A Unique Spumavirus Gag N-terminal Domain with Functional Properties of Orthoretroviral Matrix and Capsid

<div><p>The <i>Spumaretrovirinae</i>, or foamyviruses (FVs) are complex retroviruses that infect many species of monkey and ape. Although FV infection is apparently benign, trans-species zoonosis is commonplace and has resulted in the isolation of the Prototypic Foamy Virus (PFV) from human sources and the potential for germ-line transmission. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. In addition, PFV Gag interacts with the FV Envelope (Env) protein to facilitate budding of infectious particles. Presently, there is a paucity of structural information with regards FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. Therefore, in order to probe the functional overlap of FV and orthoretroviral Gag and learn more about FV egress and replication we have undertaken a structural, biophysical and virological study of PFV-Gag. We present the crystal structure of a dimeric amino terminal domain from PFV, Gag-NtD, both free and in complex with the leader peptide of PFV Env. The structure comprises a head domain together with a coiled coil that forms the dimer interface and despite the shared function it is entirely unrelated to either the capsid or matrix of Gag from other retroviruses. Furthermore, we present structural, biochemical and virological data that reveal the molecular details of the essential Gag-Env interaction and in addition we also examine the specificity of Trim5α restriction of PFV. These data provide the first information with regards to FV structural proteins and suggest a model for convergent evolution of <i>gag</i> genes where structurally unrelated molecules have become functionally equivalent.</p></div

The effect of phosphorylation on SAMHD1 enzyme activity.

<p>(<b>A</b>) Steady-state kinetic analysis of GTP stimulated hydrolysis of TTP, by SAMHD1 (115–626) (black) and phospho-SAMHD1(115–626) (blue). The dependence of the rate of on substrate concentration is plotted. Error bars represent SEM of three independent measurements. Solid lines are the best fit to the data using the Michaelis-Menten expression. The derived constants K_M and k_cat are shown inset. (<b>B and C</b>) Effect of activator depletion on SAMHD1 and pSAMHD1 triphosphohydrolase activity. (<b>B</b>) IEX-HPLC analysis of nucleotide composition after incubation of SAMHD1(115–626) with GTP (upper), and pSAMHD1(115–626) (middle) or SAMHD1(115–626) (lower) with dATP and GTP for 0 and 300 seconds. (<b>C</b>) IEX-HPLC analysis of the hydrolysis of ddGTP added to the samples depleted of dATP in (<b>B</b>). The plot shows the quantification of the time dependent hydrolysis derived from integration of the ddGTP peak at each time point. Error bars represent the SEM from 3 independent experiments.</p

Different G based nucleotides can be accommodated in the SAMHD1 allosteric site.

<p>(<b>A</b>) The contents and conformation of the allosteric sites for structures of SAMHD1(115–583)-ddGTP (top), SAMHD1(115–583[R164A])-dGTP (middle) and SAMHD1(115–626)-GTP (bottom) are shown. Nucleotides are shown in stick representation, SAMHD1 residues making contacts with the nucleotides are labelled. (<b>B</b>) SEC-MALLS analysis of SAMHD1(115–626) incubated with ddGTP/dATP(blue), dGTP/dATP (green) or GTP/dATP (orange). The chromatograms are the output from the differential refractometer. The points are the weight-averaged molar masses determined at 1-second intervals throughout elution of chromatographic peaks.</p

Solution oligomeric state and steady state kinetics of SAMHD1.

<p>(<b>A</b>) SEC-MALLS analysis of SAMHD1 monomer-dimer-tetramer equilibrium for SAMHD1(115–583) (red) and SAMHD1(115–626) (black). The chromatograms are the output from the differential refractometer. The points are the weight-averaged molar masses determined at 1-second intervals throughout elution of chromatographic peaks. Dashed line chromatograms are apo-protein, solid lines are upon addition of 0.5 mM dGTP (<b>B</b>) Tetramer stability. SAMHD1(115–626) was incubated with 0.1 mM GTP and 0.5 mM dATP for the specified time intervals and then the oligomeric state analysed by SEC-MALLS. (<b>C</b>) Analysis of dATP hydrolysis during the time course shown in <b>B</b>. The chromatogram (black) is the recorded UV absorbance at 260 nm from the SEC-MALLS column for the five-minute incubation time point. Reference chromatograms (dashed lines) for substrate dATP (red) and product dA (blue) are overlaid. (<b>D</b>) Steady-state kinetic analysis of GTP stimulated hydrolysis of TTP by SAMHD1. The dependence of the rate of on substrate concentration for SAMHD1(115–626) (black) and SAMHD1(115–583) (red) are plotted. Solid lines are the best fit to the data using the Michaelis-Menten expression. Error bars represent the standard error of the mean (SEM) of three independent measurements. The derived constants K_M and k_cat for the reaction are displayed inset.</p

Model for phospho-regulation of SAMHD1 restriction.

<p>In the absence of dNTPs Apo-SAMHD1 is found in a monomer-dimer equilibrium regardless of the phosphorylation state (<b>1</b>). At high dNTP levels, typically in cycling cells, constitutively abundant GTP combines with dNTPs to fill allosteric sites. In both phosphorylated and un-phosphorylated SAMHD1 this results in the formation of an activated tetramer (<b>2</b>) that in the non-phosphorylated protein also includes additional intra-tetramer CtD interactions forming a stable activated tetramer (<b>3</b>). Under these conditions, both activated and stable activated tetramers hydrolyse the dNTP pool at comparable rates. At lower dNTP levels, the stabilisation afforded by the CtD interactions maintains enzyme activity in non-phosphorylated SAMHD1 by preventing the loss of dNTP-activator from the allosteric site. However, in phospho-SAMHD1, activating dNTPs dissociate from the allosteric site (<b>4</b>) resulting in disassembly of the tetramer and down-regulation of triphosphohydrolase activity. At very low levels, such as in differentiated myeloid cells, CtD-stabilised tetramers still retain activating dNTPs in the allosteric site (<b>5</b>) and SAMHD1 remains catalytically competent. It can therefore rapidly respond to any increase in intracellular dNTPs to maintain the dNTP levels below the threshold required for HIV-1 replication.</p

Structures of SAMHD1 tetramers.

<p><b>(A</b>) Crystal structures of SAMHD1(41–583) (left) and SAMHD1(115–583) (right) tetramers. Individual monomers are coloured cyan, green, magenta and orange. Bound ddGTP molecules in the active and allosteric sites are shown in yellow and green stick representation respectively. Metal ions are shown as spheres. (<b>B</b>) The conserved dimer-dimer interface of SAMHD1(41–583) and SAMHD1(115–583) (left) and the non-conserved SAMHD1(115–583) dimer-dimer interface (right). For clarity only two SAMHD1 monomers, comprising one dimer-dimer interface are shown in each panel. Active site bound ddGTP and residues that make interactions at the subunit interface are shown as sticks. Dashed lines represent hydrogen bonding interactions. (<b>C</b>) A comparison of the bound nucleotide conformation and metal configuration of SAMHD1 tetramers is shown. Nucleotides are shown in stick representation, metal ions as spheres: Fe (brown), Mn (purple), Mg (green) and Zn (blue). SAMHD1 residues that chelate metal ions and/or triphosphate are labelled. (I) SAMHD1(41–583)-ddGTP, (II) SAMHD1(115–583)-ddGTP, (III) SAMHD1(113–626)-dGTPαS [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005194#ppat.1005194.ref013" target="_blank">13</a>] (PDB ID 4BZC), (IV) SAMHD1(113–626 HD206/207RN)-dGTP [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005194#ppat.1005194.ref013" target="_blank">13</a>] (PDB ID 4BZB), (V) SAMHD1(109–626)-dGTP [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005194#ppat.1005194.ref014" target="_blank">14</a>] (PDB ID 4MZ7).</p

Restriction activity of SAMHD1 deletion constructs.

<p>(<b>A</b>) Schematic of SAMHD1 mutants showing the N-terminal domain in red with nuclear localisation signal (NLS), the sterile alpha motif (SAM) domain in yellow, the HD domain in green and the C-terminal domain (CtD) in blue. Positions of domain junctions along with active site point mutation residues are numbered above. The identifier (A–I) and amino acids present in each mutant are indicated on the left. (<b>B</b>) Restriction of HIV-1 in differentiated cells. U937 cells were transduced with full-length SAMHD1, catalytically inactive SAMHD1(HD206-7AA) or domain mutant VLPs co-expressing YFP. Cells were differentiated after three days and 72 hours later, infected with HIV-1GFP virus. The bar graph shows the degree of restriction compared to SAMHD1 negative cells. Red bars highlight restriction competent SAMHD1. The black bar is the negative control, SAMHD1(HD206-7AA). Error bars represent the range for n ≥ 8 independent experiments. (<b>C</b>) Restriction of HIV-1 in in cycling cells. U937 cells were transduced and infected as in <b>B</b> but without differentiation. The bar graph shows the degree of restriction with error bars representing the range for n = 3 independent experiments. Red bars represent constructs that are able to restrict HIV-1 in differentiated U937 cells. (<b>D</b>) Inhibition of second-strand reverse transcription products by SAMHD1. U937 cells were transduced with SAMHD1 or domain mutant SAMHD1, differentiated and infected with DNase-treated HIV-1GFP. Cells were harvested at the indicated time points post-infection and second-strand reverse transcription products were detected by qPCR. Graph shows a representative experiment (n ≥ 6). Error bars are the standard deviation within the qPCR replicate (<b>E</b>) SAMHD1 restriction of HIV-1 RT mutants. U937 cells were transduced with SAMHD1, differentiated as in <b>B</b>, and then infected with HIV-1GFP virus carrying the indicated mutations in RT. The bar graph shows relative restriction compared to wild type (WT) HIV-1GFP. Error bars represent the range for n = 3 independent experiments. (<b>F</b>) Analysis of cellular dNTP levels. U937 parental cells or cells stably expressing either full-length SAMHD1 or mutant F/ SAMHD(1–583) were differentiated by adding PMA and 72 hours later dNTPs were extracted for quantification. The bar graph shows the relative amounts of each indicated dNTP in each cell line. Error bars represent the range for n = 3 independent experiments.</p

The effect of phosphorylation on SAMHD1 restriction and tetramerisation.

<p>Restriction of HIV-1 infection by SAMHD1 Thr592 mutants in (<b>A</b>) differentiated and (<b>B</b>) cycling U937 cells. Cells were transduced with the indicated SAMHD1 phosphomimetic mutants, differentiated (<b>A</b> only), infected and analysed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005194#ppat.1005194.g001" target="_blank">Fig 1</a>. Bar charts show the fold restriction. Error bars represent standard deviation of the mean, (n = 6) for <b>A</b> and (n = 3) for <b>B</b>. (<b>C</b>) SEC-MALLS analysis of wild type SAMHD1(115–626) (black) and phospho-SAMHD1(115–626) (blue) incubated with 0.1 mM GTP and 0.5 mM dATP. The chromatograms are the output from the differential refractometer. The red points are the weight-averaged molar masses determined for the two peaks in the wild type protein. (<b>D</b>) Crystal structure of pSAMHD1(115–626) tetramer. Individual monomers are coloured cyan, green, magenta and orange. Active site bound Fe metal ions are shown as spheres. (<b>E</b>) Structural alignment of pSAMHD1(115–626) (cyan and magenta) and SAMHD1(115–583) (grey) tetramers. For clarity only two SAMHD1 monomers from each structure, comprising one dimer-dimer interface are shown. The structures were aligned on the upper dimer (cyan onto grey) to demonstrate the small displacement in the pSAMHD1(115–626) dimer-dimer interface with respect to that of the SAMHD1(115–583) structure (magenta onto grey). The α13 helix-pairs at the dimer-dimer interfaces are indicated by the double-arrows (left) SAMHD1(115–583) and (right) pSAMHD1(115–626). The active site Fe and ddGTP in SAMHD1(115–583) are also shown in sphere and in stick representation respectively.</p

X-ray data collection and structure refinement statistics.

<p>*Values in parentheses are for highest-resolution shell</p><p>X-ray data collection and structure refinement statistics.</p

Particle production and infectivity of PFV-Gag central domain mutants.

<p>(<b>A</b>) Western blot analysis of producer cell lysates (<b>Cell</b>) and pelleted viral supernatants (<b>Virus</b>) with polyclonal antibodies specific for PFV-Gag (α-Gag) and PFV Env-LP (α-Env-LP) or monoclonal antibodies specific for PFV-PR/RT (α-PR/RT) and integrase (α-IN). Residue substitutions in Gag are indicated above each track, (<i>wt</i>) wild type virus, (<i>wt +iRT</i>) wild type virus with defective reverse transcriptase. In the right-hand panel %<i>wt</i> are different <i>wt</i> control loadings and arrows indicate the migration of Gag, Env and Pol proteins. (<b>B</b>) Relative amounts of released Gag quantified from Western blots data from two independent experiments. (<b>C</b>) Relative infectivity of extracellular 293T cell culture supernatants using an eGFP marker gene transfer assay, determined 3 days post infection. Means and standard deviations of three independent experiments are shown. The values obtained using the wild type Gag packaging vector were arbitrarily set to 100%. Absolute titres of these supernatants were 1.8 x 10⁶ to 1.1 x 10⁷ ffu/ml.</p