Supplementary MaterialsS1 Fig: Expressions of molecules from antiviral signal pathway are changed after DENV infection in 293T cells by RNA-Seq analysis. another 24 h. Cell lysates and supernatants were harvested for Western blot and TCID50 assays, respectively. (B) The knockdown effectiveness of two shRNAs (shm69-1 and shm69-2) focusing on mouse TRIM69 was recognized in B16F10 cells. The mRNA level (remaining) and protein (right) of mouse TRIM69 were analyzed. (C) The viral proteins and computer virus titers were tested in mTRIM69 silenced B16F10 cells.(TIF) ppat.1007287.s003.tif (1.0M) GUID:?9919349E-ECD0-4DC0-B93B-47C96AD61847 S4 Fig: TRIM69 does not restrict H1N1 and HSV-1 infection. (A) H1N1 and HSV-1 nucleotide copies were comparable in TRIM69 overexpressed cells and Rabbit polyclonal to HGD control cells. (B) Viral titers of H1N1 and HSV-1 from supernatants of control or TRIM69 overexpressed cells. (C) Viral weight of H1N1 in peripheral blood cells in control and TRIM69 silenced mice as determined by qRT-PCR (remaining). TRIM69 knockdown effectiveness in peripheral blood cells were confirmed by qRT-PCR (right). NS, not significant. The data demonstrated are representative of 3 self-employed experiments. (D) Survival curve of H1N1 infected wide type and TRIM69 silenced mice (n = 5). Mice were infected with intranasal illness of 2×105 H1N1and monitored daily for survival rates.(TIF) ppat.1007287.s004.tif (464K) GUID:?064DF1BD-3207-4909-9427-D332931C2C7C S5 Fig: TRIM69 is not involved in IFN signal pathway. (A) TRIM69 or TRIM69 CA did not impact SeV-stimulated IFN-activation. IFN-was recognized in TRIM69 transfected 293T Cycloheximide inhibitor cells stimulated with SeV. (C) TRIM69 overexpression did not influence IFN-for 12 h and harvested to test the ISRE-luciferase activity. (D) Knockdown of TRIM69 did not influence IFN-or SeV-stimulated ISRE promoter activity. shNC or sh69-2 was co-transfected with ISRE-luc and pRL for 24 h, then cells were stimulated with IFN-or SeV for Cycloheximide inhibitor 12 h, and their luciferase activities were recognized. (E) The RNA levels of Cig5 and IFIT1 were detected in TRIM69 transfected 293T cells stimulated with SeV. NS, not significant. The data demonstrated are representative of at least 3 self-employed experiments.(TIF) ppat.1007287.s005.tif (410K) GUID:?D9C479D3-04EF-4E29-8AC7-8C92791277D3 S6 Fig: MS analysis of target proteins by TRIM69 co-IP assay in 293T infected with DENV-2. (A) The map showed distribution of IP proteins from Flag or TRIM69-Flag. (B) Target proteins immunoprecipitated by TRIM69-Flag were shown.(TIF) ppat.1007287.s006.tif (380K) GUID:?EEAE588E-539D-46E2-BE38-29C2FF091ED1 S7 Fig: TRIM69 reduces the amount of NS2B3 and influences its function. Cycloheximide inhibitor (A) TRIM69 reduced the protein level of NS2B3 complex, therefore reduced the cleavage effectiveness on STING. (B) Overexpression of TRIM69 did not interfere with the connection between NS2B and NS3. Cells were co-transfected with NS2B, NS3 and TRIM69 (or control vector) for 48h, and then treated with MG132. The connection between NS2B and NS3 were analyzed by immunoprecipitation and western blots.(TIF) ppat.1007287.s007.tif (411K) GUID:?741876FC-51B5-4191-BBCE-2D1227D12FD0 S8 Fig: mTRIM69 interacts with DENV NS3 in mouse cells. (A) Co-localization of mTRIM69-Myc (Green) and NS3-Flag (Red) in mouse B16F10 cells as analyzed by confocal microscopy. (B) Co-IP of endogenous mTRIM69 and NS3 from lysates of B16F10 cells infected with DENV-2 for 48 h.(TIF) ppat.1007287.s008.tif (2.6M) GUID:?6EF5D4B9-8C38-4C8A-9F3B-693E91209528 S1 Table: Induction of selected of well-known (A) and predicted ISGs (B) by DENV-2 infection. (DOCX) ppat.1007287.s009.docx (41K) GUID:?F0D5664C-Abdominal9A-4150-B4A1-3E13F5BF10DC Data Availability StatementAll relevant data are within the paper and its Supporting Info files. Abstract In order to get rid of viral infections, hundreds of interferon-stimulated genes (ISGs) are induced type I interferons (IFNs). However, the functions and mechanisms of most ISGs are mainly unclear. A tripartite motif (TRIM) protein encoding gene is definitely induced by dengue computer virus (DENV) illness as an ISG. TRIM69 restricts DENV replication, and its RING domain, which has the E3 ubiquitin ligase activity, is critical for its antiviral activity. An study further confirmed that.