TRIM5 is an innate immune sensor for the retrovirus capsid lattice

Abstract

TRIM5 is a RING domain-E3 ubiquitin ligase that restricts infection by human immunodeficiency virus (HIV)-1 and other retroviruses immediately following virus invasion of the target cell cytoplasm. Antiviral potency correlates with TRIM5 avidity for the retrovirion capsid lattice and several reports indicate that TRIM5 has a role in signal transduction, but the precise mechanism of restriction is unknown. Here we demonstrate that TRIM5 promotes innate immune signalling and that this activity is amplified by retroviral infection and interaction with the capsid lattice. Acting with the heterodimeric, ubiquitin-conjugating enzyme UBC13-UEV1A (also known as UBE2N-UBE2V1), TRIM5 catalyses the synthesis of unattached K63-linked ubiquitin chains that activate the TAK1 (also known as MAP3K7) kinase complex and stimulate AP-1 and NFκB signalling. Interaction with the HIV-1 capsid lattice greatly enhances the UBC13-UEV1A-dependent E3 activity of TRIM5 and challenge with retroviruses induces the transcription of AP-1 and NF-κB-dependent factors with a magnitude that tracks with TRIM5 avidity for the invading capsid. Finally, TAK1 and UBC13-UEV1A contribute to capsid-specific restriction by TRIM5. Thus, the retroviral restriction factor TRIM5 has two additional activities that are linked to restriction: it constitutively promotes innate immune signalling and it acts as a pattern recognition receptor specific for the retrovirus capsid lattice.

Figure 1. TRIM5 promotes innate immune signaling

a –c, HEK-293 cells transfected with the indicated pcDNA-based expression plasmids and luciferase reporters for AP-1 (a), NF-κB (b) or IFNB1 (c). Bars show mean luciferase activity +/− s.d. (n = 6). d, Global expression profile comparing TRIM5 KD to control KD THP-1 macrophages. Triangles indicate inflammatory genes significantly downregulated in TRIM5 KD. e, qRT-PCR for the indicated mRNAs harvested 2 to 8 hrs after LPS-treatment, depending on the peak values for that gene. Shown are the means +/− s.e.m (n = 3) relative to untreated cells. f, Concentration of the indicated proteins in the culture supernatant, 24 hrs after LPS-treatment (mean +/− s.d., n = 3). RNA and protein data are representative of at least 3 separate donors. g–j, THP-1 macrophages transduced with miR30-based lentivirus KD vectors targeting either TRIM5 (g and i), IRF3 (h), or STAT2 (i), were treated 24 hrs with the indicated compounds and challenged with VSV-G pseudotyped HIV-1 luciferase reporter virus (g–i) or with the indicated GFP reporter viruses (j). Data are expressed as fold-change compared to control KD cells, with s.e.m (n = 4). All data are representative of at least 3 independent experiments.

Figure 2. The TAK1 kinase complex interacts biochemically and functionally with TRIM5

a, HEK-293T cells were co-transfected with Myc-tagged human TRIM5α and the indicated FLAG-tagged constructs. Shown are immunoblots with anti-Myc antibody after immunoprecipitation with anti-FLAG (upper panel), or of total cell lysate (bottom panel). b, c, and f, HEK-293 cells were transfected with the indicated pcDNA-based expression plasmids and an AP-1 luciferase reporter and show the effect of TAK1 inhibitor 5z-7-oxozeaenol (b) or TAK1 KD (c). TAK1 KD and control KD THP-1 macrophages were treated with LPS for the indicated times and immunoblotted with anti-TAK1 antibody (lower panel) or anti-phospho-TAK1 antibody (upper panel) (d), or, cells were treated 24 hrs with LPS or vehicle and challenged with an HIV-1 luciferase reporter virus (e). The results in (e) are reported as fold rescue due to TAK1 KD, with respect to control KD. g and h, THP-1 cells were transduced with lentiviral vectors encoding owl monkey TRIM5Cyp, either wild-type or the H436Q mutant. Pools of each were then transduced with lentiviral KD vectors targeting either TAK1, UBC13, or control, and challenged with an HIV-1-GFP reporter vector. Infectivity was monitored by FACS (g) or by PCR for synthesis of full-length viral cDNA (h). i, HT1080 cells were transfected with dsRNA oligonucleotides targeting TRIM5, TAK1, or UEV1A and challenged with EIAV-GFP reporter vector.

Figure 3. TRIM5 acts with UBC13/UEV1A to synthesize free K63-linked Ub chains that activate TAK1

a –c, e and f, HEK-293 cells were transfected with an AP-1 luciferase reporter and the indicated pcDNA-based expression plasmids. Bars show mean +/− s.d. (n = 6). In c, HEK-293 cells had stable UBC13 KD or control KD. d, UBC13 KD or control KD THP-1 macrophages were treated for 24 hrs with LPS or vehicle and challenged with an HIV-1 luciferase reporter virus. Shown is the fold rescue due to TRIM5 KD, with respect to the control KD. g–j, Products of in vitro reactions with ATP, Ub, UBE1, UBC13/UEV1A, and the indicated E3 Ub ligases were revealed by immunoblot for total Ub (g, i, and j), K63-linked Ub chains (left panel of h), or TRIM5 (right panel of h). E3 ubiquitin ligases included purified owl monkey TRIM5Cyp (g, h, and j), or the indicated FLAG-tagged proteins immunoprecipitated from HEK-293T cells (i). j, In vitro Ub reactions like those in (i) were incubated with purified TAK1 kinase complex. Products were probed in immunoblot with the indicated antibodies.

Figure 4. Retrovirus capsid sensing by TRIM5

THP-1 cells (a and b), MDDCs (c and d), or owl monkey kidney cells (e and f), were challenged with matched pairs of VSV G-pseudotyped particles bearing retrovirion capsids that are restricted by the TRIM5 orthologue endogenous to that cell type (black bars), or unrestricted (white bars), or VSV G–derived particles that are devoid of capsid (gray bars). Restricted capsids were from N-tropic MLV (a–d) or HIV-1 (e and f). Unrestricted capsids were B-tropic MLV (a and b), N/B-tropic MLV (c and d) or SIV_MAC239 (e and f). Particles bore viral genomes in a, b, e, and f, but not in c and d. mRNA was harvested for qRT-PCR (a–c and e) and reported as fold change versus media control. Protein in the supernatant was quantitated (d and f). Bars show means +/− s.d. (n = 3), and are representative of at least 3 independent experiments. g, Immunoblots with the indicated antibodies of products from in vitro time-course with ATP, Ub, UBE1 (E1), UBC13/UEV1A (E2), and purified owl monkey TRIM5Cyp, with or without assembled HIV-1 capsid-A14C/E45C, and with or without competitive inhibitor MeIle4CsA. (h) Schematic showing entry of an HIV-1 virion core (courtesy of Pornillos and Yeager) into the target cell cytoplasm where it induces dimeric TRIM5 to form a hexameric lattice with increased E3 Ub ligase activity. With UBC13/UEV1A, TRIM5 synthesizes free K63 Ub chains that are recognized by TAB2, which multimerizes and activates the TAK1 kinase complex.