Editors' ChoiceImmunology

Promoting proteasomal degradation of the viral RNA sensor RIG-I

Sci. Signal.  08 Dec 2015:
Vol. 8, Issue 406, pp. ec363
DOI: 10.1126/scisignal.aad9959

RIG-1 is an RNA-sensing protein that elicits innate antiviral signaling, culiminating in the production of inflammatory cytokines and type I interferons. The E3 ubiquitin ligases RNF125 and c-Cbl target RIG-I for ubiquitylation, which leads to proteasomal degradation of RIG-I. The ATPase p97 cooperates with the ubiquitin-binding proteins Ufd1 and Npl4 to form a “segregase” complex that is recruited to ubiquitylated substrates and helps extract these substrates from their native complexes for degradation. Hao et al. found that Npl1 and p97 recruited RNF125 to RIG-I, thereby limiting the antiviral response. In several human and mouse cell lines, knocking down p97, Npl4, or Ufd1 or expressing a form of p97 lacking ATPase activity increased type I interferon–dependent responses to viral infection or to expression of poly(I:C), a synthetic analog of double-stranded RNA that stimulates the RIG-I–mediated antiviral response. In cultured cells expressing poly(I:C), knocking down p97, Npl4, or Ufd1 increased the abundance of RIG-I. Overexpression of wild-type p97 or Npl4, but not mutant forms of these proteins that disrupted p97-Npl4 complex formation, promoted degradation of RIG-I. Knocking down RNF125, but not c-Cbl, prevented transgenically expressed Npl4 from promoting the degradation of RIG-I and from inhibiting interferon-dependent responses. Coexpression of RNF125 enhanced Npl4-dependent ubiquitylation of RIG-I, as assayed by mass spectrometry. Knocking down Npl4 and RNF125 inhibited viral replication in cultured cells expressing wild-type RIG-I but not in cells expressing a form of RIG-I in which the RNF125-targeted site Lys181 was mutated to Arg. Coimmunoprecipitation of endogenous proteins from HEK293T cells and in vitro pulldown assays demonstrated that RNF125 interacted directly with RIG-I and p97 and that Npl4 bound to the caspase activation and recruitment domains (CARDs) of RIG-I, which contain lysine residues targeted for ubiquitylation. The observation that Npl4 bound to purified recombinant RIG-I, which is not ubiquitylated, indicated that ubiquitylation of the RIG-I CARDS was not required for binding to Npl4, suggesting that p97-Npl4 may promote degradation of RIG-I even in the absence of viral infection. Viral infection, which elicits a conformational change in RIG-I that exposes the CARDs and enables ubiquitylation at Lys172, enhanced the interaction between Npl4 and RIG-I. A mutant form of RIG-I that cannot be ubiquitylated at Lys172 exhibited reduced binding to Npl4. In vivo, pharmacological inhibitors of p97 ATPase activity reduced serum viral titers and increased the survival of mice infected with vesicular stomatitis virus. Together, these results indicate that p97 and Npl4 not only function in the ubiquitin-proteasomal degradation pathway as components of a segregase to extract ubiquitylated proteins from complexes but also function upstream of ubiquitylation by serving as adaptors to recruit E3 ubiquitin ligases to substrates.

Q. Hao, S. Jiao, Z. Shi, C. Li, X. Meng, Z. Zhang, Y. Wang, X. Song, W. Wang, R. Zhang, Y. Zhao, C. C. L. Wong, Z. Zhou, A non-canonical role of the p97 complex in RIG-I antiviral signaling. EMBO J. 34, 2903–2920 (2015). [PubMed]