Pteropus vampyrus TRIM40 Is an Interferon-Stimulated Gene That Antagonizes RIG-I-like Receptors

Abstract

Nipah virus (NiV; genus: Henipavirus; family: Paramyxoviridae) naturally infects Old World fruit bats (family Pteropodidae) without causing overt disease. Conversely, NiV infection in humans and other mammals can be lethal. Comparing bat antiviral responses with those of humans may illuminate the mechanisms that facilitate bats' tolerance. Tripartite motif proteins (TRIMs), a large family of E3-ubiquitin ligases, fine-tune innate antiviral immune responses, and two human TRIMs interact with Henipavirus proteins. We hypothesize that NiV infection induces the expression of an immunosuppressive TRIM in bat, but not human cells, to promote tolerance. Here, we show that TRIM40 is an interferon-stimulated gene (ISG) in pteropodid but not human cells. Knockdown of bat TRIM40 increases gene expression of IFNβ, ISGs, and pro-inflammatory cytokines following poly(I:C) transfection. In Pteropus vampyrus, but not human cells, NiV induces TRIM40 expression within 16 h after infection, and knockdown of TRIM40 correlates with reduced NiV titers as compared to control cells. Bats may have evolved to express TRIM40 in response to viral infections to control immunopathogenesis.

Keywords: MDA5; Nipah virus (NiV); RIG-I; RIG-I-like receptors; TRIM40; inflammatory responses in bats and humans; innate immunity; tripartite motif (TRIM) proteins; type-I interferons (IFNs).

Figure 1

Pteropodid TRIM40 is an interferon-stimulated gene. (A,B) Transfection of A549 (human lung), PVK4 (Pteropus vamyprus kidney), and ZFBK13-75 (Eidolon helvum kidney) cells with 1 μg/mL of low-molecular weight (LMW) poly(I:C). (A) Immunoblots with antibodies specific to interferon induction regulators (pTBK1 and pSTAT1) and interferon-stimulated genes (total STAT1 and RIG-I). (B) At four hours post-stimulation, RNA was collected for quantitative PCR of the TRIM40 gene. Fold induction of poly(I:C)-stimulated samples relative to mock-transfected samples is shown. (C,D) A549, PVK4, and ZFBK13-75 cells were stimulated with 500 U/mL of universal type I interferon (U-IFN). (C) Cell lysates were collected for Western blot to probe for activated STAT1 (pSTAT1 Y701) and STAT2 (pSTAT2 Y690) as well as their total protein levels. (D) At four hours post-stimulation, RNA was collected for qPCR, and log₂ fold change is presented for canonical ISGs (Mx1, OAS1, TRIM25, and TRIM56), non-IFN-regulated TRIM23, and the gene of interest, TRIM40.

Figure 2

Pteropus vampyrus TRIM40 antagonizes type I interferon and pro-inflammatory cytokine gene expression. siRNA targeting Pteropus vampyrus TRIM40 or scrambled control was transfected in PVK4 cells 48 h prior to the transfection of 1 μg/mL of low-molecular weight (LMW) poly(I:C). (A) The RNA collected at 24 h post-poly(I:C) transfection shows knockdown efficiency. (B) Lysates collected in laemmli were used for Western blot to measure activated (pSTAT2) and total STAT2. (C) RNA from lysates was used to measure fold-induction of ISGs (RIG-I, ISG56, Mx1, and OAS1) and pro-inflammatory cytokines (IL6 and TNFα). p values are reported as * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001.

Figure 3

Pteropus vampyrus TRIM40 interacts with RIG-I-like receptors RIG-I and MDA5. Cells were co-transfected with Pteropus vampyrus HA-PV-TRIM40 and FLAG-PV-RIG-I-like receptor (RLR) RIG-I or MDA5 for 30 h in 293T (A) or ZFBK13-75 cells (B). The whole cell extracts (WCE) were used to check levels of expression and immunoprecipitated (IP) with anti-FLAG antibody beads. (C) 293T cells were transfected with human (Hu) or PV HA-TRIM40 and partially purified using HA peptide. Separate 293T cells were transfected with Hu- or PV-RLRs or HA-Hu-IRF3. Lysates from FLAG-protein expressing cells were immunoprecipitated with FLAG beads and washed prior to the addition of HA-purified TRIM40. (D) ZFBK13-75 cells were co-transfected with FLAG-PV-RIG-I and HA-PV-TRIM40 for 24 h and then stimulated with 1 μg/mL low-molecular weight (LMW) poly(I:C) for 1 or 4 h, and lysates were used for WCE or IP: FLAG. (E) ZFBK13-75 cells were co-transfected with FLAG-PV-MDA5 and HA-PV-TRIM40 for 24 h and then stimulated with 1 ug/mL high-molecular weight (HMW) poly(I:C) for 1 or 4 h, and lysates were used for WCE or IP: FLAG.

Figure 4

Pteropus vampyrus TRIM40 ubiquitinates RIG-I-like receptors RIG-I and MDA5.

Figure 5

Nipah virus infection induces TRIM40 expression in Pteropus vampyrus cells. (A,B) A549 and PVK4 cells were infected with a recombinant Nipah virus (NiV) that express GFP at a multiplicity of infection of 0.05 or 1.0, and supernatants were collected throughout the 72 h post-infection period (A) with fluorescence microscopy images taken at 36 h (B). (C) Ephrin A and B gene expression in unstimulated PVK4 and A549 cells reported as relative expression normalized to GAPDH. (D) TRIM40 gene expression in A549 and PVK4 cells 16 h post-infection (h.p.i) with MOI 0.05 or 1.0 rNiV-GFP reported as fold induction (top panel), and IFNβ mRNA expression (bottom panel). p values are reported as * < 0.05, ** < 0.01, **** < 0.0001.

Figure 6

TRIM40 is proviral in bat and human cells. PVK4 cells infected with recombinant Nipah virus (rNiV) expressing GFP at a multiplicity of infection of 1.0 and measured knockdown efficiency of TRIM40 (A) and NiV titer (B) at 24 h post-infection. CRISPR-knockout or control PVK4 cells were infected with rNiV-GFP MOI 1.0 for 24 h to measure titer (C). A549 cells infected with rNiV-GFP at MOI 0.05 and measured knockdown efficiency of TRIM40 (qPCR) (D) and NiV titer (E) at 24 h post-infection. p values are reported as ** < 0.01, *** < 0.001, **** < 0.0001.

Figure 7

TRIM40 function and conservation. (A) Schematic of TRIM40′s role in regulating the RIG-I-like receptor pathway in pteropid bat and human cells. (B) Protein sub-domain organization of Pteropus vampyrus and human TRIM40. (C) Percent identity and similarity of P. vampyrus and human TRIM40.