Preference of RIG-I for short viral RNA molecules in infected cells revealed by next-generation sequencing - PubMed (original) (raw)

Preference of RIG-I for short viral RNA molecules in infected cells revealed by next-generation sequencing

Alina Baum et al. Proc Natl Acad Sci U S A. 2010.

Erratum in

Proc Natl Acad Sci U S A. 2011 Feb 15;108(7):3092

Abstract

Intracellular detection of virus infections is a critical component of innate immunity carried out by molecules known as pathogen recognition receptors (PRRs). Activation of PRRs by their respective pathogen-associated molecular patterns (PAMPs) leads to production of proinflamatory cytokines, including type I IFN, and the establishment of an antiviral state in the host. Out of all PRRs found to date, retinoic acid inducible gene I (RIG-I) has been shown to play a key role in recognition of RNA viruses. On the basis of in vitro and transfection studies, 5'ppp RNA produced during virus replication is thought to bind and activate this important sensor. However, the nature of RNA molecules that interact with endogenous RIG-I during the course of viral infection has not been determined. In this work we use next-generation RNA sequencing to show that RIG-I preferentially associates with shorter, 5'ppp containing viral RNA molecules in infected cells. We found that during Sendai infection RIG-I specifically bound the genome of the defective interfering (DI) particle and did not bind the full-length virus genome or any other viral RNAs. In influenza-infected cells RIG-I preferentially associated with shorter genomic segments as well as subgenomic DI particles. Our analysis for the first time identifies RIG-I PAMPs under natural infection conditions and implies that full-length genomes of single segmented RNA virus families are not bound by RIG-I during infection.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Immunoprecipitation of RIG-I/RNA complexes from 24-h SeV infections and enzymatic analysis of RIG-I–associated RNA. (A) A schematic representation of the experimental procedure for characterization of RNA from RIG-I IPs. (B) Western blot analysis from RIG-I pulldowns shows the efficiency of RIG-I immunoprecipitations. (C) Immunostimulatory activity of RNA from RIG-I and control (GFP) IPs upon transfection into 293T ISRE-FF cells. (D) Enrichment of immunostimulatory RNA with RIG-I IP compared with total RNA from SeV-infected cells.

Fig. 2.

(A) RNAseA treatment of RIG-I bound RNA as well as the control RNAs completely abolishes their immunostimulatory activity. (B) CIP treatment of RIG-I bound RNA as well as flu RNA and RNA from SeV-infected cells but not poly(I:C) leads to loss of all immunostimulatory activity. (C) TAP treatment of RIG-I bound RNA and purified SeV virus RNA unlike poly(I:C) leads to a complete loss in immunostimulatory activity. (D) Agilent mRNA chip of RIG-I–associated RNA reveals a distinct 550-nt band not present in the control IP.

Fig. 3.

Deep sequencing analysis of RIG-I–associated and control IP RNA from SeV virus-infected cells. (A) RNA from RIG-I IP and control (GFP) IP and total RNA from SeV-infected cells (blue, red, and teal, respectively) were subjected to Illumina deep sequencing. Obtained sequencing reads are mapped to their starting position on the virus genome; the y axis shows the number of sequences mapped to a particular position. (Upper) All sequences mapped to the entire genome are shown. (Lower) The last 484 nt are removed to allow better visualization of the rest of the genome (note the difference in the y scale between Upper and Lower). (B) Sequencing reads mapped to the genome of the DI particle show enrichment for RIG-I–associated sequences throughout the entire DI molecule. (C) Comparison of numbers of sequences that map to the DI genome or the rest of the SeV genome in the RIG-I and control (GFP) IPs. (D) Induction of ISRE-FF reporter by transfection of T7 SeV DI RNA compared with mock transfected cells.

Fig. 4.

Immunoprecipitation and deep sequencing analysis of RIG-I–associated RNA from 24-h influenza virus infections. (A) Sequencing reads from RIG-I and control (GFP) IPs were mapped to the individual PR8 influenza virus genomic segments. Each peak corresponds to the beginning position of that particular read and the number of sequences starting at that position is represented on the y axis. RIG-I enrichment (blue) can be observed on all genomic segments. (B) Transfection of RIG-I–associated RNA into the ISRE-FF reporter cells shows high immunostimulatory activity of this RNA compared with the control (GFP) IP.

Fig. 5.

Analysis of average local enrichment of influenza virus RNA in RIG-I versus control (GFP) immunoprecipitations. For each genomic segment ratios of RIG-I/GFP sequencing reads were calculated at each nucleotide position. The average of these ratios for every 100 nt was calculated and is shown.

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Preference of RIG-I for short viral RNA molecules in infected cells revealed by next-generation sequencing - PubMed (original) (raw)