RACK1 controls IRES-mediated translation of viruses.

Fighting viral infections is hampered by the scarcity of viral targets and their variability, resulting in development of resistance. Viruses depend on cellular molecules-which are attractive alternative targets-for their life cycle, provided that they are dispensable for normal cell functions. Using the model organism Drosophila melanogaster, we identify the ribosomal protein RACK1 as a cellular factor required for infection by internal ribosome entry site (IRES)-containing viruses. We further show that RACK1 is an essential determinant for hepatitis C virus translation and infection, indicating that its function is conserved for distantly related human and fly viruses. Inhibition of RACK1 does not affect Drosophila or human cell viability and proliferation, and RACK1-silenced adult flies are viable, indicating that this protein is not essential for general translation. Our findings demonstrate a specific function for RACK1 in selective mRNA translation and uncover a target for the development of broad antiviral intervention.

RNA knockdown by synthetic peptidyl-oligonucleotide ribonucleases: behavior of recognition and cleavage elements under physiological conditions.

Sequence-specific protein-based ribonucleases are not found in nature. Absolute sequence selectivity in RNA cleavage in vivo normally requires multi-component complexes that recruit a guide RNA or DNA for target recognition and a protein-RNA assembly for catalytic functioning (e.g. RNAi molecular machinery, RNase H). Recently discovered peptidyl-oligonucleotide synthetic ribonucleases selectively knock down pathogenic RNAs by irreversible cleavage to offer unprecedented opportunities for control of disease-relevant RNA. Understanding how to increase their potency, selectivity and catalytic turnover will open the translational pathway to successful therapeutics. Yet, very little is known about how these chemical ribonucleases bind, cleave and leave their target. Rational design awaits this understanding in order to control therapy, particularly how to overcome the trade-off between sequence specificity and potency through catalytic turnover. We illuminate this here by characterizing the interactions of these chemical RNases with both complementary and non-complementary RNAs using Tm profiles, fluorescence, UV-visible and NMR spectroscopies. Crucially, the level of counter cations, which are tightly-controlled within cellular compartments, also controlled these interactions. The oligonucleotide component dominated interaction between conjugates and complementary targets in the presence of physiological levels of counter cations (K+), sufficient to prevent repulsion between the complementary nucleic acid strands to allow Watson-Crick hydrogen bonding. In contrast, the positively-charged catalytic peptide interacted poorly with target RNA, when counter cations similarly screened the negatively-charged sugar-phosphate RNA backbones. The peptide only became the key player, when counter cations were insufficient for charge screening; moreover, only under such non-physiological conditions did conjugates form strong complexes with non-complementary RNAs.Communicated by Ramaswamy H. Sarma.

Artificial ribonucleases inactivate a wide range of viruses using their ribonuclease, membranolytic, and chaotropic-like activities.

Artificial ribonucleases (aRNases) are small compounds catalysing RNA cleavage. Recently we demonstrated that aRNases readily inactivate various viruses in vitro. Here, for three series of aRNases (1,4-diazabicyclo [2.2.2]octane-based and peptide-like compounds) we show that apart from ribonuclease activity the aRNases display chaotropic-like and membranolytic activities. The levels of membranolytic and chaotropic-like activities correlate well with the efficiency of various viruses inactivation (enveloped, non-enveloped, RNA-, DNA-containing). We evaluated the impact of these activities on the efficiency of virus inactivation and found: i) the synergism between membranolytic and chaotropic-like activities is sufficient for the inactivation of enveloped viruses (influenza A, encephalitis, vaccinia viruses) for 1,4-diazabicyclo [2.2.2]octane based aRNases, ii) the inactivation of non-enveloped viruses (encephalomyocarditis, acute bee paralysis viruses) is totally dependent on the synergism of chaotropic-like and ribonuclease activities, iii) ribonuclease activity plays a leading role in the inactivation of RNA viruses by aRNases Dp12F6, Dtr12 and K-D-1, iv) peptide-like aRNases (L2-3, K-2) being effective virus killers have a more specific mode of action. Obtained results clearly demonstrate that aRNases represent a new class of broad-spectrum virus-inactivating agents.

Influenza virus inactivated by artificial ribonucleases as a prospective killed virus vaccine.

The inactivation of viral particles with agents causing minimal damage to the structure of surface epitopes is a well-established approach for the production of killed virus vaccines. Here, we describe new agents for the inactivation of influenza virus, artificial ribonucleases (aRNases), which are chemical compounds capable of cleaving RNA molecules. Several aRNases were identified, exhibiting significant virucidal activity against the influenza A virus and causing a minimal effect on the affinity of monoclonal antibodies for the inactivated virus. Using a murine model of the influenza virus infection, a high protective activity of the aRNase-inactivated virus as a vaccine was demonstrated. The results of the experiments demonstrate the efficacy of novel chemical agents in the preparation of vaccines against influenza and, perhaps, against other infections caused by RNA viruses.

Inactivation of a non-enveloped RNA virus by artificial ribonucleases: honey bees and acute bee paralysis virus as a new experimental model for in vivo antiviral activity assessment.

RNA-containing viruses represent a global threat to the health and wellbeing of humans and animals. Hence, the discovery of new approaches for the design of novel vaccines and antiviral compounds attains high attention. Here we describe the potential of artificial ribonucleases (aRNases), low molecular weight compounds capable to cleave phosphodiester bonds in RNA under mild conditions, to act as antiviral compounds via destroying the genome of non-enveloped RNA viruses, and the potential of utilizing honey bee larvae and adult bees (Apis mellifera) as a novel experimental system for the screening of new antiviral compounds. Pre-incubation of an Acute bee paralysis virus (ABPV) suspension with aRNases D3-12, K-D-1 or Dp12F6 in a concentration-dependent manner increased the survival rate of bee larvae and adult bees subsequently infected with these preparations, whereas incubation of the virus with aRNases ABL3C3 or L2-3 had no effect at all. The results of RT-PCR analysis of viral RNA isolated from aRNase-treated virus particles confirmed that virus inactivation occurs via degradation of viral genomic RNA: dose-dependent inactivation of ABPV correlates well with the cleavage of viral RNA. Electron microscopy analysis revealed that the morphology of ABPV particles inactivated by aRNases remains unaffected as compared to control virus preparations. Altogether the obtained results clearly demonstrate the potential of aRNases as a new virus inactivation agents and bee larvae/ABPV as a new in vivo system for the screening of antiviral compounds.