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

RIG-I Goes Beyond Naked Recognition

It is currently unclear at which point during viral replication that RNA genomes are first recognized as nonself by the immune system. In this issue of Cell Host & Microbe, Weber et al. show that incoming nucleocapsid-bound genomes are sufficient to bind and activate innate immune sensors

Schematic representation of RNAs used in this study.

<p>The influenza A virus segment 8 cRNA is shown with NS1 and NS2/NEP coding sequences boxed. The extended lines represent the 5′ and 3′ non-coding sequences. Bars (not drawn to scale) indicate sequences (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032661#s4" target="_blank">Materials and Methods</a>) used to generate <i>in vitro</i> transcribed (IVT) RNAs.</p

Homogeneity of IVT RNAs.

<p>(A) Denaturing agarose gels of <i>in vitro</i> transcribed RNAs show products running as single bands. (B) U/A-rich vRNA was prepared by IVT and subjected to mass determination by MALDI-TOF mass spectroscopy. A single peak was observed spanning ∼1 kDa (13.8 k–14.8 k) and corresponding to the expected mass +/− ∼1–3 nucleotides. (C) To determine if the <i>in vitro</i> transcribed RNAs are ssRNA or ds NA, RNA samples were resolved on TAE PAGE, transferred onto nylon membrane and probed for dsRNA using dsRNA specific antibodies as described in Material and Methods. None of the <i>in vitro</i> transcribed RNAs nor the 41-nt long chemically synthesized ssRNA complementary strands were detected by the dsRNA-specific antibody. Only annealed 41 bp dsRNA was detected by the dsRNA-specific antibody.</p

Induction of IFN-β message is triphosphate independent.

<p>(A) The secondary structures of the IVT-RNAs shown were predicted by the program mfold (v3.2). (B) A549 cells were transfected with 3 µg of UTR RNA from the 3′ end of the cRNA or 5′ end of the vRNA and RNA was isolated 24 hr post-transfection to determine IFN-β levels by qRT-PCR. The data are shown as fold increases over levels in mock transfected cells. Error bars represent the standard deviation of triplicate qRT-PCR runs using RNAs from one of three representative experiments. Hatched bar, filled bar and empty bars represent untreated, CIP-treated and capped RNAs.</p