TRIM E3 ligases interfere with early and late stages of the retroviral life cycle

Abstract

Members of the TRIpartite interaction Motif (TRIM) family of E3 ligases have been shown to exhibit antiviral activities. Here we report a near comprehensive screen for antiretroviral activities of 55 TRIM proteins (36 human, 19 mouse). We identified approximately 20 TRIM proteins that, when transiently expressed in HEK293 cells, affect the entry or release of human immunodeficiency virus 1 (HIV), murine leukemia virus (MLV), or avian leukosis virus (ALV). While TRIM11 and 31 inhibited HIV entry, TRIM11 enhanced N-MLV entry by interfering with Ref1 restriction. Strikingly, many TRIM proteins affected late stages of the viral life cycle. Gene silencing of endogenously expressed TRIM 25, 31, and 62 inhibited viral release indicating that they play an important role at late stages of the viral life cycle. In contrast, downregulation of TRIM11 and 15 enhanced virus release suggesting that these proteins contribute to the endogenous restriction of retroviruses in cells.

Figure 1. TRIM Proteins Inhibiting or Enhancing Retroviral Entry

(A) Experimental design: HIV, N, and B-MLV viruses carried an ALV-A envelope and the target HEK293 cells expressed cognate receptor Tva950 and TRIM proteins. Effects on entry were measured at the level of gene expression using GFP as reporter. (B–D) Effects of TRIM protein expression on the entry of HIV (B), N-MLV (C), and B-MLV (D) shown as fold inhibition using a log2 scale with standard errors. The dotted line represents the statistically determined cut-off value. (E) Ratio of fold inhibition in N- and B-tropic MLV entry. (F, G) Scatter plot comparing the effects on entry for one virus against another as indicated. (H) Effect of silencing endogenous TRIM proteins 11, 25, 31, and 62 in HeLa cells using specific or control siRNA on HIV, NB- and N-MLV entry. The ratio of fold increase in entry of N versus NB-MLV is shown to the right.

Figure 2. TRIM11 Reduces the Protein Levels of TRIM5

(A) Effects of increasing amounts of transfected plasmid encoding human TRIM11 on entry of B-MLV and increasing amounts of N-MLV. (B) The effects of transient expression of TRIM11 (10 ng DNA transfected), TRIM30 (50 ng DNA transfected), and empty vector (Vector) on N-MLV entry were tested in HEK293 cells treated with either control (control si) or TRIM5 specific siRNA (H5 si). (C) Western blot (anti-HA serum) of HEK293 cells 48 h after transfection with plasmids encoding HA-tagged TRIM5 and either increasing amounts (ng DNA) of empty vector (pcDNA) or a plasmid expressing mouse TRIM11 (M11). Cells were treated with the proteasome inhibitor MG132 (10 μM) for 6 h prior to analysis as indicated. (D) An experiment as in (C) for cells expressing HA-tagged TRIM5 or TRIM15-YFP together with human TRIM11 (H11), its mutant lacking the RING domain (ΔRING) and mouse TRIM30 (M30). The lower panel shows the parallel TRIM5 GFP-levels in cells. (E) Fold increase in N-MLV entry in the HeLa cells expressing either control plasmid, wild-type human TRIM11, or its RING domain mutant (ΔRING). (F) TRIM11-YFP (5 ng, red) and TRIM5-CFP (50 ng, green) were transfected into HEK293 and the localization of both proteins monitored by confocal microscopy 24 h post-transfection.

Figure 3. TRIM Proteins Interfering with Virus Production and Release

(A) Experimental design: HEK293 cells transfected with viral plasmids encoding full-length HIV or MLV and carrying a GFP-reporter gene in the presence or absence of plasmids encoding individual TRIM proteins. 48 h after transfection, producer cells and culture supernatants were harvested. Producer cells were subjected to FACS analysis to measure viral gene expression. The infectivity of culture supernatants was determined by infecting target cells and measuring GFP-positive cells 36 h after initiation of infection using FACS analysis. (B, C) Effects of TRIM protein expression in HEK293 cells on the production and release of HIV (B) and MLV (C). Graphs represent the fold inhibition (log10 scale) in released viral infectivity (grey bars) and in viral gene expression (black bars). All experiments were performed at least in triplicates and the bars represent standard errors from the mean values. The dotted line represents the statistically determined cut-off value of 6. MLV Gag released into culture supernatant was analysed by western blot using antibodies against p30 MLV capsid and presented beneath the graph in (C). Partition lines indicate individual gels. (D, E) Scatter plots of data shown in panels (B) and (C) depicting fold inhibitions in released virus infectivity plotted against viral gene expression from the LTR upon TRIM expression for HIV (D) and MLV (E).

Figure 4. Inhibitory and Enhancing Roles of Individual TRIM Proteins on Viral Release

(A, B) Fold increase in HIV and MLV infectivity released from HEK293 (A) and HeLa (B) cells treated with control siRNA (contsi) or siRNA targeting endogenously expressed human TRIM proteins 11, 15, 25, 31, and 62. (C) Fold increase in HIV gene expression and released infectivity in HEK293 cells transfected with increasing amounts of plasmid (0–100 ng) expressing TRIM62 or its E3 mutant (62E3m). (D) Infectivity of MLV released from HEK293 cells is shown as fold inhibition in presence of plasmids expressing TRIM11, 15, 25, 62, or their E3 mutants (E3m).

Figure 5. The Antiviral Activity of TRIM15 Resides in Its B-box

(A) Fold inhibition in MLV released infectivity in presence of indicated TRIM15-YFP derivatives. The vectors pcDNA or pEYFP were used as controls. Top schematic displays the TRIM15 domain structure. (B) Western blot using GFP antibodies to detect MLV Gag-GFP that co-immunoprecipitated with antibodies against TRIM15. (C) Western blot using antibodies to GFP to identify TRIM15-YFP derivatives that co-immunoprecipitated with MLV Gag. (D) MLV Gag-CFP (green), full length MLV and TRIM15-YFP (red) were transfected into HEK293 cells and monitored 24 h later. Green arrows indicate the accumulation of Gag at the plasma membrane. Red arrows point to cytoplasmic TRIM15 bodies. (E) An experiment as described in (D) was performed for HIV Gag-YFP (red) and TRIM15-CFP (green).

Figure 6. Anti-retroviral Activities of TRIM Proteins Identified in This Study

(A) A Java TREEVIEW graphic display of all TRIM proteins activities against early and late stages of the retroviral life cycle. Input fold inhibition values were log2 transformed and shown as red/green color-coding for inhibitory and enhancing activities, respectively. Grey stands for experiments not carried out. (B–D) Scatter plot analyses were performed as indicated.