The Double-Stranded RNA-Binding Protein PACT Functions as a Cellular Activator of RIG-I to Facilitate Innate Antiviral Response

Department of Biochemistry and State Key Laboratory for Liver Research, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong.
Cell host & microbe (Impact Factor: 12.33). 04/2011; 9(4):299-309. DOI: 10.1016/j.chom.2011.03.007
Source: PubMed


RIG-I, a virus sensor that triggers innate antiviral response, is a DExD/H box RNA helicase bearing structural similarity with Dicer, an RNase III-type nuclease that mediates RNA interference. Dicer requires double-stranded RNA-binding protein partners, such as PACT, for optimal activity. Here we show that PACT physically binds to the C-terminal repression domain of RIG-I and potently stimulates RIG-I-induced type I interferon production. PACT potentiates the activation of RIG-I by poly(I:C) of intermediate length. PACT also cooperates with RIG-I to sustain the activation of antiviral defense. Depletion of PACT substantially attenuates viral induction of interferons. The activation of RIG-I by PACT does not require double-stranded RNA-dependent protein kinase or Dicer, but is mediated by a direct interaction that leads to stimulation of its ATPase activity. Our findings reveal PACT as an important component in initiating and sustaining the RIG-I-dependent antiviral response.

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    • "We cannot exclude that another mechanism may be involved as well. PACT-Dicer interaction [74] or PACT induction of RIG-I upon Sendai virus infection [75] seems unlikely here because it would lead to viral restriction or enhanced innate immune response respectively, which we do not observe during HIV-1 infection or after PACT overexpression of HIV-1-expressing cells (Figures 1, 3, 4, 6). Therefore, ADAR1-PACT interaction is currently the most likely mechanism, which may contribute, at least in part, to the change in PACT activity during HIV-1 infection. "
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    ABSTRACT: HIV-1 translation is modulated by the activation of the interferon (IFN)-inducible Protein Kinase RNA-activated (PKR). PKR phosphorylates its downstream targets, including the alpha subunit of the eukaryotic translation Initiation Factor 2 (eIF2alpha), which decreases viral replication. The PKR Activator (PACT) is known to activate PKR after a cellular stress. In lymphocytic cell lines, HIV-1 activates PKR only transiently and not when cells replicate the virus at high levels. The regulation of this activation is due to a combination of viral and cellular factors that have been only partially identified. PKR is transiently induced and activated in peripheral blood mononuclear cells after HIV-1 infection. The addition of IFN reduces viral replication, and induces both the production and phosphorylation of PKR. In lymphocytic Jurkat cells infected by HIV-1, a multiprotein complex around PKR contains the double-stranded RNA binding proteins (dsRBPs), adenosine deaminase acting on RNA (ADAR)1 and PACT. In HEK 293 T cells transfected with an HIV-1 molecular clone, PACT unexpectedly inhibited PKR and eIF2alpha phosphorylation and increased HIV-1 protein expression and virion production in the presence of either endogenous PKR alone or overexpressed PKR. The comparison between different dsRBPs showed that ADAR1, TAR RNA Binding Protein (TRBP) and PACT inhibit PKR and eIF2alpha phosphorylation in HIV-infected cells, whereas Staufen1 did not. Individual or a combination of short hairpin RNAs against PACT or ADAR1 decreased HIV-1 protein expression. In the astrocytic cell line U251MG, which weakly expresses TRBP, PACT mediated an increased HIV-1 protein expression and a decreased PKR phosphorylation. In these cells, a truncated PACT, which constitutively activates PKR in non-infected cells showed no activity on either PKR or HIV-1 protein expression. Finally, PACT and ADAR1 interact with each other in the absence of RNAs. In contrast to its previously described activity, PACT contributes to PKR dephosphorylation during HIV-1 replication. This activity is in addition to its heterodimer formation with TRBP and could be due to its binding to ADAR1. HIV-1 has evolved to replicate in cells with high levels of TRBP, to induce the expression of ADAR1 and to change the function of PACT for PKR inhibition and increased replication.
    Retrovirology 09/2013; 10(1):96. DOI:10.1186/1742-4690-10-96 · 4.19 Impact Factor
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    • "In fact it is known that type I IFNs post-transcriptionally repress Dicer protein production , in contrast to IFN␥, which induces Dicer expression (Wiesen and Tomasi, 2009). Furthermore, Dicer requires dsRNA and associated proteins for optimal RNase activity which itself results in the production of type-I IFNs (Kok et al., 2011). It is therefore not surprising that Dicer gene knock-out mice exhibit aberrant IFN expression and are more susceptible to virus infection (Ostermann et al., 2012). "
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    • "Expression of VP35 Disrupts the Association of PACT with RIG-I PACT interacts with RIG-I to augment RIG-I signaling (Kok et al., 2011). To understand how VP35 interferes with PACT-mediated RIG-I signaling, we first confirmed VP35-PACT and RIG-I-PACT interaction by coimmunoprecipitation assay (Figures 3A and 3B). "
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    ABSTRACT: The cytoplasmic pattern recognition receptor RIG-I is activated by viral RNA and induces type I IFN responses to control viral replication. The cellular dsRNA binding protein PACT can also activate RIG-I. To counteract innate antiviral responses, some viruses, including Ebola virus (EBOV), encode proteins that antagonize RIG-I signaling. Here, we show that EBOV VP35 inhibits PACT-induced RIG-I ATPase activity in a dose-dependent manner. The interaction of PACT with RIG-I is disrupted by wild-type VP35, but not by VP35 mutants that are unable to bind PACT. In addition, PACT-VP35 interaction impairs the association between VP35 and the viral polymerase, thereby diminishing viral RNA synthesis and modulating EBOV replication. PACT-deficient cells are defective in IFN induction and are insensitive to VP35 function. These data support a model in which the VP35-PACT interaction is mutually antagonistic and plays a fundamental role in determining the outcome of EBOV infection.
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