A Mass Spectrometry-Based Profiling of Interactomes of Viral DDB1- and Cullin Ubiquitin Ligase-Binding Proteins Reveals NF-κB Inhibitory Activity of the HIV-2-Encoded Vpx

Front Immunol. 2018 Dec 19;9:2978. doi: 10.3389/fimmu.2018.02978. eCollection 2018.


Viruses and hosts are situated in a molecular arms race. To avoid morbidity and mortality, hosts evolved antiviral restriction factors. These restriction factors exert selection pressure on the viruses and drive viral evolution toward increasingly efficient immune antagonists. Numerous viruses exploit cellular DNA damage-binding protein 1 (DDB1)-containing Cullin RocA ubiquitin ligases (CRLs) to induce the ubiquitination and subsequent proteasomal degradation of antiviral factors expressed by their hosts. To establish a comprehensive understanding of the underlying protein interaction networks, we performed immuno-affinity precipitations for a panel of DDB1-interacting proteins derived from viruses such as mouse cytomegalovirus (MCMV, Murid herpesvirus [MuHV] 1), rat cytomegalovirus Maastricht MuHV2, rat cytomegalovirus English MuHV8, human cytomegalovirus (HCMV), hepatitis B virus (HBV), and human immunodeficiency virus (HIV). Cellular interaction partners were identified and quantified by mass spectrometry (MS) and validated by classical biochemistry. The comparative approach enabled us to separate unspecific interactions from specific binding partners and revealed remarkable differences in the strength of interaction with DDB1. Our analysis confirmed several previously described interactions like the interaction of the MCMV-encoded interferon antagonist pM27 with STAT2. We extended known interactions to paralogous proteins like the interaction of the HBV-encoded HBx with different Spindlin proteins and documented interactions for the first time, which explain functional data like the interaction of the HIV-2-encoded Vpr with Bax. Additionally, several novel interactions were identified, such as the association of the HIV-2-encoded Vpx with the transcription factor RelA (also called p65). For the latter interaction, we documented a functional relevance in antagonizing NF-κB-driven gene expression. The mutation of the DDB1 binding interface of Vpx significantly impaired NF-κB inhibition, indicating that Vpx counteracts NF-κB signaling by a DDB1- and CRL-dependent mechanism. In summary, our findings improve the understanding of how viral pathogens hijack cellular DDB1 and CRLs to ensure efficient replication despite the expression of host restriction factors.

Keywords: DNA damage-binding protein (DDB1); NF-κB; cytomegalovirus; hepatitis B virus (HBV); human immunodeficiency virus (HIV); interaction partner; interferon; mass spectrometry (MS).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cytomegalovirus / immunology
  • DNA-Binding Proteins / immunology
  • DNA-Binding Proteins / metabolism
  • Fibroblasts
  • Gene Expression Regulation / immunology
  • HEK293 Cells
  • HIV-2 / genetics
  • HIV-2 / immunology*
  • HIV-2 / metabolism
  • Hepatitis B virus / immunology
  • Host-Pathogen Interactions / genetics
  • Host-Pathogen Interactions / immunology
  • Humans
  • Immunoprecipitation / methods
  • Mass Spectrometry / methods
  • Mice
  • Muromegalovirus / immunology
  • NIH 3T3 Cells
  • Primary Cell Culture
  • Protein Binding / immunology*
  • Protein Interaction Mapping / methods
  • Transcription Factor RelA / genetics*
  • Transcription Factor RelA / immunology
  • Transcription Factor RelA / metabolism
  • Ubiquitin-Protein Ligases / immunology
  • Ubiquitin-Protein Ligases / metabolism
  • Viral Regulatory and Accessory Proteins / genetics
  • Viral Regulatory and Accessory Proteins / immunology
  • Viral Regulatory and Accessory Proteins / metabolism*
  • Virus Diseases / immunology*
  • Virus Diseases / virology


  • DDB1 protein, human
  • DNA-Binding Proteins
  • RELA protein, human
  • Transcription Factor RelA
  • VPX protein, Human immunodeficiency virus 2
  • Viral Regulatory and Accessory Proteins
  • CULL-RING ligase, human
  • Ubiquitin-Protein Ligases