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.

SUMO Binding by the Epstein-Barr Virus Protein Kinase BGLF4 Is Crucial for BGLF4 Function

“…To validate the interaction between BGLF4 and SUMO2, we first performed an in vitro pulldown assay using GST-SUMO2, a GST-SUMO2 tandem trimer [GST-(SUMO2) 3 ], and lysate from Flag-BGLF4-transfected cells. We found that both GST-SUMO2 and GST-(SUMO2) 3 , but not the GST control, bound BGLF4 (Fig. 1A, upper).…”

“…SUMOylation regulates the transcriptional activity of ZTA and RTA (10,13,14,45,47,80). Noncovalent SUMO-protein interactions can also occur through a SUMO interaction motif (SIM) in the target proteins (3,57,90,91,93). EBNA3C contains a SIM motif and upregulates EBNA2-mediated gene activation by binding to a SUMOylated protein (84).…”

“…Cell lysate (500 l) containing Flag-tagged wildtype (WT) or mutant BGLF4 was precleared with 50 l of 50% glutathione-Sepharose 4B at 4°C for 1 to 2 h. Twenty l of GST, GST-SUMO1, GST-SUMO2, or GST-(SUMO2) 3 bound to glutathione-Sepharose 4B was added to the precleared lysate, rotated on a vertical wheel at 4°C for 2 h, washed 4 times with lysis buffer, and boiled in 2ϫ SDS sample buffer. GST-(SUMO2) 3 expresses 3 tandem copies of SUMO2 fused to GST (107).…”

TRIM5α Promotes Ubiquitination of Rta from Epstein–Barr Virus to Attenuate Lytic Progression

“…TRIM5α contains B boxes, a coiled-coil domain, and a RING domain, and can function as an E3 ubiquitin ligase (Reymond et al, 2001). In addition, TRIM5α contains a SPRY/B30.2 domain with two SUMO-interaction motifs (SIMs), which are required for N-MLV restriction (Arriagada et al, 2011). This SPRY/B30.2 domain can also directly bind with the HIV capsid, and is believed to be critical for HIV restriction (Sawyer et al, 2005; Stremlau et al, 2005; Luban, 2007; Ganser-Pornillos et al, 2011).…”

“…Moreover, TRIM5α-mediated ubiquitin conjugation is required for HIV-1 capsid destabilization and inhibition of reverse transcription (Campbell et al, 2016). Interestingly, SIM mutations in TRIM5α not only lead to loss of restriction capability against N-MLV (Arriagada et al, 2011), but the mutations also prevent TRIM5α from shuttling into the cell nucleus, thus rendering it unable to restrict incoming HIV retrovirion cores (Brandariz-Nunez et al, 2013). …”