SUMO-interacting motifs of human TRIM5α are important for antiviral activity

Abstract

Human TRIM5α potently restricts particular strains of murine leukemia viruses (the so-called N-tropic strains) but not others (the B- or NB-tropic strains) during early stages of infection. We show that overexpression of SUMO-1 in human 293T cells, but not in mouse MDTF cells, profoundly blocks N-MLV infection. This block is dependent on the tropism of the incoming virus, as neither B-, NB-, nor the mutant R110E of N-MLV CA (a B-tropic switch) are affected by SUMO-1 overexpression. The block occurred prior to reverse transcription and could be abrogated by large amounts of restricted virus. Knockdown of TRIM5α in 293T SUMO-1-overexpressing cells resulted in ablation of the SUMO-1 antiviral effects, and this loss of restriction could be restored by expression of a human TRIM5α shRNA-resistant plasmid. Amino acid sequence analysis of human TRIM5α revealed a consensus SUMO conjugation site at the N-terminus and three putative SUMO interacting motifs (SIMs) in the B30.2 domain. Mutations of the TRIM5α consensus SUMO conjugation site did not affect the antiviral activity of TRIM5α in any of the cell types tested. Mutation of the SIM consensus sequences, however, abolished TRIM5α antiviral activity against N-MLV. Mutation of lysines at a potential site of SUMOylation in the CA region of the Gag gene reduced the SUMO-1 block and the TRIM5α restriction of N-MLV. Our data suggest a novel aspect of TRIM5α-mediated restriction, in which the presence of intact SIMs in TRIM5α, and also the SUMO conjugation of CA, are required for restriction. We propose that at least a portion of the antiviral activity of TRIM5α is mediated through the binding of its SIMs to SUMO-conjugated CA.

Figure 1. Overexpression of SUMO-1 blocks N-tropic MLV infection in 293T cells.

A. 293T cells stably expressing an empty vector, HA-SUMO-1, HA-SUMO-2 or HA-SUMO-3 were generated. The presence of the overexpressed HA-tagged SUMO paralogs was detected by Western blot using an antibody directed against the HA-tag (upper panel), using actin as a control (bottom panel). B–D. The 293T empty vector control cell line or HA-SUMO-1, HA-SUMO-2 or HA-SUMO-3 overexpressing cell lines were mock treated or infected with increasing amount of VSV-G pseudotyped B-MLV luc (B), N-MLV luc (C) or NB-MLV luc (D). Forty-eight hours after infection, luciferase activity was measured. One representative experiment of five independent experiments is shown. Error bars indicate standard deviation among triplicates in the same experiment.

Figure 2. Overexpression of SUMO-1 in MDTF and TE671 cells.

A. Stable MDTF and TE671 cells expressing an empty vector or HA-SUMO-1 were generated, and overexpressed HA-tagged SUMO-1 and actin were detected by Western blot. B. The MDTF empty vector control or HA-SUMO-1 over-expressing cell lines were mock treated or infected with increasing amounts of VSV-G pseudotyped N-MLV luc. C. The TE671 empty vector control cell line or HA-SUMO-1 overexpressing cell line were mock treated or infected with increasing amount of VSV-G pseudotyped N-MLV luc. Forty-eight hours after infection, luciferase activity was measured. One representative experiment of three independent experiments is shown. Error bars indicate standard deviation among triplicates in the same experiment.

Figure 3. Characterization of SUMO-1 block of N-MLV infection in 293T cells.

A. The 293T empty vector control or HA-SUMO-1 cell lines were infected with increasing amounts of a mutant version of N-MLV luc in which amino acid 110 of the CA protein was mutated from arginine to glutamic acid (R110E). Luciferase activity was measured forty-eight hours post-infection. One representative experiment of three independent experiments is shown. B. The 293T empty vector control or HA-SUMO-1 cell lines were infected with increasing amounts of VSV-G-pseudotyped N-MLV expressing a GFP reporter, and four hours post-infection, cells were superinfected with a fixed amount of VSV-G-pseudotyped N-MLV luc. Luciferase activity was measured forty-eight hours post-infection. One representative experiment of four independent experiments is shown. C. The 293T empty vector control or HA-SUMO-1 overexpressing cell lines were infected with undiluted (1∶1) or 10-fold diluted (1∶10) VSV-G-pseudotyped N-MLV luc. Mock treated cells were included as a negative control. Total DNA was isolated twenty hours after infection, and the amount of viral DNA synthesized in the infected cells was measured by quantitative PCR. Primers specific for the minus-strand strong stop (MSS) DNA (left panel), Luciferase gene (middle panel) or LTR-LTR junction (right panel) were used. The values were normalized to GAPDH DNA and expressed as fold over empty vector. Error bars indicate standard deviation among triplicates in the same experiment (A and B) or 3 different experiments (C).

Figure 4. SUMO-1 block of N-MLV infection on 293T cells is mediated by TRIM5α.

A. Schematic representation of the target site of five different shRNAs directed to the human TRIM5α messenger RNA. B. The 293T empty vector control cell line stably expressing a control scrambled (scr) shRNA (white bar) or the HA-SUMO-1 overexpressing cell line stably expressing a control scrambled shRNA, or different shRNAs directed to human TRIM5α mRNA (black bars), were generated. RNA was extracted, and the mRNA levels of TRIM5α were determined by quantitative PCR. The values were normalized to GAPDH mRNA and expressed as fold over empty vector-scr. C. The 293T expressing the control scr shRNA or the different shRNAs directed to TRIM5α mRNA were infected with a fixed multiplicity of N-MLV luc. Forty-eight hours after infection luciferase activity was measured. D. The 293T expressing the control scr shRNA or the shRNA4 were transiently transfected with an empty plasmid or a plasmid encoding human TRIM5α with a FLAG-tag as indicated. Twenty-four hours after transfection, cells were infected with a single dose of N-MLV luc. Forty-eight hours after infection, luciferase activity was measured, and Western blots were performed on the same extract to verify the expression of FLAG-TRIM5α using an anti-FLAG antibody (upper panel), an anti-HA antibody to verify the expression of HA-SUMO-1 (middle panel) and an anti-actin antibody as a control (bottom panel). Error bars indicate standard deviation among triplicates in the same experiment.

Figure 5. SUMO interacting motifs present in human TRIM5α are important for restriction of N-MLV.

A. Amino acid sequence of human TRIM5α. The RING (blue), B-box (green), coiled-coil (yellow), B30.2 (pink) domains, and the location of a consensus SUMO conjugation site (green letter) and three putative SUMO interacting motifs (SIM) (red letters) are indicated. The variable regions of the B30.2 domain are depicted with horizontal bars above. B. The substitution mutations are indicated. WT: wild-type amino acid sequence; mut: mutated amino acid sequence. C. The 293T empty vector cell line expressing scr shRNA or the HA-SUMO-1 expressing scr shRNA were transfected with an empty plasmid, and the HA-SUMO-1 expressing shRNA4 (shRNA4-TRIM5α) cells were transiently transfected with an empty plasmid or plasmids encoding FLAG-tagged wild-type human TRIM5α or the C15A/C18A, K10R, SIM1mut, SIM2mut and SIM3mut mutants. Twenty-four hours after transfection, cells were infected with a single dose of N-MLV luc. Forty-eight hours after infection, luciferase activity was measured and Western blotting was performed using the same extract to verify the expression of the different FLAG-TRIM5α proteins using an anti-FLAG antibody (upper panel) and an anti-GAPDH antibody as a control (bottom panel). D. MDTF (upper panel) and CRFK (bottom panel) cells stably expressing an empty vector, and Myc-tagged wild-type human TRIM5α or K10R, SIM1mut, SIM2mut and SIM3mut mutants were generated. The presence of the TRIM5α-Myc proteins was detected by Western blot using a Myc-antibody; the presence of actin was used as a control. E. The MDTF empty vector control cell line and the indicated TRIM5α-Myc overexpressing cell lines were mock treated or infected with increasing amounts of N-MLV luc. Forty-eight hours after infection, luciferase activity was measured. One representative experiment of four independent experiments is shown. F. The CRFK empty vector control and the various TRIM5α-Myc overexpressing cell lines were mock treated or infected with increase dose of N-MLV luc. Forty-eight hours after infection luciferase activity was measured. One representative experiment of three independent experiments is shown. Error bars indicate standard deviation among triplicates in the same experiment.

Figure 6. SUMO interacting motifs present in TRIM5α orthologs are important for restriction activity.

A. CRFK cells stably expressing an empty vector, and FLAG-tagged wild-type rhesus TRIM5α or K10R, SIM1mut, SIM2mut and SIM3mut mutants were generated. The presence of the FLAG-TRIM5α proteins was detected by Western blot using a FLAG-antibody; the presence of GAPDH was used as a control. B. The CRFK empty vector control cell line and the indicated FLAG-TRIM5α overexpressing cell lines were mock treated or infected with increasing amounts of N-MLV luc. Forty-eight hours after infection, luciferase activity was measured. One representative experiment of three independent experiments is shown. Error bars indicate standard deviation among triplicates in the same experiment.

Figure 7. CA mutations altering putative SUMO conjugation site reduce SUMO-1 and TRIM5α-mediated restriction.

A. The ability of human TRIM5α to interact with SUMO-1 is shown by GST-pulldown. 293T cell were transiently transfected with an empty plasmid or a plasmid encoding a FLAG-tagged version of human TRIM5α, forty-eight hours after transfection total extract were prepared and assayed for interaction with GST or GST-SUMO1 produced in bacteria. Input corresponds to 5% of the amount used in the interaction assay. B. Amino acid sequence of the UBC9 binding region of CA protein. The lysine residues conserved between Mo-CA and N-CA are highlighted, and the consensus binding sequence is underlined. The position and identity of the substituted amino acids in N-CA are indicated for each mutant. C. The 293T empty vector and HA-SUMO-1 cell lines were infected with wild type N-MLV luc or N-CA mutant viruses. D. MDTF empty vector and wild-type human TRIM5α cell lines were infected with wild-type N-MLV luc or the N-CA mutant viruses. Forty-eight hours after infection, luciferase activity was measured, and the fold restriction between the two cell lines was calculated for different experiments, presented as average fold restriction. Error bars indicate standard deviation among 6 different experiments. * indicates p<0.01.