The role of SUMO and PIAS proteins in retrovirus replication and restriction

Since its discovery in 1997, the SUMO (small ubiquitin-like modifier) protein has emerged as a key regulator of protein function. SUMOylation regulates a substrate's function by altering its intracellular localization, protein-protein interactions, or stability and enzymatic activity. Proteins targeted for SUMOylation include transcription factors, proteins associated with DNA recombination, replication and repair, and proteins involved in signal transduction. In recent years numerous viral proteins have been found to interact with the SUMOylation machinery. This research investigates the role of SUMO and the SUMOylation machinery in retrovirus replication and restriction.;First, we demonstrate the antiviral effect of overexpression of SUMO1 on N-tropic MLV (murine leukemia virus) infection. We determined that the SUMO1 block of N-tropic MLV viral transduction is specific to 293T cells and is not observed in murine cell lines, Mus Dunni tail fibroblasts (MDTF) or Sc-1 cells. Further characterization of the SUMO1-enhanced restriction of N-tropic MLV reveals that the viral block, like that mediated by Fv1, is dependent on capsid residue 110 and mutation of this residue from arginine to glutamine renders the virus resistant to the SUMO1 antiviral effect. We also demonstrate that the viral block occurs early in the virus life cycle, before reverse transcription, and this block can be saturated. The SUMO1 antiviral effect on N-tropic MLV appears to involve a 293T specific factor that is not present in MDTF or Sc-1 cells and could possibly be enhancing the activity of a restriction factor, such as the TRIM5alpha (Tripartite motif 5alpha) protein.;In order to determine whether SUMO1 is enhancing the activity of the human TRIM5alpha (hTRIM5alpha) restriction factor, we generated hTRIM5alpha knockdowns and found that reduction in hTRIM5alpha expression resulted in a loss of the SUMO1 antiviral effect. Furthermore, introduction of an shRNA-resistant hTRIM5alpha construct to the SUMO1/hTRIM5alpha knockdown cells successfully restored the SUMO1 antiviral effect. We also determined that treatment of cells with the proteasome inhibitor, MG132, had no effect on the SUMO1/hTRIM5alpha block of N-tropic MLV.;Finally, we observed that overexpression of human PIAS1 and PIAS3 proteins, members of the PIAS family of SUMO E3 ligases, have no effect on B-, N-, or NB-tropic MLV viral transduction. Interestingly, human PIASy overexpression resulted in a reduction of B-, N-, and NB-tropic MLV viral transduction, as opposed to overexpression of SUMO-1, which only reduced N-tropic MLV viral transduction. Knockdown of human PIASy in 293T cells appears to have no effect on viral transduction and this could possibly be due to insufficient knockdown or functional redundancy of PIAS proteins. However, knockdown of human PIASy in 293T cells stably overexpressing SUMO1 did result in ablation of the SUMO1 block of N-tropic MLV. This suggests that hPIASy is also involved in the SUMO1-enhanced TRIM5alpha-mediated restriction of N-MLV. We also observed enhancement of B-tropic, N-tropic, and NB-tropic MLV infectivity in Balb/c cells in response to knockdown of mPIASy.;These findings have identified novel roles for SUMO1 and PIASy in viral replication and restriction. SUMO1 enhances the antiviral effects of hTRIM5alpha and specifically blocks N-tropic MLV viral transduction, in a mechanism that requires hPIASy. Human and murine PIASy negatively impact all three subclasses of MLV viral transduction. The mechanisms of SUMO1-enhanced hTRIM5alpha restriction and PIASy inhibition of viral transduction remain to be determined.