HIV 1 exploits innate signaling by TLR8 and DC SIGN for productive infection of dendritic cells

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1 HIV 1 exploits innate signaling by TLR8 and DC SIGN for productive infection of dendritic cells Sonja I Gringhuis 1,2*, Michiel van der Vlist 1,2*, Linda M van den Berg 1,2, Jeroen den Dunnen 2,3, Manja Litjens 2 and Teunis B H Geijtenbeek 1,2 1 Center of Infection and Immunity Amsterdam and Center for Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam,The Netherlands; 2 Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands; 3 Present address: Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. * These authors contributed equally to this work. 1

2 Supplementary Figure 1 Supplementary Figure 1 Silencing of Raf-1 in DCs by RNA interference and Raf-1 inhibition by GW5074. (a,b) Silencing of Raf-1 was verified by quantitative real-time PCR or flow cytometry at 72 h after transfection. (c-f) Cell viability and apoptosis after Raf-1 silencing (c,d) or Raf-1 inhibition with GW5074 (e,f) were determined by Cell-Titer Glo assay (c,e) and caspase 3/7 activity (d,f), respectively. Staurosporine (10 μm; Calbiochem) was used as a positive control. Data are presented as mean ± s.d. of four independent experiments (a) or representative for two (c-f) or four (b) independent experiments. 2

3 Supplementary Figure 2 Supplementary Figure 2 HIV-1 LAI induces Raf-1 activation. Raf-1 phosphorylation at Ser338 or Tyr was measured by flow cytometry in unstimulated DCs (thin line) or stimulated for 10 min with HIV-1 LAI in the absence (filled histogram) or presence (thick line) of blocking antibodies. Data are representative for 4 independent experiments. 3

4 Supplementary Figure 3 Supplementary Figure 3 Silencing of MyD88, TLR7 and TLR8 in DCs by RNA interference. (a-d) Silencing of target genes in MyD88-, TLR7-, TLR8- or control-silenced DCs was verified by quantitative real-time PCR or flow cytometry at 72 h after transfection. (e) Functional silencing in TLR7-silenced DCs was verified by stimulation with TLR7- specific ligand R837. Cytokine mrna expression was determined by quantitative real-time PCR after 6 h stimulation. Expression is normalized to GAPDH. Data are presented as mean ± s.d. of two (e) or four (a,c) independent experiments or representative for at least four independent experiments (b,d). 4

5 Supplementary Figure 4 Supplementary Figure 4 Co-infections enhance HIV-1 replication via Raf-1 signaling pathways in myeloid DCs. DC-SIGN + myeloid DCs were co-infected for 6 h with HIV-1 BaL in combination with Mycobacterium tuberculosis or Candida albicans in the presence or absence of Raf-1 inhibitor GW5074 and Tat-Rev mrna was determined by quantitative realtime PCR. mrna expression is normalized to GAPDH. Data are presented as mean ± s.d. of two independent experiments. 5

6 Supplementary Table 1 Expression primer sequences Gene product Forward primer Reverse primer TNF CCATGTTCGTCATGGGTGTG GGTGCTAAGCAGTTGGTGGTG IL-10 GAGGCTACGGCGCTGTCAT CCACGGCCTTGCTCTTGTT IFN-β ACAGACTTACAGGTTACCTCCGAAAC CATCTGCTGGTTGAAGAATGCTT Tat-Rev ATGGCAGGAAGAAGCGGAG ATTCCTTCGGGCCTGTCG Vpu TCTCTCGACGCAGGACTCG TCTGATGAGCTCTTCGTCGC Gag GTGTGGAAAATCTCTAGCAGTGG CGCTCTCGCACCCATCTC GAPDH CCATGTTCGTCATGGGTGTG GGTGCTAAGCAGTTGGTGGTG Abortive HIV-1 RNA GGGTCTCTCTGGTTAGACCAGATC GGTTCCCTAGTTAGCCAGAGAGC 18S TCGAGGCCCTGTAATTGGAA CCCTCCAATGGATCCTCGTT ChIP primer sequences Gene product Forward primer Reverse primer HIV-1 LTR CGAGAGCTGCATCCGGAGTA GAGGCTTAAGCAGTGGGTTCC HIV GACGCAGGACTCGGCTTG TGGCGTACTCACCAGTCGC HIV TGAGGGACAATTGGAGAAGTGA TCTGCACCACTCTTCTCTTTGC HIV AGTTGTCCCCCTAACGGACAC TCCCGAATCCTGCAAAGC HIV GCCTTAGGCATCTCCTATGGC GATGAGCTCTTCGTCGCTGTC GAPDH TACTAGCGGTTTTACGGGCG TCGAACAGGAGGAGCAGAGAGCGA 6