Vpx associates with the DCAF1/VprBP substrate receptor subunit of the Cullin4-based E3 ubiquitin ligase CRL4DCAF1
. Failure to infect macrophages and dendritic cells by HIV-2 and SIVmac viruses encoding mutant Vpx proteins deficient in association with this E3 suggested that Vpx usurps CRL4DCAF1
to overcome the inhibition of infection in myeloid cells12,13
. The inhibition of HIV-1 infection in monocyte-derived macrophages (MDM) can be overcome by co-infection with Vpx-containing SIVsm virus-like particles (SIV VLP)4,14
. These SIV VLP do not contain any viral genetic material and can be used to deliver Vpx, which is naturally packaged into virion cores through its interaction with Gag15,16
, to target cells. To assess whether Vpx-mediated relief of the inhibition of HIV-1 infection in macrophages is also linked to CRL4DCAF1
, we co-infected human MDM with VSV-G-pseudotyped HIV-1 NL4-3-derived single cycle reporter virus encoding the GFP reporter (HIV-1–GFP) in the presence of VSV-G-pseudotyped SIV VLP loaded with HIV-2 Rod Vpx (SIV VLP(Vpx)) or with a Vpx variant (VpxQ76A
), that does not bind to DCAF112
. Wild type Vpx greatly enhanced MDM transduction by HIV-1–GFP, whereas the VpxQ76A
did not have such a stimulatory effect (), and viral cDNA synthesis was inefficient both in the absence of Vpx and upon co-infection with VpxQ76A
(), as also observed with vpx
-defective single cycle HIV-2–GFP and SIVmac–GFP reporter viruses12
(Supplementary Figure 1
). This evidence supports the model in which Vpx relieves the inhibition of HIV-1 infection in MDM by targeting an unknown anti-viral inhibitory protein for proteasome-dependent degradation, via
E3 ubiquitin ligase.
Vpx-mediated relief of the inhibition of HIV-1 infection in MDM requires Vpx glutamine Q76
To identify Vpx-recruited substrates for the CRL4DCAF1
ubiquitin ligase, a proteomic screen was used to search for the cellular protein(s) that associate with the CRL4DCAF1
complex only in the presence of Vpx. Specifically, we directed the assembly of three sets of protein complexes in HEK 293T cells for their subsequent proteomic characterization (). First, to capture the cellular proteins bound by Vpx, we transiently expressed a functional SIVmac 239 Vpx protein tagged with FLAG- and HA- epitopes in tandem. Second, to trap proteins recruited by Vpx to CRL4DCAF1
, we assembled quaternary complexes comprising HA-Cullin4, DDB1, DCAF1 and FLAG-Vpx, in cells transiently expressing epitope-tagged Cullin4 and Vpx subunits. The placement of epitope tags on Cullin4 and Vpx, subunits that are the most distally located in the quaternary complex17
, ensured that partial complexes lacking one or more subunits, or comprising CRL4 substrate receptors other than DCAF1, would not co-purify with the dually-tagged complexes. Third, as a negative control, we assembled and purified a ternary HA-Cullin4, DDB1, FLAG-DCAF1 complex, lacking Vpx. All protein complexes were purified by two sequential immunoprecipitations of epitope tags, under native conditions, and their subunit composition was analyzed by Multidimensional Protein Identification Technology (MudPIT)18
Vpx recruits SAMHD1 to DDB1-DCAF1 module of CRL4DCAF1 E3 complex
The most abundant of the candidate proteins uncovered in this screen was the innate immune SAM-domain HD-domain containing protein 1 (SAMHD1), which co-purified with both Vpx (6 spectra) and Vpx-CRL4DCAF1
complex (37 spectra), but not with CRL4DCAF1
complex alone (Supplementary Table I
). Interestingly, SAMHD1
mutations are associated with Aicardi-Goutieres syndrome (AGS), an autoimmune disease caused by an abnormal innate immune response to endogenous nucleic acids7,8
. Besides SAMHD1, approximately 150 cellular proteins were found associated with Cullin4 in the presence of Vpx. Because these proteins were low quality hits represented by three or fewer spectra, and/or were found previously to be frequent contaminants, they were not considered to be viable candidates.
To assess whether SAMHD1 binding and recruitment to the DCAF-linked complex are conserved functions of Vpx, we investigated HIV-2 Rod Vpx, which, as SIVmac 239 Vpx, is also functional6,12,13
. As a negative control we selected the Vpr accessory proteins of HIV-1 and SIVmac, because they are closely related to Vpx, sharing roughly 25% and 50% amino acid identity, and can load cellular substrate proteins onto CRL4DCAF1
but, unlike Vpx, Vpr does not alleviate the inhibition of lentivirus infection in MDM. Human epitope-tagged SAMHD1 was co-expressed with Vpx, or Vpr, in HEK 293T cells and Vpx/Vpr immune complexes were analyzed by Western blotting. SAMHD1 co-precipitated efficiently with both Vpx variants, but not with Vpr (). Next, we confirmed that Vpx specifically links SAMHD1 to the CRL4DCAF1
E3 complex. SAMHD1 was co-expressed with wild type SIVmac Vpx, a VpxQ76A
variant that does not bind DCAF1, or HIV-1 Vpr that does not bind SAMHD1, in HEK 293T cells. Analysis of SAMHD1 immune complexes revealed that SAMHD1 directed assembly of a protein complex containing Vpx, and the DCAF1- and DDB1- subunits of CRL4DCAF1
complex (). In contrast, neither VpxQ76A
, which exhibited reduced binding to SAMHD1, nor Vpr, formed such complexes. We conclude that Vpx specifically binds to, and recruits SAMHD1 to the DCAF1-DDB1 module of CRL4DCAF1
E3 ubiquitin ligase.
Because SAMHD1 was originally identified in HEK 293T cells, it was important to confirm that it is both expressed and targeted by Vpx for degradation via
in MDM. We therefore performed Western blot analysis of MDM lysates and measured steady state SAMHD1 levels following MDM infection with SIV VLP loaded with either Vpx or the VpxQ76A
variant control (). We observed that wild type Vpx readily depleted endogenous SAMHD1 levels. In contrast, VpxQ76A
, which is defective for DCAF1 binding12
, did not decrease SAMHD1 levels. Importantly, the effect of Vpx was blocked when macrophages were exposed to the proteasome inhibitor MG132 prior to and during infection with Vpx-loaded SIV VLP (). We conclude that Vpx programs SAMHD1 for destruction in MDM.
Vpx programs SAMHD1 for proteasomal degradation in MDM, via CRL4DCAF1 E3
Our above biochemical and functional studies with VpxQ76A variants suggested that Vpx uses the CRL4DCAF1 E3 ubiquitin ligase to program SAMHD1 degradation. To test this directly, we first depleted the DCAF1 subunit of CRL4DCAF1 by RNA interference (RNAi), and then characterized the effects of this knockdown on Vpx-mediated SAMHD1 depletion in MDM. MDM were transfected with siRNA targeting DCAF1 or with non-targeting control siRNAs (NT1, NT2). DCAF1-specific siRNA caused a large reduction in DCAF1 levels (). Two days following the initiation of RNAi, the cells were infected with Vpx-loaded SIV VLP and the SAMHD1 levels were assessed 2 days later. In the absence of Vpx (left panels) there was no significant change in SAMHD1 levels following treatment with either the DCAF1, or control siRNAs. Significantly, Vpx induced an almost complete degradation of SAMHD1 in cells treated with the control siRNAs. By contrast, in the DCAF1 depleted cells degradation of SAMHD1 induced by Vpx was significantly inhibited providing further evidence for our hypothesis that Vpx utilizes CRL4DCAF1 E3 to program SAMHD1 degradation.
Then we set out to investigate the role of SAMHD1 in HIV-1 infection in MDM. Unfortunately, RNAi-mediated knockdown of SAMHD1 was only weakly effective in MDM (, left panel). We believe that SAMHD1 levels in MDM are resistant to depletion by RNAi because the half-life of SAMHD1 is long, and RNAi can only prevent new protein synthesis; thus, due to slow turnover rate, it cannot achieve depletion of existing SAMHD1 protein. Hence, we developed a two-step protocol to manipulate SAMHD1 levels over a wide concentration range by using a combination of RNAi and SIV VLP to deliver to cells low doses of Vpx. In the first step, macrophages were infected with low doses of Vpx-loaded SIV VLP to deplete the pre-existing SAMHD1 pool. In the second step we blocked SAMHD1 re-synthesis by RNAi.
As shown in , freshly isolated CD14+
monocytes cultured in the presence of M-CSF were infected with increasing, but suboptimal, doses of Vpx-loaded SIV VLP. RNAi targeted to SAMHD1 or a non-targeting RNAi was initiated 4 days later. Three days following the initiation of RNAi the macrophages were challenged with HIV-1–GFP reporter virus. We observed that RNAi to SAMHD1 stimulated MDM transduction by several-fold, compared to non-targeting RNAi, throughout the entire titration range for Vpx, and the permissiveness to HIV-1–GFP transduction showed good, but not simple, inverse correlation with SAMHD1 levels in MDM (, see also Supplementary Figures 2, 3 and 4
). The most dramatic increases in MDM permissiveness were revealed at low doses of Vpx that on their own barely stimulated HIV-1 transduction. Quantification of viral reverse transcription intermediates showed that depleting SAMHD1 levels stimulated synthesis of viral full length cDNA by approximately 10-fold (), and again this effect was well pronounced even at the lowest doses of Vpx. Specifically, it can be clearly seen that the “early” (“E”) vs. “late” (“L”) cDNA PCR readouts showed selective repression of “late” reverse transcription products at high but not low SAMHD1 levels. Furthermore, MDM transduction by Vpx-defective HIV-2vpx-
–GFP and SIVmacvpx-
–GFP reporter constructs was also enhanced up to 12-fold, by siRNA to SAMHD1 (). This evidence clearly demonstrates that SAMHD1 inhibits MDM infection by HIV-1 and other primate lentiviruses by disrupting synthesis of viral cDNA.
SAMHD1 inhibits HIV-1 infection in macrophages
In summary, our results show that SAMHD1 interferes with HIV infection of macrophages by preventing efficient viral cDNA synthesis. The Vpx accessory protein of HIV-2/SIVsm lineage viruses removes this inhibition by targeting SAMHD1 for proteasome-dependent degradation via
E3 ubiquitin ligase (Supplementary Figure 5
). Since HIV-2 and related simian viruses have evolved the Vpx function to counteract SAMHD1, they are able to infect macrophages much more readily than HIV-1. Indeed, vpx
-deficient SIVsm/mac viruses are attenuated in simian models of AIDS, and show evidence for defective macrophage-dependent dissemination at early stages of infection and the apparent absence of macrophage-driven encephalitis at later stages22,23
. Our discovery that the SAMHD1 protein inhibits HIV infection in macrophages unveils a novel and unexpected link between unconventional cell-intrinsic innate immune mechanisms and the anti-viral defence. Notably, in addition to SAMHD1, two nucleases TREX1 and RNAse H2 have been linked to Aicardi-Goutieres syndrome8
. Recent studies of TREX1 suggest that it plays a role in the control of retrotransposon and endogenous retroviral elements, and in inhibiting the innate immune response to HIV-1 DNA in T cells and macrophages by clearing excess of viral cDNA9,24
. However, the reported effects of TREX1 on HIV-1 were not associated with an overt restriction of HIV-1 infection in either cell type, in contrast to our findings with SAMHD1.
One unanswered question is how myeloid cell specificity of SAMHD1-mediated inhibition of HIV-1 infection is achieved? SAMHD1 is expressed in HEK 293T cells, undifferentiated THP-1 cells and other non-myeloid cell types that do not possess a Vpx-sensitive mechanism restricting primate lentivirus infection (Supplementary Figures 6 and 7
). One possible explanation is that SAMHD1-mediated restriction may require a myeloid cell-specific molecule. Alternatively, cell type specific differences in early post-entry events involved in viral core uncoating and/or reverse transcription complex function may make HIV/SIV prone to SAMHD1 specifically in myeloid cells25,26
. Of note, we observed that SAMHD1 levels are depleted by Vpx in both MDM and monocyte-derived dendritic cells in a Vpx glutamine Q76 dependent manner (data not shown) implicating SAMHD1 as the key AGS protein inhibiting lentivirus infection in myeloid cells. Whether SAMHD1 functions autonomously, what are the other key components of a putative SAMHD1 pathway, and what are the salient features of SAMHD1 that endow this protein with anti-HIV activity, requires further investigation.