SAMHD1 restricts human immunodeficiency virus-1 (HIV-1) infection of dendritic and other myeloid cells at an early stage in the replication cycle. SIVsm/HIV-2 lineage viruses counteract SAMHD1-mediated restriction by encoding Vpx, a virion-packaged accessory protein that targets SAMHD1 for degradation. We show that SAMHD1 restricts HIV-1 infection of monocyte-derived macrophages (MDM) by hydrolyzing the cellular deoxynucleotide triphosphates (dNTP), reducing their level to below that required for the synthesis of the viral genomic DNA. Vpx prevented the SAMHD1-mediated decrease in dNTP. The restriction was partially alleviated in MDM by the addition of exogenous deoxynucleosides. HIV-1 with a V148I mutation in reverse transcriptase that lowers its affinity for dNTP was particularly sensitive to SAMHD1-mediated restriction. Nucleotide starvation could serve as a mechanism to protect cells from infection by a wide variety of infectious agents that replicate through a DNA intermediate.

To replicate in their hosts, viruses have to navigate the complexities of the mammalian cell, co-opting mechanisms of cellular physiology while defeating restriction factors that are dedicated to halting their progression. Primate lentiviruses devote a relatively large portion of their coding capacity to counteracting restriction factors by encoding accessory proteins dedicated to neutralizing the antiviral function of these intracellular inhibitors. Research into the roles of the accessory proteins has revealed the existence of previously undetected intrinsic defenses, provided insight into the evolution of primate lentiviruses as they adapt to new species and uncovered new targets for the development of therapeutics. This Review discusses the biology of the restriction factors APOBEC3, SAMHD1 and tetherin and the viral accessory proteins that counteract them.

bSterile alpha motif domain-and HD domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphohydrolase that restricts the replication of lentiviruses in myeloid cells by hydrolyzing the cellular deoxynucleotide triphosphates to a level below that which is required for reverse transcription. Human immunodeficiency virus type 2 (HIV-2) and some simian immunodeficiency viruses (SIVs) encode the accessory protein viral protein X (Vpx) that counteracts SAMHD1. Vpx recruits SAMHD1 to a cullin4A-RING E3 ubiquitin ligase (CRL4), which targets the enzyme for proteasomal degradation. Vpx and SAMHD1 both localize to the nucleus of the cell. We identified the nuclear localization sequence (NLS) of SAMHD1 as a conserved KRPR sequence at amino acid residues 11 to 14. SAMHD1 lacking a functional NLS localized to the cytoplasm but retained its triphosphohydrolase and antiviral activities. However, cytoplasmic SAMHD1 was resistant to Vpx-induced degradation, and its antiviral activity was not counteracted by Vpx. Cytoplasmic SAMHD1 interacted with Vpx and retained it in the cytoplasm. The inhibition of nuclear export with leptomycin B did not impair the ability of Vpx to degrade SAMHD1. These findings suggest that SAMHD1 is targeted by Vpx for ubiquitination and degradation in the nucleus.

BackgroundSAMHD1 is a triphosphohydrolase that restricts the replication of HIV-1 and SIV in myeloid cells. In macrophages and dendritic cells, SAMHD1 restricts virus replication by diminishing the deoxynucleotide triphosphate pool to a level below that which supports lentiviral reverse transcription. HIV-2 and related SIVs encode the accessory protein Vpx to induce the proteasomal degradation of SAMHD1 following virus entry. While SAMHD1 has been shown to restrict HIV-1 and SIV, the breadth of its restriction is not known and whether other viruses have a means to counteract the restriction has not been determined.ResultsWe show that SAMHD1 restricts a wide array of divergent retroviruses, including the alpha, beta and gamma classes. Murine leukemia virus was restricted by SAMHD1 in macrophages yet removal of SAMHD1 did not alleviate the block to infection because of an additional block to viral nuclear import. Prototype foamy virus (PFV) and Human T cell leukemia virus type I (HTLV-1) were the only retroviruses tested that were not restricted by SAMHD1. PFV reverse transcribes predominantly prior to entry and thus is unaffected by the dNTP level in the target cell. It is possible that HTLV-1 has a mechanism to render the virus resistant to SAMHD1-mediated restriction.ConclusionThe results suggest that SAMHD1 has broad anti-retroviral activity against which most viruses have not found an escape.

APOBEC3A (A3A), one of the seven-member APOBEC3 family of cytidine deaminases, lacks strong antiviral activity against lentiviruses but is a potent inhibitor of adeno-associated virus and endogenous retroelements. In this report, we characterize the biochemical properties of mammalian cell-produced and catalytically active E. coli-produced A3A. The enzyme binds to single-stranded DNA with a Kd of 150 nM and forms dimeric and monomeric fractions. A3A, unlike APOBEC3G (A3G), deaminates DNA substrates nonprocessively. Using a panel of oligonucleotides that contained all possible trinucleotide contexts, we identified the preferred target sequence as TC (A/G). Based on a three-dimensional model of A3A, we identified a putative binding groove that contains residues with the potential to bind substrate DNA and to influence target sequence specificity. Taking advantage of the sequence similarity to the catalytic domain of A3G, we generated A3A/A3G chimeric proteins and analyzed their target site preference. We identified a recognition loop that altered A3A sequence specificity, broadening its target sequence preference. Mutation of amino acids in the predicted DNA binding groove prevented substrate binding, confirming the role of this groove in substrate binding. These findings shed light on how APOBEC3 proteins bind their substrate and determine which sites to deaminate.