Abstract
TRIM5α is an antiviral, cytoplasmic, E3 ubiquitin (Ub) ligase that assembles on incoming retroviral capsids and induces their premature dissociation. It inhibits reverse transcription of the viral genome and can also synthesize unanchored polyubiquitin (polyUb) chains to stimulate innate immune responses. Here, we show that TRIM5α employs the E2 Ub-conjugating enzyme Ube2W to anchor the Lys63-linked polyUb chains in a process of TRIM5α auto-ubiquitination. Chain anchoring is initiated, in cells and in vitro, through Ube2W-catalyzed monoubiquitination of TRIM5α. This modification serves as a substrate for the elongation of anchored Lys63-linked polyUb chains, catalyzed by the heterodimeric E2 enzyme Ube2N/Ube2V2. Ube2W targets multiple TRIM5α internal lysines with Ub especially lysines 45 and 50, rather than modifying the N-terminal amino group, which is instead αN-acetylated in cells. E2 depletion or Ub mutation inhibits TRIM5α ubiquitination in cells and restores restricted viral reverse transcription, but not infection. Our data indicate that the stepwise formation of anchored Lys63-linked polyUb is a critical early step in the TRIM5α restriction mechanism and identify the E2 Ub-conjugating cofactors involved.
Highlights
TRIM5a is an innate immune effector that belongs to the tripartite (TRIM) protein superfamily
Human TRIM5a (TRIM5ahu) acts differently on two closely related variants of murine leukemia virus (MLV) that differ in their capsid sequences: TRIM5ahu restricts N-tropic MLV (N-MLV) but not B-tropic MLV (B-MLV) (Towers et al, 2000; Besnier et al, 2003; Keckesova et al, 2004; Perron et al, 2004; Yap et al, 2004)
VSV-G-pseudotyped N- and B-MLV vectors expressing GFP to assess endogenous TRIM5ahu activity. These two viral vectors were infectious in feline CrFK cells, which do not express a restricting TRIM5a protein (McEwan et al, 2009) (Supplementary Fig S1A), but were differentially infectious in human HeLa cells, which endogenously express TRIM5a (Fig 1A, Mock). siRNAs targeting 38 different human E2 or E2 variant (UEV) proteins, which collectively encompass the majority of known human E2 enzymes (Markson et al, 2009), were tested for their ability to restore N-MLV DNA synthesis (Fig 1A)
Summary
TRIM5a is an innate immune effector that belongs to the tripartite (TRIM) protein superfamily. The conserved tripartite architecture comprises a really interesting new gene (RING) E3 ubiquitin (Ub) ligase domain, one or two B-box domains, and a coiled-coil, followed by variable protein interaction domain(s). Certain TRIM proteins, including TRIM5a (Stremlau et al, 2004), TRIM21 (Mallery et al, 2010), and TRIM56 (Wang et al, 2011), have been shown to target viral replication . An emerging theme is that antiviral TRIM proteins both inhibit viral infection and initiate innate immune signaling cascades that lead to inflammatory cytokine production and an antiviral state (Pertel et al, 2011; McEwan et al, 2013; Uchil et al, 2013; Versteeg et al, 2013). The molecular mechanisms that underlie these multiple activities are not yet well defined and are of great interest for understanding the innate immune detection of viruses
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