. To address this problem, we used here as a model the interaction between a replicative cis-acting RNA element (oriL) of poliovirus and its ligand (viral protein 3CD). The mutational tolerance of a conserved tripeptide of 3CD, directly involved in this interaction, was investigated. Randomization of the relevant codons and reverse genetics were used to define the space of viability-compatible sequences. Surprisingly, at least 11 different amino acid substitutions in this tripeptide were not lethal. Several altered viruses exhibited wild-type-like phenotypes, whereas debilitated (but viable) genomes could increase their fitness by the acquisition of reversions or compensatory mutations. Together with our study on the tolerance of oriL (Prostova et al., 2015), the results demonstrate that at least 42 out of 51 possible nucleotide replacements within the two relevant genomic regions are viability-compatible. These results provide new insights into structural aspects of an important viral function as well as into the general problems of viral mutational robustness and evolution.

Mutational robustness and resilience of a replicative cis-element of RNA virus: Promiscuity, limitations, relevance.

Since replication of RNA-viruses is generally a low-fidelity process, it would be advantageous, if specific interactions of their genomic cis-elements with dedicated ligands are relatively tolerant to mutations. The specificity/promiscuity trade-off of such interactions was addressed here by investigating structural requirements of the oriL (also known as the clover leaf-like element), of poliovirus RNA, a replicative cis-element containing a conserved essential tetraloop functionally interacting with the viral protein 3CD. The sequence of this tetraloop and 2 adjacent base-pairs was randomized in the viral genome, and viable viruses were selected in susceptible cells. Strikingly, each position of this octanucleotide in 62 investigated viable viruses could be occupied by any nucleotide (with the exception of one position, which lacked U), though with certain sequence preferences, confirmed by engineering mutant viral genomes whose phenotypic properties were found to correlate with the strength of the cis-element/ligand interaction. The results were compatible with a hypothesis that functional recognition by 3CD requires that this tetraloop should stably or temporarily adopt a YNMG-like (Y=U/C, N=any nucleotide, M=A/C) fold. The fitness of "weak" viruses could be increased by compensatory mutations "improving" the tetraloops. Otherwise, the recognition of "bad" tetraloops might be facilitated by alterations in the 3CD protein. The virus appeared to tolerate mutations in its cis-element relaying on either robustness (spatial structure degeneracy) or resilience (a combination of dynamic RNA folding, low-fidelity replication modifying the cis-element or its ligand, and negative selection). These mechanisms (especially resilience involving metastable low-fit intermediates) can also contribute to the viral evolvability.

Interactions between viral and prokaryotic pathogens in a mixed infection with cardiovirus and mycoplasma.

In the natural environment, animal and plant viruses often share ecological niches with microorganisms, but the interactions between these pathogens, although potentially having important implications, are poorly investigated. The present report demonstrates, in a model system, profound mutual effects of mycoplasma and cardioviruses in animal cell cultures. In contrast to mycoplasma-free cells, cultures contaminated with Mycoplasma hyorhinis responded to infection with encephalomyocarditis virus (EMCV), a picornavirus, but not with poliovirus (also a picornavirus), with a strong activation of a DNase(s), as evidenced by the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) immunofluorescence assay and electrophoretic analysis of host DNA. This degradation was reminiscent of that observed upon apoptosis but was caspase independent, judging by the failure of the specific pan-caspase inhibitor Q-VD-OPh to prevent it. The electrophoretic mobility of the enzyme responsible for DNA degradation and dependence of its activity on ionic conditions strongly suggested that it was represented by a DNase(s) of mycoplasma origin. In cells not infected with EMCV, the relevant DNase was dormant. The possibility is discussed that activation of the mycoplasma DNase might be linked to a relatively early increase in permeability of plasma membrane of the infected cells caused by EMCV. This type of unanticipated virus-mycoplasma "cooperation" may exemplify the complexity of pathogen-host interactions under conditions when viruses and microorganisms are infecting the same host. In the course of the present study, it was also demonstrated that pan-caspase inhibitor zVAD(OMe).fmk strongly suppressed cardiovirus polyprotein processing, illustrating an additional pitfall in investigations of viral effects on the apoptotic system of host cells.

Significance of the C-terminal amino acid residue in mengovirus RNA-dependent RNA polymerase.

Replication of picornavirus genomes is accomplished by the virally encoded RNA-dependent RNA polymerase (RdRP). Although the primary structure of this enzyme exhibits a high level of conservation, there are several significant differences among different picornavirus genera. In particular, a comparative alignment indicates that the C-terminal sequences of cardiovirus RdRP (known also as 3D(pol)), are 1-amino-acid residue (arginine or tryptophan) longer than that of the enterovirus or rhinovirus enzymes. Here, it is shown that alterations of the last codon of the RdRP-encoding sequence of mengovirus RNA leading to deletion of the C-terminal Trp460 or its replacement by Ala or Phe dramatically impaired viral RNA replication and, in the former case, resulted in a quasi-infectious phenotype (i.e., the mutant RNA might generate a low yield of pseudorevertants acquiring a Tyr residue in place of the deleted Trp460). The replacement of Trp460 by His or Tyr did not appreciably alter the viral growth potential. Homology modeling of three-dimensional structure of mengovirus RdRP suggested that Trp460 may be involved in interaction between the thumb and palm domains of the enzyme. Specifically, Trp460 of the thumb may form a hydrogen bond with Thr219 and hydrophobically interact with Val216 of the palm. The proposed interactions were consistent with the results of in vivo SELEX experiment, which demonstrated that infectious virus could contain Ser or Thr at position 219 and hydrophobic Val, Leu, Ile, as well as Arg (whose side chain has a nonpolar part) at position 216. A similar thumb-palm domain interaction may be a general feature of several RdRPs and its possible functional significance is discussed.

Nonreplicative homologous RNA recombination: promiscuous joining of RNA pieces?

Biologically important joining of RNA pieces in cells, as exemplified by splicing and some classes of RNA editing, is posttranscriptional, whereas in RNA viruses it is generally believed to occur during viral RNA polymerase-dependent RNA synthesis. Here, we demonstrate the assembly of precise genome of an RNA virus (poliovirus) from its cotransfected fragments, which does not require specific RNA sequences, takes place before generation of the viral RNA polymerase, and occurs in different ways: Apparently unrestricted ligation of the terminal nucleotides, joining of any one of the two entire fragments with the relevant internal nucleotide of its partner, or internal crossovers within the overlapping sequence. Incorporation of the entire 5' or 3' partners into the recombinant RNA is activated by the presence of terminal 3'-phosphate and 5'-OH, respectively. Such postreplicative reactions, fundamentally differing from the known site-specific and structurally demanding cellular RNA rearrangements, might contribute to the origin and evolution of RNA viruses and could generate new RNA species during all stages of biological evolution.