Molecular mechanisms of the interplay between Vpx and SAMHD1

SAMHD1, a dGTP-regulated deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase, down-regulates dNTP pools in terminally differentiated and quiescent cells, thereby inhibiting HIV-1 infection at the reverse transcription step. HIV-2 and SIV counteract this restriction via a virion-associated virulence accessory factor, Vpx (Vpr in some SIVs), which loads SAMHD1 onto CRL4-DCAF1 E3 ubiquitin ligase for poly-ubiquitination, programming it for proteasome-dependent degradation. However, the detailed molecular mechanisms of SAMHD1 recruitment to the E3 ligase have not been defined. Further, whether divergent, orthologous Vpr/Vpx proteins, encoded by distinct HIV/SIV strains, bind SAMHD1 in a similar manner, at a molecular level, is not known

Evolutionary Toggling of Vpx/Vpr Specificity Results in Divergent Recognition of the Restriction Factor SAMHD1

SAMHD1 is a host restriction factor that blocks the ability of lentiviruses such as HIV-1 to undergo reverse transcription in myeloid cells and resting T-cells. This restriction is alleviated by expression of the lentiviral accessory proteins Vpx and Vpr (Vpx/Vpr), which target SAMHD1 for proteasome-mediated degradation. However, the precise determinants within SAMHD1 for recognition by Vpx/Vpr remain unclear. Here we show that evolution of Vpx/Vpr in primate lentiviruses has caused the interface between SAMHD1 and Vpx/Vpr to alter during primate lentiviral evolution. Using multiple HIV-2 and SIV Vpx proteins, we show that Vpx from the HIV-2 and SIVmac lineage, but not Vpx from the SIVmnd2 and SIVrcm lineage, require the C-terminus of SAMHD1 for interaction, ubiquitylation, and degradation. On the other hand, the N-terminus of SAMHD1 governs interactions with Vpx from SIVmnd2 and SIVrcm, but has little effect on Vpx from HIV-2 and SIVmac. Furthermore, we show here that this difference in SAMHD1 recognition is evolutionarily dynamic, with the importance of the N- and C-terminus for interaction of SAMHD1 with Vpx and Vpr toggling during lentiviral evolution. We present a model to explain how the head-to-tail conformation of SAMHD1 proteins favors toggling of the interaction sites by Vpx/Vpr during this virus-host arms race. Such drastic functional divergence within a lentiviral protein highlights a novel plasticity in the evolutionary dynamics of viral antagonists for restriction factors during lentiviral adaptation to its hosts. © 2013 Fregoso et al

A Novel T-Cell Engaging Bi-specific Antibody Targeting the Leukemia Antigen PR1/HLA-A2

Despite substantial advances in the treatment of acute myeloid leukemia (AML), only 30% of patients survive more than 5 years. Therefore, new therapeutics are much needed. Here, we present a novel therapeutic strategy targeting PR1, an HLA-A2 restricted myeloid leukemia antigen. Previously, we have developed and characterized a novel T-cell receptor-like monoclonal antibody (8F4) that targets PR1/HLA-A2 and eliminates AML xenografts by antibody-dependent cellular cytotoxicity (ADCC). To improve the potency of 8F4, we adopted a strategy to link T-cell cytotoxicity with a bi-specific T-cell-engaging antibody that binds PR1/HLA-A2 on leukemia and CD3 on neighboring T-cells. The 8F4 bi-specific antibody maintained high affinity and specific binding to PR1/HLA-A2 comparable to parent 8F4 antibody, shown by flow cytometry and Bio-Layer Interferometry. In addition, 8F4 bi-specific antibody activated donor T-cells in the presence of HLA-A2+ primary AML blasts and cell lines in a dose dependent manner. Importantly, activated T-cells lysed HLA-A2+ primary AML blasts and cell lines after addition of 8F4 bi-specific antibody. In conclusion, our studies demonstrate the therapeutic potential of a novel bi-specific antibody targeting the PR1/HLA-A2 leukemia-associated antigen, justifying further clinical development of this strategy

SIVrcm Vpx utilizes a unique interface to degrade SAMHD1.

<p>(A) Alignment of the amino acids in Vpx that have been previously shown to be important for SIVmac Vpx-mediated degradation of SAMHD1, highlighted in in black with amino acid numbers above. (B) Wild type and mutant Vpx constructs were tested for their ability to degrade SAMHD1 by cotransfection in 293T cells, and analyzed by western blotting. HIV-2_{12 Mnd 17} and Mac_{12 Mnd 17} (or HIV-2_{12 RCM 17} and Mac_{12 RCM 17}) indicates the amino acids 12 through 17 of HIV-2 Vpx and SIVmac Vpx that have been changed to the corresponding amino acids found in SIVmnd2 Vpx (or SIVrcm Vpx), while RCM_{12 HIV-2 17} and Mnd2_{12 HIV-2 17} indicates that amino acids 12 through 17 of SIVrcm and SIVmnd2 Vpx have been changed to the corresponding amino acids found in HIV-2/SIVmac Vpx. – indicates no Vpx empty vector control.</p

Vpx specificity for N- versus C-terminal binding of SAMHD1 is independent of the SAMHD1 homologue.

<p>(A) Empty vector control, C-terminal binding FLAG-SIVmac Vpx, or N-terminal binding FLAG-SIVmnd2 Vpx were co-transfected in 293T cells with HA-SAMHD1, either WT or ΔC, from multiple primate species, and degradation of SAMHD1 was measured by western blotting. (B) Same as in (A) except chimeric SAMHD1 proteins were used instead of WT or ΔC.</p

N- and C-terminal binding Vpx proteins degrade SAMHD1 through a conserved mechanism.

<p>(A) Schematic representation of wild type (WT) SAMHD1, C-terminally truncated SAMHD1 (ΔC, shown in green), and N-terminally truncated SAMHD1 (ΔN, shown in blue). Amino acid numbers of the truncations are shown, with dotted line indicating the truncated region of SAMHD1. (B) HA-SAMHD1 (WT, ΔC, and ΔN) were transiently co-expressed in 293T cells with the autologous FLAG-Vpx and immunoprecipitated from whole cell extracts with anti-HA resin. HA-SAMHD1, FLAG-Vpx, DCAF, and DDB1 were detected in immune complexes (top panels) or extracts (bottom panels) by western blotting. * denotes the antibody light-chain. (C) <i>In vitro</i> ubiquitylation of WT, ΔC, and ΔN rhesus SAMHD1, in the presence or absence of SIVmac Vpx (left panel) or SIVrcm Vpx (right panel). SAMHD1 and Vpx were incubated with Cul4, DCAF1c, RBX1, UBA1 (an E1 enzyme), UbcH5b (an E2 enzyme) and FLAG-tagged ubiquitin for increasing time (0, 15, 30 min), and ubiquitylation of each SAMHD1 construct was analyzed by western blotting.</p

SIVmnd2 and SIVrcm Vpx require the N-terminus of SAMHD1 for degradation.

<p>(A) Schematic representation of WT SAMHD1 and chimeric proteins. Amino acids in the N-terminus that differ between human (Hu) SAMHD1 and mandrill (Mnd) or RCM SAMHD1 are shown as tick-marks, while codons that differ and have been shown to be evolving under strong positive selection (amino acids 32, 36, and 107) are shown with arrowheads. The regions of the chimeric SAMHD1 protein coming from the human gene are shown in purple, while the regions coming from mandrill or RCM SAMHD1 are shown in grey. (B) 293T cells were transfected with HA-tagged WT or chimeric SAMHD1, plus or minus (−) FLAG-Vpx, and degradation was measured by western blotting as described in the legend for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003496#ppat-1003496-g001" target="_blank">Figure 1</a>.</p

HIV-2 and SIVmac Vpx require the C-terminus of SAMHD1 for degradation.

<p>(A) Schematic Vpx/Vpr phylogenetic tree based on <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003496#ppat.1003496-Lim1" target="_blank">[21]</a> of select lentiviral Vpx and Vpr proteins that degrade SAMHD1. (B) Schematic representation of SAMHD1 (WT) and C-terminal truncation (ΔC), including the SAM and HD domains shown in grey. The deletion at the C-terminus of SAMHD1 (green dotted line) truncates the protein at amino acid 611. Amino acid numbers are shown to indicate the relative boundaries of the domains. (C) 293T cells were transfected with HA-SAMHD1, either WT or ΔC (green), plus or minus (−) FLAG-Vpx from autologous viruses, and degradation was measured by western blotting. SAMHD1 levels are shown on the top blot, Vpx levels are shown in the middle blot, and tubulin levels are shown in the bottom blot (as a loading control). Note the differences between degradation of the WT SAMHD1 versus the ΔC SAMHD1 in the presence of some Vpx proteins, but not others.</p