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. 2011 Sep 11;7(10):712-9.
doi: 10.1038/nchembio.645.

Chemical inhibition of RNA viruses reveals REDD1 as a host defense factor

Miguel A Mata  1 , Neal Satterly, Gijs A Versteeg, Doug Frantz, Shuguang Wei, Noelle Williams, Mirco Schmolke, Samuel Peña-Llopis, James Brugarolas, Christian V Forst, Michael A White, Adolfo García-Sastre, Michael G Roth, Beatriz M A Fontoura
Affiliations

Chemical inhibition of RNA viruses reveals REDD1 as a host defense factor

Miguel A Mata et al. Nat Chem Biol. .

Abstract

A chemical genetics approach was taken to identify inhibitors of NS1, a major influenza A virus virulence factor that inhibits host gene expression. A high-throughput screen of 200,000 synthetic compounds identified small molecules that reversed NS1-mediated inhibition of host gene expression. A counterscreen for suppression of influenza virus cytotoxicity identified naphthalimides that inhibited replication of influenza virus and vesicular stomatitis virus (VSV). The mechanism of action occurs through activation of REDD1 expression and concomitant inhibition of mammalian target of rapamycin complex 1 (mTORC1) via TSC1-TSC2 complex. The antiviral activity of naphthalimides was abolished in REDD1(-/-) cells. Inhibition of REDD1 expression by viruses resulted in activation of the mTORC1 pathway. REDD1(-/-) cells prematurely upregulated viral proteins via mTORC1 activation and were permissive to virus replication. In contrast, cells conditionally expressing high concentrations of REDD1 downregulated the amount of viral protein. Thus, REDD1 is a new host defense factor, and chemical activation of REDD1 expression represents a potent antiviral intervention strategy.

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Conflict of interest statement

Authors declare no competing financial interest.

Figures

Figure 1
Figure 1. Identification of Small Molecules that Revert the Inhibition of Gene Expression Mediated by the Influenza Virus NS1 Protein and that Protect Cells from Virus-Induced Cell Death
(a) Luciferase expression in 293T cells transfected with NS1 and treated individually with 200,000 synthetic compounds (5 μM) was normalized to on-plate controls treated with 0.3% DMSO. Values are expressed as z scores, using the mean value and standard deviation of the experimental population screened on the same day. Red circle shows compound 1 studied here. (b) The most active 640 compounds were tested at three concentrations for the ability to inhibit the cytopathic effect of A/WSN/1933 influenza virus infection in HBECs. z scores for compounds assayed at 1.7 μM are plotted according to activity. (c) The structure of the most active naphthalimide from the primary screen, compound 1, an inactive analog, 2, and a more potent related compound, 3, are shown.
Figure 2
Figure 2. Compound 3 Is Less Cytotoxic, More Stable than 1, and Reverts Influenza Virus-Mediated Cytotoxicity and mRNA Export Block
(a) MDCK cells were treated for 30 h with compounds 1, 2, and 3 at various depicted concentrations and control cells were treated with the same concentration of DMSO as in the wells containing compound. Cell viability was determined by measuring cell ATP concentrations. (b) The fraction of compound remaining in cells treated with 1 or 3 as a function of incubation time was determined by mass spectrometry. (c) MDCK cells were pre-treated for 17 h with DMSO or with the indicated concentrations of 3 and subsequently mock infected or infected with A/WSN/1933 virus at m.o.i. 0.001 for 48 h. Indicated concentrations of compound were present during infection. DIC imaging was performed in a Zeiss Axiovert 200M. Bar, 90 μM. Cell survival was determined by counting live cells. (d) MDCK cells, mock-infected or infected with A/WSN/1933 in the presence or absence of 25 μM 3, were fixed and subjected to oligo-dT in situ hybridization to detect poly(A) RNA distribution in the nucleus and in the cytoplasm. Influenza proteins were detected by immunofluorescence using antibodies against influenza proteins. Yellow arrowheads point to cells with mRNA export block whereas white arrowheads point to cells that do not show blockage. Bar, 15 μM. (e) Data from triplicate experiments as depicted in (d) were quantified and the percentage of infected cells retaining mRNA in the nucleus is shown. Data represent mean values +/− s.d.
Figure 3
Figure 3. Compound 3 Inhibits Virus Replication but Does Not Induce Interferon Response
(a–c) MDCK cells mock infected or infected with the influenza virus strains shown, at m.o.i. 0.001, were untreated or treated with compounds, at the depicted concentrations, and the virus titers of culture supernatants were determined by plaque assay. (d) Levels of intracellular viral proteins were measured by immunoblot analysis with specific antibodies to the indicated proteins. (e) Human A549 cells treated with DMSO or 25 μM 3 were mock infected or infected with A/WSN/1933 at m.o.i. 0.001 and after 36 h, RNA was isolated and the expression of the interferon-responsive genes shown was quantified by real-time RT-PCR. (f) MDCK cells mock infected or infected with VSV-GFP (m.o.i = 0.001) were untreated or treated with the indicated compounds. At 24 h post-infection, virus titers were determined in the supernatants. Data represent mean values +/− s.d.
Figure 4
Figure 4. Influenza Virus Activated the mTORC1 Pathway and Naphthalimide Required the mTORC1 Inhibitor REDD1 for its Antiviral Activity
(a) A549 cells were untreated or treated with 30 μM 3 for the indicated time periods, in the absence or presence of actinomycin D (0.5 μg/ml). REDD1 mRNA levels were quantified by real time RT-PCR. (b) A549 cells were untreated or treated with 30 μM 3 (in the absence or presence of 0.5 μg/ml actinomycin D as indicated) for 18 h prior to infection and during infection. Cell extracts were obtained at 6 h post-infection and subjected to immunoblot analysis with the indicated antibodies. Densitometry analysis was performed to determine the ratio of REDD1 over loading control (Mito-70 kD) using ImageJ (Supplementary Results, Fig. S18). (c) A549 cells were untreated or treated as in (b) prior to infection and during infection. Cell extracts were subjected to immunoblot analysis with depicted antibodies (Supplementary Results, Fig. S19). (d) Phosphorylation of Akt or S6K was measured by immunoblot analysis in cell extracts of A549 cells infected with influenza virus in the presence or absence of 3. Compound was added prior and during infection as in (b) (Supplementary Results, Fig. S20). (e and f) REDD1+/+ cells were untreated or treated with 3 and mock-infected or infected at m.o.i 0.01 with A/WSN/1933 for 72 h. REDD1−/− MEFs, untreated or treated with 3, were infected with A/WSN/1933 at m.o.i. 0.001 for 48 h. Cell survival was determined by trypan blue exclusion assay and virus titers were measured by plaque assays. Low exp, low exposure; High exp, high exposure.
Figure 5
Figure 5. Viruses Activate the mTORC1 pathway via down-regulation of REDD1 expression
Extracts from cells mock infected or infected with influenza virus (a) or VSV-GFP (b) were subjected to immunoblot analysis with depicted antibodies. Densitometry analysis was performed to determine the ratio of REDD1 over loading control (Mito-70 kD) using ImageJ. (c) Wild-type or REDD1−/− MEFs were infected with VSV-GFP at m.o.i. of 0.001 for 24 h. DIC or fluorescent images of VSV-GFP are shown. Bar, 50 μM. (d) Supernatants of cells from (c) were subjected to plaque assays.
Figure 6
Figure 6. REDD1 regulates viral protein expression in a mTORC1 dependent manner
(a) REDD1+/+ and REDD1−/− cells were infected with influenza virus WSN at m.o.i. 2 for 1 h at 22 °C and then shifted to 37 °C. Viral protein levels were monitored over time by immunoblot analysis with the depicted antibodies. (b) Viral protein levels were monitored as in (a). (c) WSN-infected REDD1+/+ and REDD1−/− cells were treated with 100nM Rapamycin. Rapamycin was added one 1 h post-infection. NS1 levels were monitored over time by immunoblot analysis. (d) U2OS cells, untreated or treated with tetracycline to induce REDD1 overexpression, were infected as in (a) but with both influenza virus or VSV. NS1 or VSV-M protein levels were monitored by immunoblot analysis. (e and f) TSC2+/+ and TSC2−/− cells were pre-treated with 10 μM 3. Cells were then infected with influenza virus WSN at m.o.i. 2 for 1 h at 22 °C and then shifted to 37 °C in the absence of compound. After 1 hour post-infection, 3 was added back. Cell extracts were obtained at the depicted time points and subjected to immunoblot analysis with the indicated antibodies.
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