5'PPP-RNA induced RIG-I activation inhibits drug-resistant avian H5N1 as well as 1918 and 2009 pandemic influenza virus replication

<p>Abstract</p> <p>Background</p> <p>Emergence of drug-resistant strains of influenza viruses, including avian H5N1 with pandemic potential, 1918 and 2009 A/H1N1 pandemic viruses to currently used antiviral agents, neuraminidase inhibitors and M2 Ion channel blockers, underscores the importance of developing novel antiviral strategies. Activation of innate immune pathogen sensor Retinoic Acid Inducible Gene-I (RIG-I) has recently been shown to induce antiviral state.</p> <p>Results</p> <p>In the present investigation, using real time RT-PCR, immunofluorescence, immunoblot, and plaque assay we show that 5'PPP-containing single stranded RNA (5'PPP-RNA), a ligand for the intracytoplasmic RNA sensor, RIG-I can be used as a prophylactic agent against known drug-resistant avian H5N1 and pandemic influenza viruses. 5'PPP-RNA treatment of human lung epithelial cells inhibited replication of drug-resistant avian H5N1 as well as 1918 and 2009 pandemic influenza viruses in a RIG-I and type 1 interferon dependant manner. Additionally, 5'PPP-RNA treatment also inhibited 2009 H1N1 viral replication <it>in vivo </it>in mice.</p> <p>Conclusions</p> <p>Our findings suggest that 5'PPP-RNA mediated activation of RIG-I can suppress replication of influenza viruses irrespective of their genetic make-up, pathogenicity, and drug-sensitivity status.</p

The 3′ Untranslated Regions of Influenza Genomic Sequences Are 5′PPP-Independent Ligands for RIG-I

Retinoic acid inducible gene-I (RIG-I) is a key regulator of antiviral immunity. RIG-I is generally thought to be activated by ssRNA species containing a 5′-triphosphate (PPP) group or by unphosphorylated dsRNA up to ∼300 bp in length. However, it is not yet clear how changes in the length, nucleotide sequence, secondary structure, and 5′ end modification affect the abilities of these ligands to bind and activate RIG-I. To further investigate these parameters in the context of naturally occurring ligands, we examined RNA sequences derived from the 5′ and 3′ untranslated regions (UTR) of the influenza virus NS1 gene segment. As expected, RIG-I-dependent interferon-β (IFN-β) induction by sequences from the 5′ UTR of the influenza cRNA or its complement (26 nt in length) required the presence of a 5′PPP group. In contrast, activation of RIG-I by the 3′ UTR cRNA sequence or its complement (172 nt) exhibited only a partial 5′PPP-dependence, as capping the 5′ end or treatment with CIP showed a modest reduction in RIG-I activation. Furthermore, induction of IFN-β by a smaller, U/A-rich region within the 3′ UTR was completely 5′PPP-independent. Our findings demonstrated that RNA sequence, length, and secondary structure all contributed to whether or not the 5′PPP moiety is needed for interferon induction by RIG-I

Delineation of interfaces on human alpha-defensins critical for human adenovirus and human papillomavirus inhibition.

Human α-defensins are potent anti-microbial peptides with the ability to neutralize bacterial and viral targets. Single alanine mutagenesis has been used to identify determinants of anti-bacterial activity and binding to bacterial proteins such as anthrax lethal factor. Similar analyses of α-defensin interactions with non-enveloped viruses are limited. We used a comprehensive set of human α-defensin 5 (HD5) and human neutrophil peptide 1 (HNP1) alanine scan mutants in a combination of binding and neutralization assays with human adenovirus (AdV) and human papillomavirus (HPV). We have identified a core of critical hydrophobic residues that are common determinants for all of the virus-defensin interactions that were analyzed, while specificity in viral recognition is conferred by specific surface-exposed charged residues. The hydrophobic residues serve multiple roles in maintaining the tertiary and quaternary structure of the defensins as well as forming an interface for virus binding. Many of the important solvent-exposed residues of HD5 group together to form a critical surface. However, a single discrete binding face was not identified for HNP1. In lieu of whole AdV, we used a recombinant capsid subunit comprised of penton base and fiber in quantitative binding studies and determined that the anti-viral potency of HD5 was a function of stoichiometry rather than affinity. Our studies support a mechanism in which α-defensins depend on hydrophobic and charge-charge interactions to bind at high copy number to these non-enveloped viruses to neutralize infection and provide insight into properties that guide α-defensin anti-viral activity

Common misconceptions of defensin antiviral activity.

<p>Studies of viral interactions with defensins have dispelled many misconceptions about the key properties of defensins that dictate their activity. The in vivo relevance of these mechanisms for viral pathogenesis has yet to be firmly established. Image of the HD5 dimer was generated using Pymol (PDB: 1ZMP).</p

Purification and characterization of rPenton.

<p><i>A</i>, immunoblots of input (I), bound (B), and flow-through (F/T) fractions from a representative rPenton purification were probed for fiber (top) or penton base (PB, bottom). Viral proteins were included as positive controls (C). <i>B</i>, fractions from this rPenton purification were separated by SDS-PAGE and stained with coomassie dye. Sizes of molecular weight standards (M) are indicated. * indicates a prominent non-viral protein impurity. <i>C</i>, electron micrographs of the resulting purified rPenton. Scale bar is 100 nm. <i>D</i>, background-corrected SPR sensorgrams of CAR-Ig at 35 nM bound to immobilized rPenton (black, 2115 RU), fiber (gray, 889 RU), or penton base (light gray, 1061 RU). Three individual traces for each ligand are shown.</p

Binding of alanine scan mutants to rPenton.

<p><i>A–C</i>, background-corrected SPR sensorgrams of defensins, each at 333 nM, binding to immobilized rPenton (2115 RU). Three individual traces for each mutant are shown. The wild type HD5 sensorgram (solid black) is identical in <i>A–C</i> for comparison with (<i>A</i>) T7A (dotted black), T12A (solid gray), and E21A (dotted gray); (<i>B</i>) I22A (dotted black), R28A (solid gray), and V19A (dotted gray); and (<i>C</i>) L29A (dotted black), L26A (solid gray), and Y27A (dotted gray). <i>D</i>, Representative steady-state binding curves for wild type HD5 (solid black), L29A (dotted black), L26A (solid gray), and Y27A (dotted gray). Dissociation constants (<i>E</i>) and maximum binding (B_max) values (<i>F</i>) for alanine mutants. Data are best-fit values ± SE.</p