Ubiquitin E3 Ligase c-Cbl Is a Host Negative Regulator of Nef Protein of HIV-1

Abstract

Nef is an accessory protein encoded by human immunodeficiency virus type-1 (HIV-1) and plays important roles in regulating HIV-1 infection and viral replication. Interestingly, HIV-1 Nef can promote degradation of numerous host proteins to disrupt cellular antiviral immune response. However, how HIV-1 Nef is degraded by host factors remains largely unexplored. Here, we identified c-Cbl as a host ubiquitin E3 ligase of HIV-1 Nef. We found that c-Cbl interacts with Nef and reduces protein levels of HIV-1 Nef. Further studies demonstrated that c-Cbl promoted Lys48-linked polyubiquitination of HIV-1 Nef, thus attenuating protein stability of HIV-1 Nef. Importantly, cellular c-Cbl ubiquitinated and degraded Nef proteins produced by HIV-1 NL4-3 virions, and ultimately attenuated HIV-1 virulence for infection of THP1 cells. This study reveals a ubiquitination and proteasome-dependent degradation mechanism of HIV-1 Nef protein, and could provide potential strategies for fighting against HIV-1.

Ubiquitin-protein ligase E3A (UBE3A) mediation of viral infection and human diseases

“…Therefore, for the smooth transitioning of the HIV-1 life cycle from early to late viral gene expression, the stage-specifically expressed proteins should be degraded in a stage-specific and coordinated manner. In fact, viral proteins in HIV-1-infected cells exploit cellular machinery to decay viral and cellular proteins at every step of the HIV-1 life cycle: Nef/Env/Vpu degrade CD4 critical for the virus entry and superinfection ( Aiken et al., 1994 ; Garcia and Miller, 1992 ; Lama et al., 1999 ; Levesque et al., 2004 ; Mariani and Skowronski, 1993 ; Ruiz et al., 2010 ; Wildum et al., 2006 ; Willey et al., 1992 ), The tripartite motif-containing protein 5α (TRIM5α)-mediated degradation of Gag is integral to species tropism ( Arriagada et al., 2011 ; Battivelli et al., 2010 ; Chatterji et al., 2006 ; Sakuma et al., 2007 ; Shi and Aiken, 2006 ; Zhang et al., 2008 ), Vif/APOBEC3G determines the susceptibility of HIV-1 ( Desimmie et al., 2014 ; Ehrlich and Yu, 2006 ; Mangeat et al., 2003 ; Mariani et al., 2003 ; Stopak et al., 2003 ; Yu et al., 2003 ), c-Cbl interacts with Nef and decays it ( Zhang et al., 2020 ), and Vpu-triggered degradation of Tetherin is essential for release of the assembled virus particles ( Harris et al., 2012 ; McNatt et al., 2009 ; Miyagi et al., 2009 ; Neil et al., 2008 ), etc. Further, it is reported that HIV-1 Nef plays an important role in regulating the stability of another key HIV-1 regulatory protein, Tat ( Sugiyama et al., 2011 ).…”

Critical Role of Notch‐1 in Mechanistic Target of Rapamycin Hyperactivity and Vascular Inflammation in Patients With Takayasu Arteritis

“…and immunoprecipitation. Immunoblotting and coimmunoprecipitation were performed as previously described (11,22). The antibodies used were anti-mTOR (sc-517464; Santa Cruz Biotechnology), anti-phosphorylated S6 ribosomal protein (sc-293144; Santa Cruz Biotechnology), anti-S6 ribosomal protein (sc-74459; Santa Cruz Biotechnology), anti-Notch-1 (sc-373891; Santa Cruz Biotechnology), antiphosphorylated Akt (Ser 473 ) (9271; Cell Signaling Technology), anti-Akt (9272; Cell Signaling Technology), and anti-lysosome-associated membrane protein 1 (sc-20011; Santa Cruz Biotechnology).…”

Novel role of HIV-1 Nef in regulating the ubiquitination of cellular proteins

“…The function of HIV-1 Nef is multifarious, as the protein plays a critical role in: T cell activation for virus replication ( Sleckman et al., 1992 ; Salghetti et al., 1995 ; Brown et al., 1999 ; Kim et al., 1999 ; Schibeci et al., 2000 ; Wang et al., 2000 ); membrane trafficking of molecules, such as CD4 ( Guy et al., 1987 ; Garcia and Miller, 1991 ; Anderson et al., 1993 ; Mariani and Skowronski, 1993 ; Rhee and Marsh, 1994 ), major histocompatibility complex class (MHC I) ( Schwartz et al., 1996 ; Le Gall et al., 1997 ), and others ( Aiken et al., 1994 ; Schwartz et al., 1996 ) to facilitate HIV-1 infectivity and immune escape; stability of viral and cellular proteins via the ubiquitin proteasome system ( Sugiyama et al., 2011 ; Kmiec et al., 2018 ; Pyeon et al., 2019 ; Ali et al., 2020 ; Qiu et al., 2020 ; Zhang et al., 2020 ); chemotaxis ( Swingler et al., 1999 ; Choe et al., 2002 ; Park and He, 2009 ; Stolp et al., 2009 ; Stolp et al., 2012 ; Park and He, 2013 ; Rossi et al., 2016 ; Lamas-Murua et al., 2018 ); and intercellular communications through exosomes ( Lenassi et al., 2010 ; Narayanan et al., 2013 ; McNamara et al., 2018 ; Mukhamedova et al., 2019 ; Chen et al., 2020 ; Dubrovsky et al., 2020 ) and conduits/filopodia ( Xu et al., 2009 ; Nobile et al., 2010 ; Tan et al., 2013 ; Park et al., 2014 ). Molecular processes, such as signaling cascades, gene expression, etc., leading to these biological changes by HIV-1 Nef have been comprehensively investigated, as reviewed ( Renkema and Saksela, 2000 ; Abraham and Fackler, 2012 ; Felli et al., 2017 ; Heusinger and Kirchhoff, 2017 ; Buffalo et al., 2019 ; Staudt et al., 2020 ).…”

“…Substitution of all the lysine residues in Nef with arginine abolished Nef ubiquitination and subsequently CD4 downregulation ( Jin et al., 2008 ), indicating that Nef ubiquitination is important for modulating the surface expression of CD4. Degradation of Nef is mediated by interaction with c-Cbl, a host ubiquitin (Ub) E3 ligase ( Zhang et al., 2020 ), while our previous reports show that UBE3A (E6-AP), another host Ub E3 ligase, determines stability of Nef ( Pyeon et al., 2019 ). Nef can also promote proteasomal degradation of host cellular proteins, such as p53 tumor suppressor using UBE3A ( Ali et al., 2020 ) and CXCR4 by recruiting the HECT domain E3 ligases, AIP4 or NEDD ( Chandrasekaran et al., 2014 ).…”