SUMO-3 promotes the ubiquitin-dependent turnover of TRIM55.

Human muscle-specific RING fingers (MURFs) are members of the tripartite motif (TRIM) family of proteins characterized by their C-terminal subgroup one signature domain. MURFs play a role in sarcomere formation and microtubule dynamics. It was previously established that some TRIMs undergo post-translational modification by small ubiquitin-like modifier (SUMO). In this study, we explored the putative SUMOylation of MURF proteins as well as their interactions with SUMO. MURF proteins (TRIM54, TRIM55, and TRIM63) were not found to be SUMOylated. However, TRIM55 turnover by proteasomal and lysosomal degradation was higher upon overexpression of SUMO-3 but not of SUMO-1. Furthermore, it is predicted that TRIM55 contains two potential SUMO-interacting motifs (SIMs). We found that SIM1- and SIM2-mutated TRIM55 were more stable than the wild-type (WT) protein partly due to decreased degradation. Consistently, SIM-mutated TRIM55 was less polyubiquitinated than the WT protein, despite similar monoubiquitination levels. Using IF microscopy, we observed that SIM motifs influenced TRIM55 subcellular localization. In conclusion, our results suggest that SUMO-3 or SUMO-3-modified proteins modulate the localization, stability, and RING ubiquitin ligase activity of TRIM55.

Editing of the human TRIM5 gene to introduce mutations with the potential to inhibit HIV-1.

The type I interferon (IFN-I)-inducible human restriction factor TRIM5α inhibits the infection of human cells by specific nonhuman retroviruses, such as N-MLV and EIAV, but does not generally target HIV-1. However, the introduction of two aminoacid substitutions, R332G and R355G, in the human TRIM5α (huTRIM5α) domain responsible for retroviral capsid recognition leads to efficient HIV-1 restriction upon stable over-expression. CRISPR-Cas-based approaches to precisely edit DNA could be employed to modify TRIM5 in human cells. Toward this aim, we used a DNA transfection-based CRISPR-Cas9 genome editing protocol to successfully mutate TRIM5 to its potentially HIV-1-restrictive version by homology-directed repair (HDR) in HEK293T cells. Nine clones bearing at least one HDR-edited TRIM5 allele containing both mutations were isolated (5.6% overall efficiency), whereas another one contained only the R332G mutation. Of concern, several of these HDR-edited clones contained on-target undesired mutations, and none had all the alleles corrected. Our study demonstrates the feasibility of editing the TRIM5 gene in human cells and identifies the main challenges to be addressed in order to use this approach to confer protection from HIV-1.