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Stress-regulated targeting of the NKG2D ligand Mult1 by a membrane-associated RING-CH family E3 ligase.

ABSTRACT: NKG2D is a stimulatory receptor expressed by NK cells and some T cell subsets. Expression of the self-encoded ligands for NKG2D is presumably tightly regulated to prevent autoimmune disorders while allowing detection of infected cells and developing tumors. The NKG2D ligand Mult1 is regulated at multiple levels, with a final layer of regulation controlling protein stability. In this article, we report that Mult1 cell-surface expression was prevented by two closely related E3 ubiquitin ligases membrane-associated RING-CH (MARCH)4 and MARCH9, members of an E3 family that regulates other immunologically active proteins. Lysines within the cytoplasmic domain of Mult1 were essential for this repression by MARCH4 or MARCH9. Downregulation of Mult1 by MARCH9 was reversed by heat-shock treatment, which resulted in the dissociation of the two proteins and increased the amount of Mult1 at the cell surface. These results identify Mult1 as a target for the MARCH family of E3 ligases and show that induction of Mult1 in response to heat shock is due to regulated association with its E3 ligases.

SUBMITTER: Nice TJ

PROVIDER: S-EPMC3001296 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Stress-regulated targeting of the NKG2D ligand Mult1 by a membrane-associated RING-CH family E3 ligase.

Nice Timothy J TJ Deng Weiwen W Coscoy Laurent L Raulet David H DH

Journal of immunology (Baltimore, Md. : 1950) 20100924 9

NKG2D is a stimulatory receptor expressed by NK cells and some T cell subsets. Expression of the self-encoded ligands for NKG2D is presumably tightly regulated to prevent autoimmune disorders while allowing detection of infected cells and developing tumors. The NKG2D ligand Mult1 is regulated at multiple levels, with a final layer of regulation controlling protein stability. In this article, we report that Mult1 cell-surface expression was prevented by two closely related E3 ubiquitin ligases me ...[more]

PMID: 20870941

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Project description:An emerging class of cellular inhibitory proteins has been identified that targets viral glycoproteins. These include the membrane-associated RING-CH (MARCH) family of E3 ubiquitin ligases that, among other functions, downregulate cell-surface proteins involved in adaptive immunity. The RING-CH domain of MARCH proteins is thought to function by catalyzing the ubiquitination of the cytoplasmic tails (CTs) of target proteins, leading to their degradation. MARCH proteins have recently been reported to target retroviral envelope glycoproteins (Env) and vesicular stomatitis virus G glycoprotein (VSV-G). However, the mechanism of antiviral activity remains poorly defined. Here we show that MARCH8 antagonizes the full-length forms of HIV-1 Env, VSV-G, Ebola virus glycoprotein (EboV-GP), and the spike (S) protein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) thereby impairing the infectivity of virions pseudotyped with these viral glycoproteins. This MARCH8-mediated targeting of viral glycoproteins requires the E3 ubiquitin ligase activity of the RING-CH domain. We observe that MARCH8 protein antagonism of VSV-G is CT dependent. In contrast, MARCH8-mediated targeting of HIV-1 Env, EboV-GP and SARS-CoV-2 S protein by MARCH8 does not require the CT, suggesting a novel mechanism of MARCH-mediated antagonism of these viral glycoproteins. Confocal microscopy data demonstrate that MARCH8 traps the viral glycoproteins in an intracellular compartment. We observe that the endogenous expression of MARCH8 in several relevant human cell types is rapidly inducible by type I interferon. These results help to inform the mechanism by which MARCH proteins exert their antiviral activity and provide insights into the role of cellular inhibitory factors in antagonizing the biogenesis, trafficking, and virion incorporation of viral glycoproteins. Viral envelope glycoproteins are an important structural component on the surface of enveloped viruses that direct virus binding and entry and also serve as targets for the host adaptive immune response. In this study, we investigate the mechanism of action of the MARCH family of cellular proteins that disrupt the trafficking and virion incorporation of viral glycoproteins across several virus families. This research provides novel insights into how host cell factors antagonize viral replication, perhaps opening new avenues for therapeutic intervention in the replication of a diverse group of highly pathogenic enveloped viruses.

Project description:UnlabelledThe rotavirus nonstructural protein NSP1 acts as an antagonist of the host antiviral response by inducing degradation of key proteins required to activate interferon (IFN) production. Protein degradation induced by NSP1 is dependent on the proteasome, and the presence of a RING domain near the N terminus has led to the hypothesis that NSP1 is an E3 ubiquitin ligase. To examine this hypothesis, pulldown assays were performed, followed by mass spectrometry to identify components of the host ubiquitination machinery that associate with NSP1. Multiple components of cullin RING ligases (CRLs), which are essential multisubunit ubiquitination complexes, were identified in association with NSP1. The mass spectrometry was validated in both transfected and infected cells to show that the NSP1 proteins from different strains of rotavirus associated with key components of CRL complexes, most notably the cullin scaffolding proteins Cul3 and Cul1. In vitro binding assays using purified proteins confirmed that NSP1 specifically interacted with Cul3 and that the N-terminal region of Cul3 was responsible for binding to NSP1. To test if NSP1 used CRL3 to induce degradation of the target protein IRF3 or ?-TrCP, Cul3 levels were knocked down using a small interfering RNA (siRNA) approach. Unexpectedly, loss of Cul3 did not rescue IRF3 or ?-TrCP from degradation in infected cells. The results indicate that, rather than actively using CRL complexes to induce degradation of target proteins required for IFN production, NSP1 may use cullin-containing complexes to prevent another cellular activity.ImportanceThe ubiquitin-proteasome pathway plays an important regulatory role in numerous cellular functions, and many viruses have evolved mechanisms to exploit or manipulate this pathway to enhance replication and spread. Rotavirus, a major cause of severe gastroenteritis in young children that causes approximately 420,000 deaths worldwide each year, utilizes the ubiquitin-proteasome system to subvert the host innate immune response by inducing the degradation of key components required for the production of interferon (IFN). Here, we show that NSP1 proteins from different rotavirus strains associate with the scaffolding proteins Cul1 and Cul3 of CRL ubiquitin ligase complexes. Nonetheless, knockdown of Cul1 and Cul3 suggests that NSP1 induces the degradation of some target proteins independently of its association with CRL complexes, stressing a need to further investigate the mechanistic details of how NSP1 subverts the host IFN response.

Dataset Information

Stress-regulated targeting of the NKG2D ligand Mult1 by a membrane-associated RING-CH family E3 ligase.

Publications

Stress-regulated targeting of the NKG2D ligand Mult1 by a membrane-associated RING-CH family E3 ligase.

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