Structural features of influenza A virus panhandle RNA enabling the activation of RIG-I independently of 5′-triphosphate
Nucleic Acids Research
Structural features of influenza A virus panhandle RNA enabling the activation of RIG-I independently of 5 -triphosphate
Mi-Kyung Lee 1 2
Hee-Eun Kim 0
Eun-Byeol Park 3
Janghyun Lee 2
Ki-Hun Kim 2
Kyungeun Lim 2
Seoyun Yum 3
Young-Hoon Lee 2
Suk-Jo Kang 3
Joon-Hwa Lee 0
Byong-Seok Choi 2
0 Department of Chemistry and Research Institute of Natural Science, Gyeongsang National University , Jinju, Gyeongnam 52828 , Republic of Korea
1 Disease Target Structure Research Center , KRIBB, Daejeon 34141 , Republic of Korea
2 Department of Chemistry , KAIST, Daejeon 34141 , Republic of Korea
3 Department of Biological Sciences , KAIST, Daejeon 34141 , Republic of Korea
Retinoic acid-inducible gene I (RIG-I) recognizes specific molecular patterns of viral RNAs for inducing type I interferon. The C-terminal domain (CTD) of RIG-I binds to double-stranded RNA (dsRNA) with the 5 -triphosphate (5 -PPP), which induces a conformational change in RIG-I to an active form. It has been suggested that RIG-I detects infection of influenza A virus by recognizing the 5 -triphosphorylated panhandle structure of the viral RNA genome. Influenza panhandle RNA has a unique structure with a sharp helical bending. In spite of extensive studies of how viral RNAs activate RIG-I, whether the structural elements of the influenza panhandle RNA confer the ability to activate RIG-I signaling has been poorly explored. Here, we investigated the dynamics of the influenza panhandle RNA in complex with RIG-I CTD using NMR spectroscopy and showed that the bending structure of the panhandle RNA negates the requirement of a 5 -PPP moiety for RIG-I activation.
INTRODUCTION
To defend against viral infections, host immune systems
employ a multitude of highly sophisticated pathways. As
the first step in surveillance for viral infection, the pattern
recognition receptors (PRRs) recognize molecular patterns
of the viral pathogens, and then initiate a signaling
cascade which culminates with induction of type I interferons
(IFN) for antiviral activity (
1
). Retinoic acid-inducible gene
I (RIG-I)-like receptors (RLRs), recognize double-stranded
viral RNAs in the cytosol of cells (
2
). Three RLRs, RIG-I,
melanoma differentiation associated factor 5 (MDA5) and
laboratory of genetics and physiology 2 (LGP2), are known
and belong to the DExD/H-box family of helicases. Among
them, RIG-I has been reported as an important sensor of
negative-sense RNA viruses such as influenza, hepatitis C,
Sendai and vesicular stomatitis viruses, for induction of
interferon beta (IFN- ) (
3
).
RIG-I consists of two N-terminal caspase recruitment
domains (CARDs), a central RNA helicase domain, and a
C-terminal RNA-binding domain. The C-terminal domain
(CTD) of RIG-I recognizes the 5 -triphosphate (PPP) group
of non-self RNAs and undergoes a conformational change
to induce IFN- production (
4
). Structural and
biochemical studies have demonstrated that RIG-I CTD can bind to
blunt-ended double stranded (ds) RNAs containing either
PPP or a hydroxyl residue (OH) at the 5 end (
5,6
). Although
5 -PPP dsRNA binds to the CTD more strongly and
stimulates interferon production more effectively compared to 5
OH dsRNA (
7,8
), it has been shown that RIG-I can be
activated by non-triphosphorylated RNAs (
9–12
). Therefore,
the mechanism underlying the discrimination of 5 -PPP or
5 -OH ends of dsRNAs by RIG-I is not fully understood.
The genome of the influenza A virus is composed of 8
segmented, single-stranded RNAs. Each RNA segment has
a highly conserved sequence of 12 and 13 nucleotides at the
5 and 3 ends, respectively (Figure 1A) (
13
). The 5 - and 3
end sequences form a partial duplex (referred to as
panhandle RNA), and this panhandle region functions as a
promoter for viral transcription and replication by interacting
with RNA-dependent RNA polymerase (
14,15
). RIG-I was
suggested to target the panhandle structure, leading to
induction of interferons for antiviral activity (
16–19
). A
recent study reported that defective interfering (DI) RNAs
of the influenza virus, which were generated by deletion
of genomic RNA and shorter genomic segments with the
same ends, preferentially bound to RIG-I compared to the
full-length genomic RNA (
20
). However, another study
reported that 5 -PPP-containing full-length genomic RNAs
were RIG-I agonists (
17
). Thus, it remains to be determined
which type of RNA is a bona fide ligand for RIG-I
activation during the course of physiological infection. Previously,
we demonstrated that the panhandle structure had an
internal loop and an unusual bending at the 5 -terminal stem
(
21
). Whether this structural feature influences the
recognition by RIG-I remains unexplored. Thus, it is of interest
to determine which structural element––the 5 -PPP moiety
or the fine structure at the terminal sequence of the RNA
genome––is the more critical factor for recognition by the
CTD of RIG-I.
Using NMR spectroscopy, (...truncated)