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HIV-1 blocks the signaling adaptor MAVS to evade antiviral host defense after sensing of abortive HIV-1 RNA by the host helicase DDX3

Nature Immunology volume 18, pages 225–235 (2017)Cite this article

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An Erratum to this article was published on 22 March 2017

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Abstract

The mechanisms by which human immunodeficiency virus 1 (HIV-1) avoids immune surveillance by dendritic cells (DCs), and thereby prevents protective adaptive immune responses, remain poorly understood. Here we showed that HIV-1 actively arrested antiviral immune responses by DCs, which contributed to efficient HIV-1 replication in infected individuals. We identified the RNA helicase DDX3 as an HIV-1 sensor that bound abortive HIV-1 RNA after HIV-1 infection and induced DC maturation and type I interferon responses via the signaling adaptor MAVS. Notably, HIV-1 recognition by the C-type lectin receptor DC-SIGN activated the mitotic kinase PLK1, which suppressed signaling downstream of MAVS, thereby interfering with intrinsic host defense during HIV-1 infection. Finally, we showed that PLK1-mediated suppression of DDX3–MAVS signaling was a viral strategy that accelerated HIV-1 replication in infected individuals.

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Figure 1: Raf-1 activation suppresses type I IFN responses after HIV-1 infection of DCs.
Figure 2: Type I interferon responses after HIV-1 infection are mediated by DDX3–MAVS.
Figure 3: DDX3 bound to capped abortive HIV-1 RNA products induces type I interferon responses via MAVS.
Figure 4: HIV-1 infection blocks TRAF3 recruitment to MAVS and subsequent IRF3 activation.
Figure 5: PLK1 activation during HIV-1 infection impedes TRAF3 recruitment to DDX3–MAVS and type I interferon responses.
Figure 6: Type I interferon responses by DCs suppress HIV-1 replication in infected individuals.
Figure 7: PLK1-mediated suppression of DDX3 signaling after HIV-1 infection is impeded by mutation of MAVS.
Figure 8: HIV-1 infection attenuates DDX3–MAVS-mediated DC maturation.

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  • 23 January 2017

    In the version of this article initially published, the fifth author's surname was spelled incorrectly (as 'Sarrami-Fooroshani'). The correct spelling is 'Sarrami-Forooshani'. Also, the cells for the third plot in Figure 1d were identified incorrectly as 'Intesrinal DCs'. The correct label is 'Intestinal DCs'. The errors have been corrected in the PDF and HTML versions of this article.

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Acknowledgements

We thank L. Westera, C. Buskens and W. Bemelman (AMC, Amsterdam, the Netherlands) for the intestinal tissue cell suspensions and C.L. Verweij (VUmc, Amsterdam, the Netherlands) for the IFNB, ISG15 and Mx2 primer sequences. HIV-2 virus, MT-2 cells, AZT and gp120 were obtained through the NIH AIDS Research and Reference Reagent Program. Supported by Aids Fonds (2012042; S.I.G.), the Netherlands Organization for Scientific Research (NWO) VICI 918.10.619; T.B.H.G.) and the European Research Council (Advanced grant 670424; T.B.H.G.).

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Authors and Affiliations

  1. Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands

    Sonja I Gringhuis, Nina Hertoghs, Tanja M Kaptein, Esther M Zijlstra-Willems, Ramin Sarrami-Forooshani, Joris K Sprokholt, Nienke H van Teijlingen, Neeltje A Kootstra, Thijs Booiman, Karel A van Dort, Carla M S Ribeiro, Agata Drewniak & Teunis B H Geijtenbeek

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Contributions

S.I.G. designed and supervised research, and performed experiments; N.H., T.M.K., E.M.Z.-W., R.S.-F., J.K.S., N.H.v.T., C.M.S.R. and A.D. performed experiments; N.H. generated the 293T CRISPR knockout cell lines; N.A.K. and T.B. provided data from the Amsterdam cohort studies (ACS) on HIV-1 infection and AIDS; K.A.v.D. assisted with virus isolation; and S.I.G. and T.B.H.G. interpreted results and wrote the paper.

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Correspondence to Sonja I Gringhuis or Teunis B H Geijtenbeek.

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Integrated supplementary information

Supplementary Figure 1 Raf-1 inhibition does not affect TLR4-induced type I interferon responses.

Real-time PCR (RT-PCR) analyses of IFNB, ISG15, TRIM5, TRIM22 and APOBEC3G mRNA in moDCs 6 h after stimulation with TLR4 ligand LPS, in the absence or presence of Raf-1 inhibitor GW5074. Mean ± SD; n = 3.

Supplementary Figure 2 DDX3 sensing pathway is independent from RIG-I–MDA5 and cGAS pathways in DCs.

(a-d) RT-PCR analyses of IFNB mRNA in moDCs 4 h after infection with HIV-1BaL (a,b) or stimulation with RIG-I–MDA5 ligand poly(I:C)-LyoVec, TLR4 ligand LPS (d), cGAS ligand HSV DNA coupled to LyoVec or STING ligand 3’3’-cGAMP (d), in the absence or presence of Raf-1 inhibitor GW5074 (a,b), reverse transcription inhibitor AZT or integrase inhibitor raltegravir (RAL) (b) and after silencing (siRNA) of indicated proteins (a-d). Mean ± SD; n = 4 (a,c,d), 3 (b). ** p < 0.01, * p < 0.05 (Student’s t-test). The data shown in (b) are an addendum to the data presented in Fig. 2b.

Supplementary Figure 3 Depletion of protein expression in human 293T cells by CRISPR–Cas9-directed genome editing.

Immunoblot (IB) analyses of depletion (Δ) of indicated proteins by transfection of 293T cells with control or specific guide RNA and Cas9-expressing plasmids.

Supplementary Figure 4 Raf-1 and MST1 but not PLK1 inhibit HIV-1 transcription, whereas Raf-1 blocks HIV-2 but not HTLV-1 transcription.

(a,b) RT-PCR analyses of tat-rev (HIV-1, HIV-2) or tax-rex (HTLV-1) mRNA in moDCs 6 h after infection with HIV-1BaL (a), HIV-2 or HTLV-1 (b), in the absence or presence of Raf-1 inhibitor GW5074 (a,b), and after silencing (siRNA) of Raf-1, PLK1 or MST1 (b). Mean ± SD; n = 6 (a), 2 (b).

Supplementary Figure 5 IRF3 activation is suppressed by a MST1–PLK1 cascade after HIV-1 infection.

Immunoblot (IB) analyses of Ser396 phosphorylation of IRF3 in nuclear extracts (NE) of moDCs 3 h after HIV-1BaL infection, after silencing (siRNA) of MST1 or PLK1. RNA polymerase II (RNAP2) served as loading control. Representative of 2 independent experiments.

Supplementary Figure 6

(a-c) Flow cytometry analyses of DDX3 (a) or MAVS (b,c) expression in control-, DDX3- or MAVS-silenced (siRNA) moDCs 48 h after transfection of plasmids with RNAi-resistant cDNAs encoding wild-type DDX3 (a) or wild-type MAVS (b,c). In (c), minor genotype moDCs were transfected. FI, fluorescence intensity; FSC, forward scatter. Representative of 4 (a,b) or 3 (c) independent experiments. (d) RT-PCR analyses of IFNB mRNA in control- or MAVS-silenced minor genotype moDCs 4 h after infection with HIV-1BaL, in the absence or presence of Raf-1 inhibitor GW5074, after complementation of MAVS expression via transfection of plasmids with RNAi-resistant cDNA encoding wild-type (QS, Q198,S409) MAVS. Mean ± SD; n = 4. ** p < 0.01, * p < 0.05 (Student’s t-test).

Supplementary Figure 7 DC-SIGN signaling via Raf-1–MST1–PLK1 suppresses signaling downstream of MAVS after sensing of abortive HIV-1 transcripts by the RNA helicase DDX3.

DC-SIGN signaling via Raf-1-MST1-PLK1 suppresses signaling downstream of MAVS after sensing of abortive HIV-1 transcripts by RNA helicase DDX3.

Supplementary Figure 8 Silencing of protein expression in human DCs by RNA interference.

(a-q) Realtime PCR (left panels), flow cytometry (middle panels) and immunoblot (right panels) analsyes of indicated mRNA and proteins in moDCs silenced for indicated proteins using specific siRNAs and non-targeting siRNA as a control (left panels; mean ± SD, n ≥ 7; middle panels; FI, fluorescence intensity; representative of at least 3 independent experiments; right panels; representative of 2 independent experiments).

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Gringhuis, S., Hertoghs, N., Kaptein, T. et al. HIV-1 blocks the signaling adaptor MAVS to evade antiviral host defense after sensing of abortive HIV-1 RNA by the host helicase DDX3. Nat Immunol 18, 225–235 (2017). https://doi.org/10.1038/ni.3647

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