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

NMR and refinement statistics for PFV-Gag Central domains.

<p>NMR and refinement statistics for PFV-Gag Central domains.</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

Hydrodynamic parameters of PFV-Gag Central domains.

<p>Hydrodynamic parameters of PFV-Gag Central domains.</p

NMR structure of PFV-Gag CtD_CEN homodimer.

<p>(<b>A</b>) Cartoon representation of the structure of the PFV-Gag CtD_CEN dimer. Monomer-A is shown in dark blue and Monomer-B in cyan. The α-helices are labelled as for PFV-Gag(300–477) and the N- and C-termini of each monomer are indicated. (<b>B</b>) Details of the homodimer interface. Residues that contribute to the interface are shown as sticks. (<b>C</b>) C(S) distribution derived from sedimentation velocity data recorded from PFV-Gag CtD_CEN L410E/M413E mutant at 2 mg/mL (dashed line). The C(S) distribution derived from sedimentation velocity data recorded from <i>wt</i> PFV-Gag CtD_CEN at 2 mg /mL is shown also for comparison (solid line).</p

Analysis of genome content of PFV-Gag central domain mutants.

<p>Quantification of particle-associated PFV genomic RNA (vgRNA) and DNA (vgDNA) by qPCR. Mean values and standard deviation (n = 2) normalized for Gag content are shown as relative values compared to the <i>wt</i> control.</p

Alignment of PFV_CEN domains with orthoretroviral CA proteins.

<p>Panels <b>A-E</b>, are best-fit 3D structural superimpositions of PFV-NtD_CEN (light cyan) with (<b>A</b>) HIV-1 NtD_CA (orange), (<b>B</b>) RSV NtD_CA (maroon), (<b>C</b>) MLV NtD_CA (green), (<b>D</b>) HIV-1 CtD_CA (orange) and (<b>E</b>) RSV CtD_CA (maroon). Panels <b>F-J</b> are best-fit 3D structural superimpositions of PFV-CtD_CEN (dark cyan) with (<b>F</b>) HIV-1 NtD_CA (orange), (<b>G</b>) RSV NtD_CA (maroon), (<b>H</b>) MLV NtD_CA (green), (<b>I</b>) HIV-1 CtD_CA (orange) and (<b>J</b>) RSV CtD_CA (maroon). In all panels, molecules are shown in cartoon representation with α-helices displayed as cylinders. The dashed lines in panels D and I indicate the connectivity between helices 8 and 9 of HIV-CTD_CA that is disordered in the CA hexamer structures used in the alignment.</p

Conformation and solution oligomeric state of FV-Gag central domains.

<p>(<b>A</b>) C(S) distributions derived from sedimentation velocity data recorded from PFV-Gag(300–477) at 1 mg/mL (left panel); PFV-Gag NtD_CEN at 2 mg/mL (middle panel) and PFV-CtD_CEN 2 mg/mL (right panel). (<b>B</b>) Multi-speed sedimentation equilibrium profiles determined from interference data collected on PFV-Gag(300–477) at 49 μM (left panel) and PFV-Gag CtD_CEN at 42 μM (right panel). Data was recorded at the speeds indicated. The solid lines represent the global best fit to the data using either a single species or monomer-dimer equilibrium model. The lower panels show the residuals to the fit.</p

Cryo-electron microscopy analysis of wt and mutant PFV particles.

<p>(<b>A</b>) 5 nm thick slices of electron cryotomograms of five individual <i>wt</i> PFV particles. (<b>B</b>) 5 nm thick slices of electron cryotomograms of four PFV-Gag central domain mutants. Three representative images of each mutant are shown. Scale bars are 50 nm.</p

NMR structure of the central domain of PFV Gag.

<p>(<b>A</b>) Schematic representation of PFV Gag. Regions corresponding to the Gag-NtD and Gag-central domains are coloured cyan and magenta respectively. Sequence motifs and conserved regions are highlighted cytoplasmic targeting and retention sequence (CTRS) (blue), PGQA and YxxLGL (orange), Chromatin binding sequence (CBS) (green) and GR boxes (yellow). The Gag processing cleavage site is indicated with an arrow. (<b>B</b>) Sequence alignment of foamy virus Gag-central domains from mammals, old and new world monkeys (SFV). Mammalian FVs are abbreviated as follows: BFV, Bovine; EFV, Equine; FFV, Feline. Monkey species are abbreviated as follows: mac, Macaque; agm, African green monkey; spm, Spider monkey; sqm, Squirrel monkey; mar, marmoset. Numbering corresponds to the PFV sequence. Cartoons (cyan coils) above the alignment indicate the position of α-helices in the PFV-Gag NtD_CEN and CtD_CEN domain structures. The regions with greatest sequence homology are boxed and highlighted and residues that are conserved in all sequences are also coloured white. (<b>C</b>) Cartoon representation of PFV-Gag(300–477) backbone is shown in cyan. The secondary structure elements are numbered sequentially from the amino-terminus and the N- and C-termini are indicated. Helices α1 to α4 and α5 to α9 that comprise NtD_CEN and CtD_CEN respectively are indicated in the left and right hand panels. (<b>D</b>) The PFV-Gag NtD_CEN and CtD_CEN interface. The protein backbone is shown in grey cartoon representation. NtD_CEN and CtD_CEN α-helices that pack at the interface are labelled. Residues that make hydrophobic contacts are shown as sticks, blue from NtD_CEN and green from CtD_CEN.</p