Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Jul 22;286(29):26210-9.
doi: 10.1074/jbc.M111.229856. Epub 2011 Jun 3.

Retinoic acid-inducible gene I-inducible miR-23b inhibits infections by minor group rhinoviruses through down-regulation of the very low density lipoprotein receptor

Ryota Ouda  1 , Koji Onomoto, Kiyohiro Takahasi, Michael R Edwards, Hiroki Kato, Mitsutoshi Yoneyama, Takashi Fujita
Affiliations

Retinoic acid-inducible gene I-inducible miR-23b inhibits infections by minor group rhinoviruses through down-regulation of the very low density lipoprotein receptor

Ryota Ouda et al. J Biol Chem. .

Abstract

In mammals, viral infections are detected by innate immune receptors, including Toll-like receptor and retinoic acid inducible gene I (RIG-I)-like receptor (RLR), which activate the type I interferon (IFN) system. IFN essentially activates genes encoding antiviral proteins that inhibit various steps of viral replication as well as facilitate the subsequent activation of acquired immune responses. In this study, we investigated the expression of non-coding RNA upon viral infection or RLR activation. Using a microarray, we identified several microRNAs (miRNA) specifically induced to express by RLR signaling. As suggested by Bioinformatics (miRBase Target Data base), one of the RLR-inducible miRNAs, miR-23b, actually knocked down the expression of very low density lipoprotein receptor (VLDLR) and LDLR-related protein 5 (LRP5). Transfection of miR-23b specifically inhibited infection of rhinovirus 1B (RV1B), which utilizes the low density lipoprotein receptor (LDLR) family for viral entry. Conversely, introduction of anti-miRNA-23b enhanced the viral yield. Knockdown experiments using small interfering RNA (siRNA) revealed that VLDLR, but not LRP5, is critical for an efficient infection by RV1B. Furthermore, experiments with the transfection of infectious viral RNA revealed that miR-23b did not affect post-entry viral replication. Our results strongly suggest that RIG-I signaling results in the inhibitions of infections of RV1B through the miR-23b-mediated down-regulation of its receptor VLDLR.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
Artificial oligomerization of the CARD of RIG-I results in signaling to activate the IFN-β and IL-6 genes. A, schematic representation of the FK/RIG fusion protein and its oligomerization by a cross-linking reagent, AP20187. B, HeLa cells stably expressing FK/RIG were treated with AP for the period indicated and cells were harvested to examine endogenous IFN-β and IL-6 mRNA levels by real-time PCR.
FIGURE 2.
FIGURE 2.
Induction of miR-23b by RIG-I signaling, SeV and RV infection, or IFN-β treatment. HeLa cells stably expressing FK/RIG were treated with AP (A). B–E, HeLa cells were infected with SeV (B), infected with RV16 (C), RV1B (D), or treated with IFN-β (1000 units/ml) (E). The miRNA fraction was extracted at the time points indicated and the amount of miR-23b was determined by real-time PCR.
FIGURE 3.
FIGURE 3.
The effect of miR-23b transfection on viral growth. A, HeLa cells were transfected with negative control miRNA (NC miRNA) or miR-23b for 48 h, the culture supernatant (sup) was collected, and HeLa cells were treated with supernatant for 24 h and then infected with RV for 24 h. Levels of RV genomic RNA were determined by real-time PCR. B-G, HeLa cells transfected with NC miRNA or miR-23b for 48 h were infected with SeV (B), NDV (C), VSV (D), EMCV (E), RV16 (F), or RV1B (G) for an additional 24 h. The viral NP level was determined by Western blotting (B, SeV; C, NDV). Infectivity in the culture supernatant was determined by plaque assay (D, VSV; E, EMCV). Levels of RV genomic RNA were determined by real-time PCR (F, RV16; G, RV1B) using specific primer sets. H, Amido Black staining of miRNA-transfected and RV-infected cells. IFN-β mRNA levels of the RNA samples in F and G are shown in I and J, respectively. **, p < 0.005.
FIGURE 4.
FIGURE 4.
Anti-miR-23b enhanced RNA yields of RV1B. HeLa cells were transfected with NC miRNA or anti-miR-23b for 48 h and infected with RV for 24 h. A, the expression of miR-23b after anti-miR-23b transfection was determined by real-time PCR. B and C, the yield of genomic RNA of RV16 (B) and RV1B (C) was determined by real-time PCR. *, p < 0.05.
FIGURE 5.
FIGURE 5.
miR-23b targets LRP5 and VLDLR. A and B, the results of the search for target sequences of miR-23b (miRBase Target Data base). Candidate sequences in LRP5 (A) and VLDLR (B) mRNA are shown. HeLa cells were transfected with NC miRNA, miR-23b, or anti-miR-23b for 48 h and LRP5 (A) and VLDLR (B) were detected by Western blotting. C, HeLa FK/RIG cells were transfected with either NC miRNA or anti-miR-23b for 24 h and AP20187 was treated for 48 h. LRP5 and VLDLR were detected by Western blotting.
FIGURE 6.
FIGURE 6.
Knockdown of VLDLR blocks accumulation of viral RNA in RV1B-infected cells. HeLa cells were transfected with control siRNA or siRNA targeting VLDLR or LRP5 as indicated. A–C, at 48 h after the transfection, cells were mock infected or infected with RV16 or RV1B for an additional 24 h. VLDLR and LRP5 were detected by Western blotting in mock-infected cells (A). RNA levels of RV16 (B) or RV1B (C) were determined by real-time PCR.
FIGURE 7.
FIGURE 7.
miR-23b did not influence intracellular replication of RV1B. A–C, HeLa cells were co-transfected with RV genomic RNA and NC miRNA or miR-23b for 3 and 9 h. Levels of miR-23b (A), RV16 RNA (B), and RV1B RNA (C) were determined by real-time PCR.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Vaucheret H., Béclin C., Fagard M. (2001) J. Cell Sci. 114, 3083–3091 - PubMed
    1. Takaoka A., Yanai H. (2006) Cell. Microbiol. 8, 907–922 - PubMed
    1. Baulcombe D. (2004) Nature 431, 356–363 - PubMed
    1. Akira S., Takeda K. (2004) Nat. Rev. Immunol. 4, 499–511 - PubMed
    1. Yoneyama M., Kikuchi M., Matsumoto K., Imaizumi T., Miyagishi M., Taira K., Foy E., Loo Y. M., Gale M., Jr., Akira S., Yonehara S., Kato A., Fujita T. (2005) J. Immunol. 175, 2851–2858 - PubMed

MeSH terms