Fig 2: TRIM69 exhibits signatures of positive selection, and anti-VSIV activity has been lost in the Mus lineage. (A) The diagram at top illustrates the conserved domains of TRIM69 based on the Conserved Domains Database (CDD), version 3.16, from NCBI. The bar chart at bottom represents the dN/dS values of the sites with positive selection. The six sites with statistically significant positive selection (PAML M8 with Bayes Empirical Bayes [BEB], P > 0.9) are shown in red. The phylogenetic relationship between the 18 sequences spanning the hominoids is shown on the left, with the regions of the alignment with statistically significant positive sites shown numbered on the right. (B) The results of the chi-square test comparing the PAML models is shown in the table along with the proportion of sites under positive selection and the average dN/dS for those sites. (C) MT4 cells were modified to express myc-TRIM69 orthologs from multiple species in a doxycycline-inducible fashion. Western blot analysis of TRIM69 expression in cells with and without 24 h of doxycycline treatment is shown beneath the titers of VSIV-GFP (16 h postinfection) in the presence and absence of doxycycline treatment. (D) The experiment as described for panel C, examining TRIM69 from rat and divergent mice. (E) NIH 3T3 cells were modified to stably express TagRFP or human or mouse TRIM69 (LHCX) before being infected with serially diluted VSIV-GFP. (F) Western blot analysis of TRIM69 expression in the cells from the experiment described if panel E. (G to H) The cells from the experiment described in panel C were used to determine the titers of VSNJV (G) and CHNV-GFP (H). In all cases, virus titrations were carried out on at least two occasions, and typical results are shown. Mean and standard deviations are plotted, and titers are based on at least three doses (in the linear range). LRT, likelihood ratio test.

Fig 3: Overexpression of TRIM69 decreased cell apoptosis, p53 expression, and ROS production caused by UVB irradiation. (A) HLECs were transduced with TRIM69-overexpression virus (TRIM69 OE) or control vector virus (vector). Expression of TRIM69 protein was determined with western blot assays at 48 h of culture. HLECs were transduced with TRIM69 OE or vector for 24 h, then subjected to UVB irradiation (2 W/m2) for 10 min. (B) After 24 h of culture, Annexin V-FITC/PI staining was performed to analyze cell apoptosis. (C) Western blot assays were used to detect the expression of p53, Bax, and Bcl-2 protein. (D, E) DCFH-DA (D) and DHE (E) staining was performed to analyze ROS production. ***P < 0.001 vs. control; ###P < 0.001 vs. vector + UVB.

Fig 4: p53 mediated the effects of TRIM69 in UVB-induced cell apoptosis and ROS production. HLECs were divided into the following five groups and treated as described in the Materials and Methods section before UVB irradiation: control; PFTα; vector; TRIM69 OE; and TRIM69 OE + p53 OE. (A) Cell apoptosis was detected with Annexin V-FITC/PI staining. (B, C) ROS production was assessed with DCFH-DA (B) and DHE (C). (D) Western blot assays were used to detect the expression of p53, Bax, and Bcl-2 protein. ***P < 0.001 vs. control, ###P < 0.001 vs. PFTα, &&&P < 0.001 vs. TRIM69 OE.

Fig 5: Inhibition of ROS mitigated the effects of UVB irradiation on ROS production, cell apoptosis, and TRIM69 expression. HLECs were divided into the following three groups and treated as described in the Materials and Methods section: control; UVB; and UVB + NAC. (A, B) ROS production were assessed 24 h after UVB irradiation. (C) Cell apoptosis was assessed 24 h after UVB irradiation. (D) Protein expression was assessed 24 h after UVB irradiation. (E) HLECs were transduced with Fox3a overexpressing (Foxo3a OE)/vector or Fox3a shRNA (shFox3a#2)/control shRNA (shNC) and transfected with the TRIM69 promoter plasmid. The activity of the TRIM69 promoter was measured with dual luciferase reporter assays. ***P < 0.001 vs. control, ###P < 0.001 vs. UVB; &&&P < 0.001 vs. vector; $$$P < 0.001 vs. shNC.