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Molecular determinants for recognition of divergent SAMHD1 proteins by the lentiviral accessory protein Vpx.

Schwefel D, Boucherit VC, Christodoulou E, Walker PA, Stoye JP, Bishop KN, Taylor IA - Cell Host Microbe (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Structure, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.

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SAM Domain-SIVmnd-2 Vpx Interactions(A) The SAMHD1mnd SAM domain-SIVmnd-2 Vpx interface. The SAMHD1mnd SAM domain (magenta) and SIVmnd-2 Vpx (blue) are shown in cartoon representation. Residues making interactions are shown in stick representation with hydrogen bonding and salt bridges displayed as dashed lines.(B and C) Quantification of reporter expression in cells stably expressing SAMHD1mnd (1–114) degron reporter constructs with (B) SAM domain point mutations and (C) mandrill to human SAMHD1 amino acid substitutions. Cells were transduced with increasing titers of particles expressing SIVmnd-2 Vpx, and the level of transduction (YFP) and degron reporter (EGFP) expression was measured by flow cytometry.
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fig5: SAM Domain-SIVmnd-2 Vpx Interactions(A) The SAMHD1mnd SAM domain-SIVmnd-2 Vpx interface. The SAMHD1mnd SAM domain (magenta) and SIVmnd-2 Vpx (blue) are shown in cartoon representation. Residues making interactions are shown in stick representation with hydrogen bonding and salt bridges displayed as dashed lines.(B and C) Quantification of reporter expression in cells stably expressing SAMHD1mnd (1–114) degron reporter constructs with (B) SAM domain point mutations and (C) mandrill to human SAMHD1 amino acid substitutions. Cells were transduced with increasing titers of particles expressing SIVmnd-2 Vpx, and the level of transduction (YFP) and degron reporter (EGFP) expression was measured by flow cytometry.

Mentions: In addition to the ternary interface that “traps” the SAMHD1mnd N terminus within the complex a further DCAF1-independent interaction between the SAM domain of SAMHD1mnd-NtD and SIVmnd-2 Vpx is also observed. This interface has a buried surface area of 580 Å2 and comprises residues that project upward from Vpx α2 that contact SAMHD1 residues N-terminal to the SAM domain helix 1 as well as those on the underside of the SAM domain in helices 1 and 2 (Figure 5A). The center of the interface comprises a hydrophobic core created by Vpx residues P38, L41, F42, W45, and V49 and SAMHD1 residues P34, V36, L38, and F52 (Figure 5A, upper panel). In addition to this hydrophobic patch, there are electrostatic interactions between SAMHD1 R55 and Vpx D54 (Figure 5A, upper panel), SAMHD1 R69 and Vpx E50 (Figure 5A, lower panel), as well as hydrogen bonding from Vpx E39 to the backbone of the first turn of SAM domain helix 1 (Figure 5B, lower panel). The contribution of residues in the SAM domain-Vpx interface to degron function was assessed by introduction of alanine mutations at SAMHD1 residues R55 and R69 in the EGFP-fusion reporter. Both residues make extensive electrostatic interactions at the interface, and the alanine substitutions result in a loss of reporter degradation (Figure 5B). R69 is also among a group of residues in the N-terminal region of SAMHD1 that have undergone positive evolutionary selection (Lim et al., 2012) indicative of an interaction hotspot between restriction factor and viral antagonist, further underlining the importance of Vpx-SAM domain contacts for induction of SAMHD1 proteasomal degradation.


Molecular determinants for recognition of divergent SAMHD1 proteins by the lentiviral accessory protein Vpx.

Schwefel D, Boucherit VC, Christodoulou E, Walker PA, Stoye JP, Bishop KN, Taylor IA - Cell Host Microbe (2015)

SAM Domain-SIVmnd-2 Vpx Interactions(A) The SAMHD1mnd SAM domain-SIVmnd-2 Vpx interface. The SAMHD1mnd SAM domain (magenta) and SIVmnd-2 Vpx (blue) are shown in cartoon representation. Residues making interactions are shown in stick representation with hydrogen bonding and salt bridges displayed as dashed lines.(B and C) Quantification of reporter expression in cells stably expressing SAMHD1mnd (1–114) degron reporter constructs with (B) SAM domain point mutations and (C) mandrill to human SAMHD1 amino acid substitutions. Cells were transduced with increasing titers of particles expressing SIVmnd-2 Vpx, and the level of transduction (YFP) and degron reporter (EGFP) expression was measured by flow cytometry.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400269&req=5

fig5: SAM Domain-SIVmnd-2 Vpx Interactions(A) The SAMHD1mnd SAM domain-SIVmnd-2 Vpx interface. The SAMHD1mnd SAM domain (magenta) and SIVmnd-2 Vpx (blue) are shown in cartoon representation. Residues making interactions are shown in stick representation with hydrogen bonding and salt bridges displayed as dashed lines.(B and C) Quantification of reporter expression in cells stably expressing SAMHD1mnd (1–114) degron reporter constructs with (B) SAM domain point mutations and (C) mandrill to human SAMHD1 amino acid substitutions. Cells were transduced with increasing titers of particles expressing SIVmnd-2 Vpx, and the level of transduction (YFP) and degron reporter (EGFP) expression was measured by flow cytometry.
Mentions: In addition to the ternary interface that “traps” the SAMHD1mnd N terminus within the complex a further DCAF1-independent interaction between the SAM domain of SAMHD1mnd-NtD and SIVmnd-2 Vpx is also observed. This interface has a buried surface area of 580 Å2 and comprises residues that project upward from Vpx α2 that contact SAMHD1 residues N-terminal to the SAM domain helix 1 as well as those on the underside of the SAM domain in helices 1 and 2 (Figure 5A). The center of the interface comprises a hydrophobic core created by Vpx residues P38, L41, F42, W45, and V49 and SAMHD1 residues P34, V36, L38, and F52 (Figure 5A, upper panel). In addition to this hydrophobic patch, there are electrostatic interactions between SAMHD1 R55 and Vpx D54 (Figure 5A, upper panel), SAMHD1 R69 and Vpx E50 (Figure 5A, lower panel), as well as hydrogen bonding from Vpx E39 to the backbone of the first turn of SAM domain helix 1 (Figure 5B, lower panel). The contribution of residues in the SAM domain-Vpx interface to degron function was assessed by introduction of alanine mutations at SAMHD1 residues R55 and R69 in the EGFP-fusion reporter. Both residues make extensive electrostatic interactions at the interface, and the alanine substitutions result in a loss of reporter degradation (Figure 5B). R69 is also among a group of residues in the N-terminal region of SAMHD1 that have undergone positive evolutionary selection (Lim et al., 2012) indicative of an interaction hotspot between restriction factor and viral antagonist, further underlining the importance of Vpx-SAM domain contacts for induction of SAMHD1 proteasomal degradation.

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.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Structure, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.

Show MeSH
Related in: MedlinePlus