Research Article: Leader-Containing Uncapped Viral Transcript Activates RIG-I in Antiviral Stress Granules

Date Published: February 10, 2016

Publisher: Public Library of Science

Author(s): Seong-Wook Oh, Koji Onomoto, Mai Wakimoto, Kazuhide Onoguchi, Fumiyoshi Ishidate, Takahiro Fujiwara, Mitsutoshi Yoneyama, Hiroki Kato, Takashi Fujita, Friedemann Weber.


RIG-I triggers antiviral responses by recognizing viral RNA (vRNA) in the cytoplasm. However, the spatio-temporal dynamics of vRNA sensing and signal transduction remain elusive. We investigated the time course of events in cells infected with Newcastle disease virus (NDV), a non-segmented negative-strand RNA virus. RIG-I was recruited to viral replication complexes (vRC) and triggered minimal primary type I interferon (IFN) production. RIG-I subsequently localized to antiviral stress granules (avSG) induced after vRC formation. The inhibition of avSG attenuated secondary IFN production, suggesting avSG as a platform for efficient vRNA detection. avSG selectively captured positive-strand vRNA, and poly(A)+ RNA induced IFN production. Further investigations suggested that uncapped vRNA derived from read-through transcription was sensed by RIG-I in avSG. These results highlight how viral infections stimulate host stress responses, thereby selectively recruiting uncapped vRNA to avSG, in which RIG-I and other components cooperate in an efficient antiviral program.

Partial Text

Retinoic acid-inducible gene I (RIG-I), a DExD/H-box RNA helicase family protein, is a crucial cytosolic viral RNA (vRNA) sensor that initiates signal transduction to produce antiviral cytokines, namely, type I and III interferons (IFN-α/β and IFN-λ) [1–4]. RIG-I selectively recognizes relatively short double-stranded RNA (<100 nt), and this recognition is markedly enhanced by the presence of 5’-triphosphate [5–8]. Once RIG-I is activated, it physically associates with downstream IFN-β promoter stimulator 1 (IPS-1, also termed MAVS, VISA, or Cardif), which is anchored to the mitochondrial outer membrane [9–12]. The RIG-I/IPS-1 interaction leads to the recruitment of a variety of signaling adaptors in order to activate transcription factors including IFN regulatory factors (IRF) 3 and 7 as well as NF-κB. These transcription factors activate the genes coding for cytokines and IFNs in addition to IFN-stimulated genes (ISG). The present study revealed that NDV-derived vRNA species were sensed by two distinct mechanisms. NDV infection resulted in the generation of vRC as early as 1.5 hpi and their number subsequently increased (Fig 2B). At 6 hpi, IFNB mRNA was detected in cells exhibiting vRC. vRC contained vRNAs (-, +), vdsRNA, and RIG-I (Figs 1B and 3A and S4 Fig). These results suggested that vRC are a locale in which vRNA is sensed by RIG-I and triggers IFN-inducing signal in the early stages of the infection (before 7.5 hpi). At 7.5 hpi, avSG were detected with concomitant increases in IFNB mRNA levels (Fig 2C). Since RIG-I also co-localized with avSG (Fig 3A) and avSG were strongly correlated with the culmination of IFNB gene induction at >9 hpi, these results suggested that avSG were a second locale for vRNA detection by RIG-I. This is consistent with the result that most (77 to 92%) cells expressing IFNB mRNA were positive for vRC and avSG after 7.5 hpi (Fig 2C) and that the inhibition of avSG markedly attenuated IFNB gene expression (Figs 4B and 5). Immunostaining results clearly indicated that vRC and avSG made contact with IPS-1 (Fig 3C and S8 Fig), suggesting signal transduction to IPS-1 from two distinct types of granules.




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