Evidence for IFNα-induced, SAMHD1-independent inhibitors of early HIV-1 infection
- Caroline Goujon†1,
- Torsten Schaller†1,
- Rui Pedro Galão1,
- Sarah M Amie2,
- Baek Kim2,
- Kevin Olivieri3,
- Stuart JD Neil1 and
- Michael H Malim1Email author
© Goujon et al; licensee BioMed Central Ltd. 2013
Received: 26 November 2012
Accepted: 19 February 2013
Published: 25 February 2013
Type I interferon (IFN) treatment of some cells, including dendritic cells, macrophages and monocytic THP-1 cells, restricts HIV-1 infection and prevents viral cDNA accumulation. Sterile alpha motif and HD domain protein 1 (SAMHD1), a dGTP-regulated deoxynucleotide triphosphohydrolase, reduces HIV-1 infectivity in myeloid cells, likely by limiting dNTPs available for reverse transcription, and has been described as IFNα-inducible. Myeloid cell infection by HIV-1 is enhanced by HIV-2/SIVSM Vpx, which promotes SAMHD1 degradation, or by exogenous deoxyribonucleoside (dN) addition.
SAMHD1 expression was not substantially influenced by IFNα treatment of monocyte-derived macrophages or THP-1 cells. The contributions of SAMHD1 to the inhibition of HIV-1 infectivity by IFNα were assessed through the provision of Vpx, exogenous dN addition, or via RNAi-mediated SAMHD1 knock-down. Both Vpx and dN efficiently restored infection in IFNα-treated macrophages, albeit not to the levels seen with these treatments in the absence of IFNα. Similarly using differentiated THP-1 cells, the addition of Vpx or dNs, or SAMHD1 knock-down, also stimulated infection, but failing to match the levels observed without IFNα. Neither Vpx addition nor SAMHD1 knock-down reversed the IFNα-induced blocks to HIV-1 infection seen in dividing U87-MG or THP-1 cells. Therefore, altered SAMHD1 expression or function cannot account for the IFNα-induced restriction to HIV-1 infection seen in many cells and cell lines.
IFNα establishes an anti-HIV-1 phenotype in many cell types, and appears to accomplish this without potentiating SAMHD1 function. We conclude that additional IFNα-induced suppressors of the early stages of HIV-1 infection await identification.
KeywordsHIV-1 Interferon Restriction Macrophages SAMHD1 Vpx Deoxyribonucleosides
Type I interferon (IFN) treatment of some cell types, including macrophages, dendritic cells and the monocytic cell line THP-1, potently induces a block to HIV-1 infection at the level of viral DNA accumulation [1–4]. The identities and roles of participating IFN-induced anti-HIV-1 host factors are yet to be defined. The HIV-2/SIVSM Vpx protein greatly increases the permissivity of myeloid cells to HIV-1 infection [5, 6]. The protein sterile alpha motif (SAM) histidine/aspartic acid (HD) domain containing 1 (SAMHD1) was recently identified as a target for Vpx-induced proteasomal degradation in monocyte-derived macrophages (MDMs) and dendritic cells, as well as in quiescent CD4 T-cells [7–10]. SAMHD1 is a dGTP-regulated deoxynucleotide triphosphohydrolase that limits the pool of dNTPs available for reverse transcription, therefore reducing HIV-1 infection of myeloid cells [11, 12]; for a short review see . Interestingly, SAMHD1 has been reported to be IFNγ- and IFNα-inducible in human dendritic cells and monocytes, respectively [14, 15]. And consistent with this, it was also shown that Vpx enhances HIV-1 infection of IFNα-treated monocyte-derived dendritic cells . In this context, we sought to investigate further the role of SAMHD1 in IFNα-induced HIV-1 restriction.
The positive effect of both Vpx and dN correlated with significant increases in the intracellular dNTP pool (as shown by intracellular dATP measurement, Figure 2B displays the mean data and Additional file 2: Figure S1B shows the results for each donor). In agreement with this finding, Vpx efficiently induced the degradation of SAMHD1 (Figure 2C). Closer inspection of these data reveals that dN provision or Vpx addition promote infection to greater magnitudes in cultures treated with IFNα. While the molecular basis for this is not yet known, it is plausible that, although its expression is not increased, SAMHD1 activity might be potentiated in IFNα-treated MDMs. However, measurement of dATP levels in the presence or absence of IFNα did not reveal a significant difference in dATP concentrations, implying that the dNTP hydrolase activity of SAMHD1 is not measurably affected by IFNα treatment (Figure 2B and Additional file 2: Figure S1B). Alternatively, other restriction factor(s) might suppress HIV-1 infection more effectively when cellular dNTP concentrations are low. Of note, MDMs from different donors behave differently with respect to the rescue of HIV-1 infectivity following dN treatment, suggesting that donor-specific differences in SAMHD1 levels or activity contribute to differences in rescue by dN or Vpx (see donors 1 and 2, versus donors 3 and 4 in Additional file 2: Figure S1).
To investigate further the relationship between IFNα action and SAMHD1 function, we evaluated the effect of IFNα in THP-1 cells selectively depleted of SAMHD1 under dividing and differentiated culture conditions (Figure 3D). THP-1 cells were transduced with an HIV-1 shRNA vector targeting SAMHD1, or a control, and selected with puromycin. Dividing or PMA-treated shControl or shSAMHD1 THP-1 cells were treated with IFNα or not for 24 h, and then infected with different doses of VSV-G pseudotyped HIV-1 GFP vector (Figure 3D). As previously reported, reducing SAMHD1 protein levels increased the permissivity of PMA-treated (non-dividing) THP-1 cells by ~4-fold (Figure 3D) [7, 15]. IFNα treatment decreased HIV-1 infectivity by more than one order of magnitude, irrespective of whether SAMHD1 was present or not, or whether the cells were dividing (Figure 3A and B). Although it is possible that low residual amounts of SAMHD1 were able to exert an effect, the data collectively suggest that SAMHD1 is, if at all, only a minor contributor to the IFNα-induced early block to HIV-1 infection in these cells. Of note, similar results were obtained in challenges with VSV-G pseudotyped full length HIV-1 (not shown).
In this report, we used several primary and immortalized cell types to examine the relationship between SAMHD1 expression and function, and the inhibition of HIV-1 infection by IFNα. Taken together, our data reveal that SAMHD1, while previously established as an important regulator of HIV-1 infection [7–10], is not a major effector of IFNα-mediated HIV-1 restriction in a number of cell types and lines. In particular, by taking advantage of the ability of Vpx to degrade SAMHD1 [7, 8], dN addition to bypass its deoxynucleoside triphosphohydrolase activity, or selective SAMHD1 knock-down, our findings indicate that IFNα is often able to suppress HIV-1 infectivity independently of SAMHD1 activity. In keeping with this, several of our experiments also showed that HIV-1 inhibitory phenotypes that bear the hallmarks of SAMHD1-mediated suppression are displayed in differentiated cultures of primary myeloid cells in the presence or absence of IFNα. Interestingly, Vpx and dN provoked a substantial increase in infection of IFNα-treated MDMs, which was greater than observed in untreated MDMs. This implies that either SAMHD1 function is at least partly responsible for the IFNα-mediated restriction in this cell type and/or its ability to reduce the pool of dNTPs is necessary for the action of other restriction factor(s). For example, we speculate that SAMHD1 may allow viral RNA or RNA/DNA reverse transcription intermediates to be specifically inhibited by IFNα-induced restriction factors, whilst the presence of Vpx or dN addition results in these structures being less abundant (or protected) owing to efficient reverse transcription. Lastly, it has been suggested that Vpx binds to APOBEC3A (A3A) possibly contributing to increased HIV-1 infectivity in monocytic cells [22, 23]. Although we cannot rule out a role for A3A in the IFNα-induced block in MDMs described here, the rescue of HIV-1 infectivity by dN treatment argues against such a direct role. Moreover, A3A is not induced in U87-MG cells treated with IFNα, despite a potent block to HIV-1 infection. Our studies in U87-MG and THP-1 cells, where manipulation of SAMHD1 fails to overcome the effects of IFNα, allude to the existence of additional, possibly cell type specific, IFNα-inducible (and Vpx-independent) inhibitors of the early stages of HIV-1 infection. Validation of these models will require the identification of such cell-encoded factors.
Human immunodeficiency virus type 1
Multiplicity of infection
Sterile alpha motif (SAM) histidine/aspartic acid (HD) domain containing 1
Short hairpin RNA
Simian immunodeficiency virus
Virus like particles
Viral protein X
This work was supported by the U.K. Medical Research Council, the National Institutes of Health (AI049781, AI070072, AI077401, AI090935, DA033773), the European Community’s Seventh Framework Programme [FP7/2007-2013] under grant agreement n°PIEF-GA-2009-237501 (to CG), and the Department of Health via a National Institute for Health Research comprehensive Biomedical Research Centre award to Guy’s and St. Thomas’ NHS Foundation Trust in partnership with King’s College London and King’s College Hospital NHS Foundation Trust.
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