Molecular determinants for recognition of divergent SAMHD1 proteins by the lentiviral accessory protein Vpx.
Bottom Line: Comparison with Vpx from SIV that infects sooty mangabeys (SIVsmm) complexed with SAMHD1-DCAF1 identifies molecular determinants directing Vpx lineages to N- or C-terminal SAMHD1 sequences.Inspection of the Vpx-DCAF1 interface also reveals conservation of Vpx with the evolutionally related HIV-1/SIV accessory protein Vpr.These data suggest a unified model for how Vpx and Vpr exploit DCAF1 to promote viral replication.
Affiliation: Division of Molecular Structure, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.Show MeSH
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Mentions: To locate the Vpx-DCAF1 recognition sequence within the SAMHD1mnd N terminus, a cell-based EGFP-degron reporter degradation assay was employed (Schwefel et al., 2014). Degron fusion proteins (Figure 1A) were constructed comprising two copies of EGFP with a nuclear localization signal (NLS) fused to either the N-terminal 114 residues of SAMHD1mnd (SAMHD1mnd-NtD) or a control C-terminal region of human SAMHD1, residues 600–626, (SAMHD1hs-CtD), which is targeted for degradation by Vpx from SIVsmm (Schwefel et al., 2014). Stable cell lines expressing each reporter construct were produced and expression levels of degron fusion proteins were confirmed by western blot (Figure S1). Cells were then transduced with increasing amounts of a bicistronic IRES vector expressing Vpx from SIVmnd-2 or SIVsmm and YFP, and 48 hr later, the level of degron reporter (EGFP) and Vpx (YFP) expression was measured by flow cytometry. These data show that SIVmnd-2 Vpx induces degradation of the SAMHD1mnd-NtD reporter construct but not EGFP-SAMHD1hs-CtD (Figure 1B, left panel). In contrast, SIVsmm Vpx induces degradation of the SAMHD1hs-CtD reporter but not SAMHD1mnd-NtD (Figure 1B, right panel). To further delineate the SIVmnd-2 SAMHD1 binding determinants, reporter constructs containing residues 1–37 and 37–114 of SAMHD1mnd were prepared. Residues 1–37 comprise an N-terminal disordered sequence containing a NLS and 37–114 constitutes the SAM domain (cp. PDB: 2E8O). However, neither fragment alone was sufficient to induce degradation of the reporter construct (Figure 1C), demonstrating that both the N-terminal NLS region and SAM domain are required for SIVmnd-2 Vpx/DCAF1-mediated degradation.
Affiliation: Division of Molecular Structure, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.