Fig 2: High-throughput, siRNA-based screening to identify cellular factors regulating HIV-1 integrase stability. a Workflow for the siRNA-based screening. Cellular fluorescence, as surrogate of IN levels, was analyzed by automated, high-content fluorescent microscopy. b Results of screening. The graphs show the log10 values of the fold change of EGFP-positive cells over control in the two replicate screenings (R1 and R2). The dotted lines show 2x increase over Control (pool of results using 4 non-targeting siRNAs and mock-transfected cells). The 6 siRNAs in red are those that were in the top 10 in both screenings. The 4 siRNAs in green are those that were among the top 10 in one of screening while anyhow showing an effect ≥2 fold over control in the other screening. The effect of MG132 is shown in blue. c Confirmation of effective silencing of Pin1, TRIM33, FBOX28, RNF31, RNF125, RFPl3, and DTX by immunoblotting with the respective antibodies. Cells transfection of non-targeting siRNA1 (siNT1) was used as a control. HSC70 served as a loading control. d Representative immunoblot showing the levels of HIV-IN after knock-down of the top 10 E3 ligases from the screening. HeLa cells were transfected with siRNAs against the identified factors or with a siRNA against Pin1, followed by transfection of Flag-IN and EGFP. Forty-eight hour later the levels of IN were assessed by anti-Flag immunoblotting. Tubulin served as a loading control; β-catenin was used to confirm effect of the MG132 treatment. The bottom panel shows a western blotting for EGFP in a representative experiment to show lack of effect of any of the tested siRNAs on EGFP levels. e Quantification of the levels of HIV-1 IN after knock-down of the top 10 E3 ligases identified by the screening. Experiments were performed as in panel d. IN levels are expressed after normalization for tubulin and as fold over siNT1. Data are mean ± SEM; n = 3 independent experiments; *P < 0.05; **P < 0.01; one-way ANOVA. f Representative high-content microscopy images showing of EGFP-IN-expressing cells after depletion of four cellular ubiquitin-conjugation factors (TRIM33, FBXO28, siDTX2, and siUBE2J2) or Pin1

Fig 3: TRIM33 inhibits HIV-1 infection. a Analysis of the effect of stable knockdown of TRIM33 in multiple round infection with wild-type HIV-1. SupT1 cells were first transduced with lentiviral vector expressing different anti-TRIM33 shRNAs; after selection with puromycin, cells were infected with wild-type HIV-1BRU or HIV-1BRUIN(S57A), followed by analysis of infection over time by p24 ELISA. b Western blotting analysis showing endogenous TRIM33 levels in SupT1 cells transduced with lentiviral vectors expressing the indicated shRNAs and selected with puromycin. Tubulin was used as a loading control. c. Stable knockdown of TRIM33 enhances wild type HIV-1 replication and rescues replication of the IN(S57A) mutant. The graph show the levels of p24 in control Sup1 cells or cells previously transduced with anti-TRIM33 shRNA #4, which efficiently knocks down TRIM33 expression, after infection with the two viruses. The results shown are representative or four independent experiments. d Quantification of the levels of integrated HIV-1 DNA in SupT1 cells, expressing an shRNA control or shRNA #4 against TRIM33, and infected with wild-type HIV-1BRU or HIV-1BRUIN(S57A), at day 3 after infection. Data are mean ± SEM; n = 3 independent experiments; **P < 0.01; n.s. not significant; one-way ANOVA. e Silencing of TRIM33 protects both wild-type IN and mutant IN(S57A) from degradation. Expression plasmids for the two IN proteins were transfected into HeLa cells in which TRIM33 was knocked down by RNAi; cells were then treated with cycloheximide 30 µg/ml for the indicated time points prior to lysis. The panel shows a representative immunoblots using antibodies against IN, TRIM33 and tubulin as a loading control. f Quantification of IN levels in the presence of cycloheximide for the indicated time points in the whole cell extracts of SupT1 cells treated with siRNAs against TRIM33 or not targeting and transfected with either wt IN or IN(S57A). After normalization for Tubulin, data (mean ± SEM; n = 3 independent experiments) are expressed as a percentage of time = 0 for each treatment

Fig 4: Interaction of TRIM33 with integrase from infectious HIV-1. a Analysis of IN protein levels in TRIM33-knockdown (TRIM33 KD) cells during HIV-1 infection. Cells stably expressing an anti-TRIM33 shRNA were infected for 1 h with HIV-1; at the indicated time points after infection, cells were washed with PBS and the levels of IN were analyzed. b Western blot showing IN levels in control and TRIM33 depleted cells Beta-actin was used as a loading control; TRIM33 downregulation was verified using an anti-TRIM33 antibody. c TRIM33 interacts with IN during HIV-1 infection. SupT1 cells were infected with HIV-1 in the presence of MG132. Either TRIM33 or IN were immunoprecipitated with the respective antibodies, followed by immunoblotting to test the presence of the reciprocal protein. d Analysis of the effect of stable knockdown and overexpression of TRIM33 variants in multiple round infection with wild-type HIV-1. SupT1 cells stably expressing an hRNA targeting the TRIM33 3′-UTR (selected after transduction with a lentiviral vector and selection for puromycin) were transduced with lentiviral vectors expressing the TRIM33 coding sequence (cs) or the coding sequence of the RING(CA) mutant. After selection with hygromycin, cells were infected with wild type HIV-1BRU, followed by analysis of infection over time by p24 ELISA. e Western blotting analysis showing TRIM33 levels after transduction with shRNA targeting 3′-UTR of TRIM33 alone or after transduction of wild-type or mutant TRIM33 (TRIMM33cs or RING(CA)cs. Tubulin was used as a loading control. f real-Time RT-PCR analysis showing TRIM33 expression after transduction with the shRNA targeting the 3′-UTR of TRIM33 alone or after transduction of wild type or mutant TRIM33. g Stable knockdown of TRIM33 enhances wild-type HIV-1 replication, while overexpression of its catalytically active cDNA reduces it. The graph shows the levels of p24 in control Sup1 cells or cells previously transduced with anti-TRIM33 shRNA targeting the 3′-UTR of the protein, with or without transduction with a lentiviral vector expressing the coding sequence of wild type TRIM33 (TRIM33cs) or that of the catalytically inactive RING(CA) mutant (RING(CA)cs), after infection with wild-type virus. The results are representative of three independent experiments

Fig 5: Cellular TRIM33 binds HIV-1 integrase and regulates its degradation by poly-ubiquitination. a Endogenous TRIM33 interacts with HIV-1 IN in vivo. HEK293T cells were transfected with the plasmids indicated on the top of the panel and immunoprecipitated with anti-Flag M2 beads, followed by immunoblotting with anti-TRIM33 antibody (upper blot). The lower three blots show the levels of expression of the transfected proteins in cell lysates. b Schematic representation of the HIV-1 IN domains used for the pull-down assay in panel c. NTD N-terminal domain, CCD catalytic core domain, CTD C-terminal domain. c TRIM33 binds the HIV-1 IN C-terminal domain (CTD) in in vitro pull-down assay. The indicated fragments of the HIV-1 IN protein fused to GST or GST alone were incubated with in vitro translated [35S]-TRIM33, extensively washed, and then resolved by SDS-PAGE. The middle pictures shows the gel exposed to a phosphoimager from a representative experiment along with the quantification of the amount of bound proteins expressed as a percentage of radiolabeled input. The lower panel shows the protein gel stained with Coomassie blue to visualize proteins. Quantification in the upper graph reports mean ± SEM; n = 3 independent experiments; **P < 0.01; one-way ANOVA. d HIV-1 IN is degraded through Lys48-linked polyubiquitination. HEK293T cells were transfected with Flag-IN and HA-Ubiquitin for 48 h, as indicated. The ubiquitination profile of IN was visualized after immunoprecipitation with anti-Flag M2 beads using anti-ubiquitin, anti-K48, anti-K63, anti-HA, and anti-Flag antibodies. e TRIM33 Inhibition reduces IN polyubiquitination. HEK293T cells were co-transfected with Flag-IN and HA-Ubiquitin for 36 h, as indicated. IN polyubiquitination was visualized after immunoprecipitation with anti-Flag M2 beads using an anti-IN antibody. f Quantifications of the amount of polyubiquitinated IN protein (Ubi-IN) in whole cell extracts, normalized over total IN. Experiments were performed as in panel e