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RIG-I and dsRNA-induced IFNbeta activation.

Hausmann S, Marq JB, Tapparel C, Kolakofsky D, Garcin D - PLoS ONE (2008)

Bottom Line: Consistent with this, most viruses require RIG-I to mount an innate immune response, whereas picornaviruses require mda-5.We have examined a SeV infection whose ability to induce interferon depends on the generation of capped dsRNA (without free 5' tri-phosphate ends), and found that this infection as well requires RIG-I and not mda-5.We also provide evidence that RIG-I interacts with poly-I/C in vivo, and that heteropolymeric dsRNA and poly-I/C interact directly with RIG-I in vitro, but in different ways; i.e., poly-I/C has the unique ability to stimulate the helicase ATPase of RIG-I variants which lack the C-terminal regulatory domain.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Medicine, University of Geneva Medical School, Geneva, Switzerland.

ABSTRACT
Except for viruses that initiate RNA synthesis with a protein primer (e.g., picornaviruses), most RNA viruses initiate RNA synthesis with an NTP, and at least some of their viral (ppp)RNAs remain unblocked during the infection. Consistent with this, most viruses require RIG-I to mount an innate immune response, whereas picornaviruses require mda-5. We have examined a SeV infection whose ability to induce interferon depends on the generation of capped dsRNA (without free 5' tri-phosphate ends), and found that this infection as well requires RIG-I and not mda-5. We also provide evidence that RIG-I interacts with poly-I/C in vivo, and that heteropolymeric dsRNA and poly-I/C interact directly with RIG-I in vitro, but in different ways; i.e., poly-I/C has the unique ability to stimulate the helicase ATPase of RIG-I variants which lack the C-terminal regulatory domain.

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Poly-I/C and pppRNA induced activation of endogenous IFNβ in helicase-deficient MEFs.RIG-I−/− (panel A) and mda-5−/− MEFs (panel B) were transfected with either 5 ug of poly-I/C, 3 ug of pppRNA1 (55 nt) or empty transfection mix (ctrl) as indicated. Cell extracts were prepared at 20 hpt, and the relative levels of endogenous IFNβ mRNA were determined by qRT/PCR (Methods). The cell supernatants of the transfected cells were also harvested at this time, and used to pretreat wt MEFs for 12 h before infection with VSV-GFP. The VSV infected cells were harvested at 12 hpi and analyzed by FACS for GFP expression (panels C and D).
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pone-0003965-g004: Poly-I/C and pppRNA induced activation of endogenous IFNβ in helicase-deficient MEFs.RIG-I−/− (panel A) and mda-5−/− MEFs (panel B) were transfected with either 5 ug of poly-I/C, 3 ug of pppRNA1 (55 nt) or empty transfection mix (ctrl) as indicated. Cell extracts were prepared at 20 hpt, and the relative levels of endogenous IFNβ mRNA were determined by qRT/PCR (Methods). The cell supernatants of the transfected cells were also harvested at this time, and used to pretreat wt MEFs for 12 h before infection with VSV-GFP. The VSV infected cells were harvested at 12 hpi and analyzed by FACS for GFP expression (panels C and D).

Mentions: Although the helicase-deficient MEFs transfect poorly with plasmid DNA, they appear to be more efficiently transfected with either relatively small poly-I/C (400 bp on average) or pppRNA (55 nt)(fig 4). When RIG-I−/− MEFs are transfected with poly-I/C or pppRNA, the level of endogenous IFNβ mRNA increases only in response to poly-I/C as expected (fig 4A), as mda-5 does not respond to pppRNA [12]. The increased IFNβ mRNA apparently leads to the secretion of IFN, as pretreatment of wt MEFs with the supernatants from the above experiment efficiently prevented the growth of VSV-GFP in these cells only when poly-I/C had been transfected (fig 4C). In contrast, when mda-5−/− MEFs are transfected with poly-I/C or pppRNA, the level of endogenous IFNβ mRNA increases in response to poly-I/C as well as to pppRNA (fig 4B), and the supernatants from both these transfections have the capacity to inhibit VSV-GFP replication when used to pretreat other MEFs (fig 4D). These results further indicate that RIG-I responds to poly-I/C as well as to pppRNA.


RIG-I and dsRNA-induced IFNbeta activation.

Hausmann S, Marq JB, Tapparel C, Kolakofsky D, Garcin D - PLoS ONE (2008)

Poly-I/C and pppRNA induced activation of endogenous IFNβ in helicase-deficient MEFs.RIG-I−/− (panel A) and mda-5−/− MEFs (panel B) were transfected with either 5 ug of poly-I/C, 3 ug of pppRNA1 (55 nt) or empty transfection mix (ctrl) as indicated. Cell extracts were prepared at 20 hpt, and the relative levels of endogenous IFNβ mRNA were determined by qRT/PCR (Methods). The cell supernatants of the transfected cells were also harvested at this time, and used to pretreat wt MEFs for 12 h before infection with VSV-GFP. The VSV infected cells were harvested at 12 hpi and analyzed by FACS for GFP expression (panels C and D).
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Related In: Results  -  Collection

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pone-0003965-g004: Poly-I/C and pppRNA induced activation of endogenous IFNβ in helicase-deficient MEFs.RIG-I−/− (panel A) and mda-5−/− MEFs (panel B) were transfected with either 5 ug of poly-I/C, 3 ug of pppRNA1 (55 nt) or empty transfection mix (ctrl) as indicated. Cell extracts were prepared at 20 hpt, and the relative levels of endogenous IFNβ mRNA were determined by qRT/PCR (Methods). The cell supernatants of the transfected cells were also harvested at this time, and used to pretreat wt MEFs for 12 h before infection with VSV-GFP. The VSV infected cells were harvested at 12 hpi and analyzed by FACS for GFP expression (panels C and D).
Mentions: Although the helicase-deficient MEFs transfect poorly with plasmid DNA, they appear to be more efficiently transfected with either relatively small poly-I/C (400 bp on average) or pppRNA (55 nt)(fig 4). When RIG-I−/− MEFs are transfected with poly-I/C or pppRNA, the level of endogenous IFNβ mRNA increases only in response to poly-I/C as expected (fig 4A), as mda-5 does not respond to pppRNA [12]. The increased IFNβ mRNA apparently leads to the secretion of IFN, as pretreatment of wt MEFs with the supernatants from the above experiment efficiently prevented the growth of VSV-GFP in these cells only when poly-I/C had been transfected (fig 4C). In contrast, when mda-5−/− MEFs are transfected with poly-I/C or pppRNA, the level of endogenous IFNβ mRNA increases in response to poly-I/C as well as to pppRNA (fig 4B), and the supernatants from both these transfections have the capacity to inhibit VSV-GFP replication when used to pretreat other MEFs (fig 4D). These results further indicate that RIG-I responds to poly-I/C as well as to pppRNA.

Bottom Line: Consistent with this, most viruses require RIG-I to mount an innate immune response, whereas picornaviruses require mda-5.We have examined a SeV infection whose ability to induce interferon depends on the generation of capped dsRNA (without free 5' tri-phosphate ends), and found that this infection as well requires RIG-I and not mda-5.We also provide evidence that RIG-I interacts with poly-I/C in vivo, and that heteropolymeric dsRNA and poly-I/C interact directly with RIG-I in vitro, but in different ways; i.e., poly-I/C has the unique ability to stimulate the helicase ATPase of RIG-I variants which lack the C-terminal regulatory domain.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Medicine, University of Geneva Medical School, Geneva, Switzerland.

ABSTRACT
Except for viruses that initiate RNA synthesis with a protein primer (e.g., picornaviruses), most RNA viruses initiate RNA synthesis with an NTP, and at least some of their viral (ppp)RNAs remain unblocked during the infection. Consistent with this, most viruses require RIG-I to mount an innate immune response, whereas picornaviruses require mda-5. We have examined a SeV infection whose ability to induce interferon depends on the generation of capped dsRNA (without free 5' tri-phosphate ends), and found that this infection as well requires RIG-I and not mda-5. We also provide evidence that RIG-I interacts with poly-I/C in vivo, and that heteropolymeric dsRNA and poly-I/C interact directly with RIG-I in vitro, but in different ways; i.e., poly-I/C has the unique ability to stimulate the helicase ATPase of RIG-I variants which lack the C-terminal regulatory domain.

Show MeSH
Related in: MedlinePlus