BackgroundSAMHD1 is a restriction factor that potently blocks infection by HIV-1 and other retroviruses. We have previously demonstrated that SAMHD1 oligomerizes in mammalian cells by immunoprecipitation. Here we investigated the contribution of SAMHD1 oligomerization to retroviral restriction.ResultsStructural analysis of SAMHD1 and homologous HD domain proteins revealed that key hydrophobic residues Y146, Y154, L428 and Y432 stabilize the extensive dimer interface observed in the SAMHD1 crystal structure. Full-length SAMHD1 variants Y146S/Y154S and L428S/Y432S lost their ability to oligomerize tested by immunoprecipitation in mammalian cells. In agreement with these observations, the Y146S/Y154S variant of a bacterial construct expressing the HD domain of human SAMHD1 (residues 109–626) disrupted the dGTP-dependent tetramerization of SAMHD1 in vitro. Tetramerization-defective variants of the full-length SAMHD1 immunoprecipitated from mammalian cells and of the bacterially-expressed HD domain construct lost their dNTPase activity. The nuclease activity of the HD domain construct was not perturbed by the Y146S/Y154S mutations. Remarkably, oligomerization-deficient SAMHD1 variants potently restricted HIV-1 infection.ConclusionsThese results suggested that SAMHD1 oligomerization is not required for the ability of the protein to block HIV-1 infection.

Ice deposited in Greenland and Antarctica entraps viable and nonviable microbes, as well as biomolecules, that become temporal atmospheric records. Five sections (estimated to be 500, 10,500, 57,000, 105,000 and 157,000 years before present, ybp) from the GISP2D (Greenland) ice core, three sections (500, 30,000 and 70,000 ybp) from the Byrd ice core, and four sections from the Vostok 5G (Antarctica) ice core (10,500, 57,000, 105,000 and 105,000 ybp) were studied by scanning electron microscopy, cultivation and rRNA gene sequencing. Bacterial and fungal isolates were recovered from 10 of the 12 sections. The highest numbers of isolates were found in ice core sections that were deposited during times of low atmospheric CO2, low global temperatures and low levels of atmospheric dust. Two of the sections (GISP2D at 10,500 and 157,000 ybp) also were examined using metagenomic/metatranscriptomic methods. These results indicated that sequences from microbes common to arid and saline soils were deposited in the ice during a time of low temperature, low atmospheric CO2 and high dust levels. Members of Firmicutes and Cyanobacteria were the most prevalent bacteria, while Rhodotorula species were the most common eukaryotic representatives. Isolates of Bacillus, Rhodotorula, Alternaria and members of the Davidiellaceae were isolated from both Greenland and Antarctica sections of the same age, although the sequences differed between the two polar regions.

Mutations in the human SAMHD1 gene are known to correlate with the development of the Aicardi-Goutières Syndrome (AGS), which is an inflammatory encephalopathy that exhibits neurological dysfunction characterized by increased production of type I interferon (IFN); this evidence has lead to the concept that the SAMHD1 protein negatively regulates the type I IFN response. Additionally, the SAMHD1 protein has been shown to prevent efficient HIV-1 infection of macrophages, dendritic cells and resting CD4+ T cells. To gain insights on the SAMHD1 molecular determinants that are responsible for the deregulated production of type I IFN, we explored the biochemical, cellular and antiviral properties of human SAMHD1 mutants known to correlate with the development of Aicardi-Goutières Syndrome. Most of the studied SAMHD1 AGS mutants exhibit defects in the ability to oligomerize, decrease the levels of cellular dNTPs in human cells, localize exclusively to the nucleus, and restrict HIV-1 infection. At least half of the tested variants preserved the ability to be degraded by the lentiviral protein Vpx, and all of them interacted with RNA. Our investigations revealed that the SAMHD1 AGS variant p.G209S preserve all tested biochemical, cellular and antiviral properties, suggesting that this residue is a determinant for the ability of SAMHD1 to negatively regulate the type I interferon response in human patients with AGS. Overall, our work genetically separated the ability of SAMHD1 to negatively regulate the type I IFN response from its ability to restrict HIV-1.

SAMHD1 is a human restriction factor that prevents efficient infection of macrophages, dendritic cells and resting CD4+ T cells by HIV-1. Here we explored the antiviral activity and biochemical properties of human SAMHD1 polymorphisms. Our studies focused on human SAMHD1 polymorphisms that were previously identified as evolving under positive selection for rapid amino acid replacement during primate speciation. The different human SAMHD1 polymorphisms were tested for their ability to block HIV-1, HIV-2 and equine infectious anemia virus (EIAV). All studied SAMHD1 variants block HIV-1, HIV-2 and EIAV infection when compared to wild type. We found that these variants did not lose their ability to oligomerize or to bind RNA. Furthermore, all tested variants were susceptible to degradation by Vpx, and localized to the nuclear compartment. We tested the ability of human SAMHD1 polymorphisms to decrease the dNTP cellular levels. In agreement, none of the different SAMHD1 variants lost their ability to reduce cellular levels of dNTPs. Finally, we found that none of the tested human SAMHD1 polymorphisms affected the ability of the protein to block LINE-1 retrotransposition.

Understanding how to achieve efficient transduction of hematopoietic stem and progenitor cells (HSPCs), while preserving their long-term ability to self-reproduce, is key for applying lentiviral-based gene engineering methods. SAMHD1 is an HIV-1 restriction factor in myeloid and resting CD4+ T cells that interferes with reverse transcription by decreasing the nucleotide pools or by its RNase activity. Here we show that SAMHD1 is expressed at high levels in HSPCs cultured in a medium enriched with cytokines. Thus, we hypothesized that degrading SAMHD1 in HSPCs would result in more efficient lentiviral transduction rates. We used viral like particles (VLPs) containing Vpx, shRNA against SAMHD1, or provided an excess of dNTPs or dNs to study this question. Regardless of the method applied, we saw no increase in the lentiviral transduction rate. The result was different when we used viruses (HR-GFP-Vpx+) which carry Vpx and encode GFP. These viruses allow assessment of the effects of Vpx specifically in the transduced cells. Using HR-GFP-Vpx+ viruses, we observed a modest but significant increase in the transduction efficiency. These data suggest that SAMHD1 has some limited efficacy in blocking reverse transcription but the major barrier for efficient lentiviral transduction occurs before reverse transcription.