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

Abstract

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.

The Human Cytomegalovirus pUL145 Isoforms Act as Viral DDB1-Cullin-Associated Factors to Instruct Host Protein Degradation to Impede Innate Immunity

“…However, MCMV does not code for a pUL145 homolog and instead induces STAT2 degradation by the protein pM27. The HCMVencoded protein pUL27, the homolog of pM27, hardly binds DDB1 and is dispensable for STAT2 degradation (Landsberg et al, 2018;Le et al, 2008). This suggests that MCMV may fail to predict functions of homologous HCMV proteins (e.g., pUL27) but discloses general principles of CMV immune evasion like STAT2 degradation and DDB1/CRL exploitation.…”

“…Our experiments showed similarities between the newly identified HCMV antagonist, pUL145, and the known MCMV-encoded STAT2 inhibitor pM27. Since pM27 recruits DDB1 to exploit cellular CRLs for poly-ubiquitination and proteasomal degradation of STAT2 (Landsberg et al, 2018;Trilling et al, 2011), and proteasomal inhibitors as well as the CRL inhibitory drug MLN4924 restore STAT2 in HCMV-infected cells Le et al, 2008), we tested if pUL145 also exploits DDB1 and associated CRLs for STAT2 degradation. To analyze this in the infection context, we constructed a recombinant HCMV based on the AD169varS background, which expresses an HA-tagged version of pUL145 under the control of the cellular EF1 promoter (AD169varS:UL145-HA).…”

Cellular Cullin RING Ubiquitin Ligases: Druggable Host Dependency Factors of Cytomegaloviruses

“…By this simple yet elegant mechanism, viruses exploit the cell intrinsic ubiquitin-proteasome system to defeat antiviral immunity. Well-known examples of viral exploitation of DDB1 and/or CRLs include the hepatitis B virus (HBV) protein HBx [84,89,90,91,92], the HIV-1- and HIV-2-encoded protein Vpr [93,94,95,96,97,98,99,100,101,102], HIV-2 Vpx [103,104,105,106,107,108,109], parainfluenza virus (PIV) V proteins [78,110,111,112], bovine herpesvirus 1 (BoHV-1) VP8 [113,114], murine gamma herpesvirus (MHV68) M2 [115], as well as the cytomegalovirus proteins pM27 [116,117,118], pUL35 [119], and pUL145 [120].…”

Convergent Evolution of HLA-C Downmodulation in HIV-1 and HIV-2

“…A similar example of convergent evolution was previously described for the inhibition of NF-κB activation. While HIV-1 and related lentiviruses use Vpu to suppress NF-κB-driven immune activation ( 37 , 41 ), HIV-2 and other vpu -deficient viruses use their accessory protein Vpr, Vpx, or Nef to achieve this ( 76 , 77 ). The evolution of independent evasion mechanisms in different primate lentiviral lineages illustrates the selection pressure exerted by the respective immune responses.…”