Cryo-electron tomography of wt PFV.

<p>Central slices through wt PFV virions having a complete capsid (Class A), an incomplete capsid (Class B), no capsid (Class C) or miscellaneous particles (Class D). For each class, one virus has its capsid, intermediate shell, viral membrane and glycoproteins colored in red, green, blue and yellow respectively. The interior of Class C virus having no characteristic features is left uncolored. For each class A virus, the corresponding radial density profile is shown and the various peaks corresponding to the glycoprotein (G), viral membrane (M), intermediate shell (I) and capsid (C) are labeled on one plot. Scale bar is 60 nm.</p

Subtomogram averaging of PFV glycoprotein.

<p>A–C- 0.8 nm thick tomographic slice perpendicular to the glycoprotein long axis from the wt, iNAB and iFuse mutants and its corresponding schematic of interlocked hexagonal assemblies of trimers. Numbers are indicated at the center of each hexagon and triangles represent the position of each trimer of Env in the hexagonal network. The wt virus shows ordered region of glycoprotein (represented on the corresponding schematic on the right) next to less ordered one while both mutants have more ordered hexagonal networks. D–I- Subtomogram averaging on glycoproteins of wt (top), iNAB (middle) and iFuse (bottom) mutants. D–F and G–I show a side view of a single trimer and a top view of intertwined hexagonal assemblies respectively. The two arrows in (G) point to less well defined peripheral trimers on wt particles. Scale bars are 100 Å.</p

<i>In Situ</i> single particle 3D reconstruction of PFV glycoprotein by cryo-EM.

<p>A- Side view of the sharpened 3D reconstruction of an hexagonal assembly of trimeric glycoproteins from the iFuse mutant (6-fold symmetry applied, ~10 Å resolution at FSC = 0.143). Spikes are colored alternately yellow and salmon, viral membrane is gray. B- Gray scale section at the level of the dotted line in (A). The triangle and hexagon symbols indicate the position of each trimer and of the 6-fold axis orthogonal to the section plane respectively. The two white arrowheads delimit the region where the trimers are interacting with each other. C, D- Side views (full (C) and cut-away (D) views) of a single PFV Env trimer (sharpened map) after 3-fold symmetry application (~9 Å resolution at FSC = 0.143). The densities corresponding to the extracellular domains and the viral membrane are colored salmon and gray respectively in C. The three central helices attributed to gp48 fusion peptide are represented by three green α helices of 22 residues long each. The TMHs are represented by three inner (colored blue) and three outer (colored orange) α helices. In D, the densities surrounding the three central helices and the three inner and outer TMHs are colored green, blue and orange respectively while the remaining of the spike is gray colored. E, F and G- Grayscale sections through the reconstruction shown in (C): E- central side view as in (D), F and G- top views at the level of the dotted lines in (E) through the three helix bundle and the TMHs respectively (the three outer and inner TMHs are labeled 1, 2, 3 and 4, 5, 6 respectively). Scale bars are 50 Å in A, B, D, E.</p

Ultrastructure of PFV virus interior.

<p>A- Central slice through a wt PFV virus imaged by cryo-ET. The white and red arrows point to some of the interactions/connections between capsid / intermediate shell and intermediate shell / viral membrane respectively. The blue parenthesis highlights local organization in the intermediate shell. B- Same as (A) except that this virus has no glycoprotein sticking out of the membrane but it still has a capsid with an intermediate shell (colored in green). C- Tomogram section of PFV iNAB mutant showing viral particles without any internal ordered structure. Black arrows point internal densities located close to the viral membrane but distinct from the intermediate shell of wt and iFuse PFV. D- 2D classification on the capsid alone of iFuse mutant viruses imaged by cryo-EM. Five 2D class averages are shown which could be interpreted as 2D projections of a pseudo-icosahedral object viewed along the 5, 5, 3, 2 and 5-fold symmetry axes respectively. Scale bars are 60 nm for each panel.</p

Analysis of PFV infecting HT1080 cells.

<p>A, B- Low magnification view of PFV infected cells showing budding at the plasma membrane (indicated by * symbol) (A) and viruses in intracellular vacuoles (symbol V) (B). Free (non-enveloped) cytoplasmic capsids can be seen as well (labeled Cap in (B)). C, D- Higher magnification view of viruses budding at the plasma membrane (C) and viruses possibly budding in vacuole (D–zoom in the rectangular region of (B)). E- 0.8 nm thick tomogram slice through five different viruses budding at the plasma membrane. The red arrowheads point to the intermediate shell between the capsid and the plasma membrane. Scale bars in A—B, C—D and E are 500, 200 and 60 nm respectively.</p

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

Integration site profiles of WT and Gag iSTP mutant viruses.

<p>(A) Percent of WT (gray bar), S224A (backslash), and T225A (horizontal slash) integrations within RefSeq genes. An independent HIV-1 dataset (black) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005860#ppat.1005860.ref085" target="_blank">85</a>] was included for comparison. (B) Percent of integrations within lamina-associated domains (LADs). (C) Average gene density in 1 Mb regions surrounding the integration sites. The data from three wt and two S224A and T225A integration site libraries (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005860#ppat.1005860.t001" target="_blank">Table 1</a>) were combined, with error bars indicating the resulting standard deviation. Dotted, horizontal lines represent the percent of integrations from the <i>in vitro</i> integration dataset. Please refer to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005860#ppat.1005860.s007" target="_blank">S7 Fig</a> for statistical analyses.</p

Effect of enzymatic PLK inhibition on the titers of PFV, HIV-1 and MLV virions.

<p>(A) Experimental outline. HT1080 target cells were infected with serial dilutions of the individual virus supernatants as indicated in the presence of the vehicle control (DMSO) or one of two BI-2536 concentrations. (B) Cell cycle profiles of mock infected cell populations of the three experimental groups determined by propidium iodide staining 25 h from the start of treatment (with either DMSO or BI-2536). (C) Virus infectivity was determined 24 h post-infection by flow cytometry analysis of infected target cell populations. The values obtained using wt variants of PFV, HIV-1, or MLV supernatants in combination with vehicle control treatment were arbitrarily set to 100%. Absolute titers of wt supernatants ranged between 5.0 x 10⁵ and 8.0 x 10⁵ (PFV), 1.9 x 10⁶ and 3.9 x 10⁶ (HIV), and 1.5 x 10⁷ and 2.5 x 10⁷ eGFP ffu/ml (MLV). Relative means and standard deviations from three independent experiments are shown. Differences between means of the respective wt viruses in combination with vehicle control and the individual mutants or treatment regimen with BI-2536 were analyzed by Welch’s t test (*, p<0.05; **, p<0.01).</p

Integration efficiency and dynamics of PFV wt and STP mutant virions.

<p>(A) Virus infectivity was determined at different time points post-infection by flow cytometry analysis of infected target cell populations as indicated. The values obtained using wt PFV Gag expression plasmids were arbitrarily set to 100%. Relative means and standard deviations normalized for Gag content (except mock) from two independent experiments in duplicates are shown. Absolute titers of wt supernatants ranged between 5.7 x 10⁴ and 9.3 x 10⁴ (1 day p.i.), 8.6 x 10⁵ and 1.5 x 10⁶ eGFP ffu/ml (18 day p.i.). (B) Comparison of wt and STP mutant integration efficiencies. HT1080 target cells were infected with wt, T225A, iRT, iIN and ΔEnv (mock) supernatants. Ten days post-infection, genomic DNA was isolated from target cells and provirus numbers integrated into the host cell genome were quantified by Alu-qPCR and normalized to ß-actin copy numbers. The values obtained using wt PFV Gag expression plasmids were arbitrarily set to 100%. Relative means and standard deviations from three independent experiments in duplicates are shown. (C) Integration dynamics of wt and STP mutant viruses analyzed by inhibition of viral infectivity by dolutegravir addition at different time points post-infection and maintenance until flow cytometric determination of viral titers 10 days post-infection. (D) The values obtained for each respective virus type without DTG were arbitrarily set to 100%. Relative means and standard deviations from three independent experiments are shown.</p

Analysis of PFV wt, iSTP- and pmSTP virions in single-round- and multiple-round infection experiments.

<p>(A) PFV virions were produced by transient transfection of 293T cells with the four-component PFV vector system, containing either the wt Gag or one of the denoted iSTP- and pmSTP Gag variants. Titers of harvested viruses were determined by flow cytometry analysis of infected HT1080 target cells three days post-infection. The mean values and standard deviation for each supernatant were calculated from samples of cells infected with serial virus dilutions as described in Material and Methods. The values obtained using wt PFV Gag expression plasmids were arbitrarily set to 100%. Relative means and standard deviations normalized for Gag content (except mock) from independent experiments (n = 4–9) are shown. Differences between means of wt virus and the individual mutants were analyzed by Welch’s t test (**, p<0.01). Absolute titers of wt supernatants ranged between 1.2 x 10⁶ and 1.2 x 10⁷ eGFP ffu/ml. (B) Replication-competent PFV virions were produced by transient transfection of proviral expression vectors, containing either the wt Gag or one of the denoted iSTP- and pmSTP Gag variants into 293T cells. Viruses were harvested two days post-transfection and used to infect HT1080 PLNE target cells. Titers were determined by flow cytometry analysis one day post-infection. The values obtained using wt PFV Gag expression plasmids were arbitrarily set to 100%. Relative means and standard deviations normalized for Gag content (except mock) from independent experiments (n = 3–8) are shown. Differences between means of wt virus and the individual mutants were analyzed by Welch’s t test (**, p<0.01). Absolute titers of wt supernatants ranged between 1.7 x 10⁴ and 7 x 10⁴ eGFP ffu/ml. (C) Titers of iSTP- and pmSTP mutant PFV particles relative to wt over multiple rounds of target cell infection. Viruses were produced and harvested as described in panel B and Gag content normalized amounts of viral supernatants were used to infect HT1080 PLNE in serial dilutions. At different time points post-infection (as indicated on the x-axis) cells were harvested for flow cytometry analysis to determine viral titers. The values obtained using wt PFV supernatants at each time point were arbitrarily set to 100%. Relative means and standard deviations from two independent experiments are shown.</p