Fig 1: Reciprocal co-immunoprecipitation validation of pIIIa-host protein interactions.(A, B) Viral pIIIa expressing Flp-In 293 T-Rex and empty vector control cell extracts subjected to co-immunoprecipitation assay. (D, E) HAdV-D37 infected HEK293 cells (MOI of 1, 15 hpi), and naïve control cell lysates were immunoprecipitated with anti-USP9x and anti-RANBP2 antibodies. Blots were developed with a-USP9x, a-RANBP2, a-pIIIa-Flag, and a-pIIIa (B) as indicated. Immunoprecipitations with IgG controls (C, F) are shown for both virus infected and inducible systems. Data is representative of 3 biological replicates.
Fig 2: Human adenovirus (HAdV) pIIIa expression and affinity purification-MS/MS analysis.(A) Surface representation of HAdV-D26 (PDB: 5TX1) colored by radius [33] with expanded zoom into the vertex interior view, rendered using UCSF Chimera v1.14. Protein IIIa (pIIIa) (brick red), penton base (green), peripentonal hexons (pale gold), and protein VIII (purple). (B) HAdV-D37 pIIIa mRNA expression levels in HEK293 cells at multiplicity of infection (MOI) of 1 through 24 hrs post infection (hpi). (B and C) Viral pIIIa mRNA expression levels seen in virus-infected cells and stable pIIIa expression in Flp-In 293 T-REx cell lines with tetracycline induction. B and C data shown represents the mean ± standard deviation. (D) Western blot showing pIIIa expression in HEK293 cells infected at an MOI of 1 (upper panel), and pIIIa-Flag protein expression in Flp-In 293 T-Rex cells upon tetracycline induction (lower panel). (E) pIIIa-FLAG and FLAG-control immunoprecipitated protein complexes were TCA precipitated, and LC-MS/MS performed. The obtained mass spectrometry protein interactions analyzed using the CRAPome database identified two high confidence interactors: USP9x and RANBP2. (F) Compared to the experimental and database controls, further analysis of FLAG-pIIIa interactions showed a 32.4 and 3.5 fold change (FC) difference for USP9x and RANBP2, respectively, with a SAINT probability score 1 (100%). Data for B to F were obtained from at least 3 replicate experiments.
Fig 3: Identified pIIIa-host interactions are crucial across different HAdV species.(A) Multiple sequence alignment of representative pIIIa amino acids across HAdV A-G species using BioEdit. Sequence conservation across types are color coded. (B) Co-immunoprecipitation of HAdV-D37 and HAdV-C5 infected cells (MOI of 1 and 5, respectively, 24 hpi) show pIIIa-USP9x and pIIIa-RANBP2 interactions for both viruses. (C) HAdV-C5 viral replication in USP9x and RANBP2-siRNA treated HEK293 cells at an MOI of 5 and 24 hpi. Data shown are mean ± standard deviation for 3 replicates (P>0.05 by unpaired t-test, two-tailed).
Fig 4: USP9x has no role in pIIIa or RANBP2 deubiquitination but deregulates their expression.(A) Flp-In 293 T-Rex cells were treated with negative control (NC) or USP9x-siRNA for 48 hours. Following MG132 proteasome inhibitor treatment (10 µmol/L for 4 hrs), pIIIa expression was induced by tetracycline (20 ng/mL for 8hrs). GAPDH control is shown for each set. (B) HEK293 cells were treated with negative control (NC) and USP9x-siRNA for 48 hours. Following MG132 proteasome inhibitor treatment (10 µmol/L for 4 hrs), cells were infected with HAdV-D37 at an MOI of 1 for 4 hrs. USP9x-siRNA treated cells showed increased pIIIa and RANBP2 expressions compared to NC-siRNA treated cells, irrespective of MG132 treatment. (C) HCT116 wild-type and (D) HCT116 USP9x (-/-) cells were treated with MG132 and infected (MOI of 5) for 4 and 8 hrs, respectively. Neither condition significantly stabilized pIIIa or RANBP2 steady state concentrations. The final data are presented as the mean ± SD of at least triplicate experiments. Statistical significance was performed with unpaired t-test (two-tailed). Only comparisons where P<0.05 are shown.
Fig 5: Human adenovirus pIIIa exploits RANBP2 nuclear import function.(A) Cytoplasmic and nuclear fractions of stable pIIIa expressing Flp-In 293 T-Rex cells treated with USP9x or RANBP2 siRNA for 48 hours, and then induced with tetracycline (20 ng/µL), and blotted for, pIIIa-Flag, RANBP2, USP9x and GAPDH protein expression (B) Schematic of the role of pIIIa-RANBP2 interaction in the adenovirus replication cycle. (C) Following Flp-In 293 T-Rex cells pretreated with USP9x or RANBP2-siRNA were tetracycline induced (20ng/mL) for 8 hrs for pIIIa expression. FLAG-pIIIa (green), phalloidin (red), and nuclei (blue) The dotted ellipse corresponds to the nucleus area. Scale bar: 10 µm. (D) ImageJ analysis done on 30 cells per group and green fluorescence intensity was measured in the nucleus of each cell. The final data are presented as the mean ± SD of at least triplicate experiments. Statistical significance was performed with two-way ANOVA followed by Tukey multiple comparison test. Only comparisons where P<0.05 are shown.
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