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ABSTRACT The type 1 interferon-regulated E3 ubiquitin ligase MARCH1 reduces surface expression of HIV-1 envelope glycoproteins (Env) and their packaging into nascent virions, a condition that restricts viral infectivity. However, how HIV-1 counters this restriction, notably during infection of macrophages, remains unclear. Here, we show that the HIV-1 accessory protein Vpu increases the levels of microRNAs-25 and -93 to target MARCH1 mRNA. By recruiting β-TRCP, a component of the SCFβ-TRCP E3 ligase complex that targets phosphorylated β-catenin for degradation, Vpu increases β-catenin levels, which, in concert with TCF4/LEF, drives transcription of the MARCH1-targeting microRNAs. This potentiates HIV-1 infectivity as a result of increased Env incorporation into nascent virions. Pharmacological targeting of the β-catenin pathway inhibits Vpu-mediated upregulation of microRNAs-25 and -93 and restores MARCH1 restriction on HIV-1 infectivity. Overall, our findings highlight a novel mechanism by which HIV-1 counteracts MARCH1 by downregulating its expression via Vpu-mediated induction of microRNAs-25 and -93. IMPORTANCE In order to efficiently produce infectious viral particles, HIV must counter several restrictions exerted by host cell antiviral proteins. MARCH1 is a member of the MARCH protein family that restricts HIV infection by limiting the incorporation of viral envelope glycoproteins into nascent virions. Here, we identified two regulatory RNAs, microRNAs-25 and -93, induced by the HIV-1 accessory protein Vpu, that downregulate MARCH1 mRNA. We also show that Vpu induces these cellular microRNAs in macrophages by hijacking the cellular β-catenin pathway. The notion that HIV-1 has evolved a mechanism to counteract MARCH1 restriction on viral infectivity underlines the importance of MARCH1 in the host antiviral response.

The membrane-associated RING-CH-type finger (MARCH) proteins of E3 ubiquitin ligases have emerged as critical regulators of immune responses. MARCH proteins target immune receptors, viral proteins as well as components in innate immune response for polyubiquitination and degradations via distinct routes. This review summarizes the current progress about MARCH proteins and their regulation on immune responses.

The membrane-associated RING-CH-type finger (MARCH) proteins of E3 ubiquitin ligases have emerged as critical regulators of immune responses. MARCH proteins target immune receptors, viral proteins as well as components in innate immune response for polyubiquitination and degradations via distinct routes. This review summarizes the current progress about MARCH proteins and their regulation on immune responses. [ABSTRACT FROM AUTHOR]

The MARCH family of RING-finger E3 ubiquitin ligases comprise 11 members that have been reported to play a variety of roles in the downregulation of cell-surface proteins involved in adaptive immunity. The RING-CH domain of MARCH proteins is thought to ubiquitinate the cytoplasmic tails (CTs) of target proteins, leading to protein degradation through either lysosomal or proteasomal pathways. Three MARCH proteins (MARCH1, 2, and 8) have recently been reported to target the HIV-1 envelope glycoprotein (Env) and vesicular stomatitis virus G glycoprotein (VSV-G), thereby impairing the infectivity of HIV-1 virions bearing HIV-1 Env or VSV-G. However, the mechanism of antiviral activity remains poorly defined. Our data show that MARCH proteins antagonize the full-length forms of HIV-1 Env, VSV-G, and Ebola glycoprotein (GP), and impair the infectivity of HIV-1 virions bearing these viral glycoproteins. This Env-targeting activity of the MARCH proteins requires the E3 ubiquitin ligase activity of the RING-CH domain. We observe that the MARCH protein targeting of VSV-G is, to a large extent, CT-dependent. In striking contrast, the MARCH-protein targeting of HIV-1 Env and Ebola GP does not require the CT. Confocal microscopy data demonstrate that MARCH proteins are able to trap the viral glycoproteins in an intracellular compartment. We observe that the endogenous expression of MARCH8 in T-cell lines and PBMCs is inducible by type I interferons (a and b) and is also upregulated by HIV-1 infection. Current studies are aimed at identifying the cellular target for MARCH-mediated ubiquitination in the context of their antiviral activity. These results will clarify the mechanism by which MARCH proteins antagonize viral glycoproteins and provide insights into the antiviral role of cellular inhibitory factors in Env biogenesis, trafficking, and virion incorporation.

Importance: In order to efficiently produce infectious viral particles, HIV must counter several restrictions exerted by host cell antiviral proteins. MARCH1 is a member of the MARCH protein family that restricts HIV infection by limiting the incorporation of viral envelope glycoproteins into nascent virions. Here, we identified two regulatory RNAs, microRNAs-25 and -93, induced by the HIV-1 accessory protein Vpu, that downregulate MARCH1 mRNA. We also show that Vpu induces these cellular microRNAs in macrophages by hijacking the cellular β-catenin pathway. The notion that HIV-1 has evolved a mechanism to counteract MARCH1 restriction on viral infectivity underlines the importance of MARCH1 in the host antiviral response., Competing Interests: The authors declare no conflict of interest.

Allogeneic, off-the-shelf (OTS) chimeric antigen receptor (CAR) cell therapies have the potential to reduce manufacturing costs and variability while providing broader accessibility to cancer patients and those with other diseases. However, host-versus-graft reactivity can limit the durability and efficacy of OTS cell therapies requiring new strategies to evade adaptive and innate-immune responses. Human herpes virus-8 (HHV8) maintains infection, in part, by evading host T and natural killer (NK) cell attack. The viral K3 gene encodes a membrane-tethered E3 ubiquitin ligase that discretely targets major histocompatibility complex (MHC) class I components, whereas K5 encodes a similar E3 ligase with broader specificity, including MHC-II and the MHC-like MHC class I polypeptide-related sequence A (MIC-A)- and sequence B (MIC-B)-activating ligands of NK cells. We created γ-retroviruses encoding K3 and/or K5 transgenes that efficiently transduce primary human T cells. Expression of K3 or K5 resulted in dramatic downregulation of MHC-IA (human leukocyte antigen [HLA]-A, -B, and -C) and MHC class II (HLA-DR) cell-surface expression. K3 expression was sufficient for T cells to resist exogenously loaded peptide-MHC-specific cytotoxicity, as well as recognition in one-way allogeneic mixed lymphocyte reactions. Further, in immunodeficient mice engrafted with allogeneic T cells, K3-transduced T cells selectively expanded in vivo. Ectopic K5 expression in MHC class I - , MIC-A + /B + K562 cells also reduced targeting by primary NK cells. Coexpression of K3 in prostate stem cell antigen (PSCA)-directed, inducible MyD88/CD40 (iMC)-enhanced CAR-T cells did not impact cytotoxicity, T cell growth, or cytokine production against HPAC pancreatic tumor target cells, whereas K5-expressing cells showed a modest reduction in interleukin (IL)-2 production without effect on cytotoxicity. Together, these results support application of these E3 ligases to advance development of OTS CAR-T cell products., (Copyright © 2020. Published by Elsevier Inc.)

Interleukin 6 (IL6) is a cytokine that regulates a number of important immune and inflammatory pathways. We used the ability of IL6 to inhibit the clonal proliferation of the mouse M1 myeloid leukemia cell line in agar to positively screen a cDNA expression library for proteins that inhibited IL6 activity. We found three clones completely resistant to IL6 that contained the cDNA for the Membrane-Associated RING-CH E3 ubiquitin ligase MARCH2. MARCH2 is a member of a family of membrane-bound E3 ubiquitin ligases that target cell surface receptors for degradation. MARCH2 overexpressing M1 clones retained responsiveness to the related cytokines leukemia inhibitory factor and oncostatin M and we showed that its inhibitory effect was a result of selective down-regulation of the IL6 receptor alpha chain and not the shared receptor subunit, gp130 or other signalling molecules. This activity of MARCH2 was also shared with related proteins MARCH4, MARCH9 and an isoform of MARCH3. The transmembrane domains and C-terminal domains, as well as a functional RING domain, of MARCH proteins were all required for substrate recognition and down-regulation. Genetic deletion of individual MARCH proteins in mice had no or little effect on IL6Rα levels but combined deletions of MARCH2,3 and 4 displayed elevated steady-state levels of IL6Rα in selected haemopoietic cell subsets including CD8+ and CD4+ T cells. These studies extend the potential immunosuppressive roles of MARCH proteins to include down-regulation of IL6 inflammatory responses., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)