MPER insertion within a narrow cavity between hexons at the icosahedral 2-fold axis.

<p>Density for two hexons (blue and purple) simulated from the final MDFF frame shown surrounding experimentally determined cryoEM density (gray) for one MPER insertion. A ribbon representation of the final MDFF model of the MPER insertion is overlaid, colored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049607#pone-0049607-g002" target="_blank">Figure 2</a>. A tilted view is shown to emphasize the confined nature of the cavity between the hexons.</p

CryoEM density showing α-helices for two hexons and two MPER insertions.

<p>(A) Coordinates from the final frame of an MDFF simulation show α-helices of both hexon and MPER docked within density rods of the cryoEM structure (gray). (B) Perpendicular view showing one of the two MPER density rods. (C) Additional view showing the second of the two MPER density rods at the icosahedral 2-fold axis with the final MDFF coordinates, which were not icosahedrally constrained. The isosurface threshold levels were chosen to highlight the well resolved density rods within each panel. Ribbon representations are shown for the hexon backbone (purple and blue), the MPER sequence (red), and the linker regions (green). The icosahedral 2-fold axis is indicated with an oval symbol.</p

CryoEM structure of the Ad-HVR2-GP41-L15 vector at subnanometer resolution.

<p>(A) Full virion viewed along an icosahedral 3-fold axis. Density assigned to the MPER insertion within the top facet is colored in red and gold, with red representing the MPER density within one asymmetric unit. This Ad vector is based on human Ad type 5, which has long and flexible fibers (>300 Å). Only short portions of the fiber (out to a radius of 463 Å) have been reconstructed (3 fibers are indicated with arrows). (B) Enlarged view with the 12 MPER density regions within one asymmetric unit numbered 1–12. Interacting MPER density regions from adjacent asymmetric units are numbered in parentheses. The four hexons are labeled H1–H4 and the penton base is labeled P. The icosahedral 2- and 3-fold axes are indicated with oval and triangle symbols respectively. Scale bars represent 100 Å.</p

MPER forms a stable helical bundle at 3-mer sites.

<p>(A) Overlay of MDFF refined model (ribbons) and the cryoEM density (gray) at the icosahedral 3-fold axis. The ribbon coloring scheme is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049607#pone-0049607-g002" target="_blank">Figure 2</a>. (B) Enlarged view of the helical packing interface viewed along the bundle axis with aromatic sidechains displayed (gold).</p

MPER interactions at 2-mer sites are weak and transient.

<p>Overlay of MDFF refined model (ribbons) and the cryoEM density (gray) at the strongest 2-mer site (between hexons H3 and H4 of a neighboring asymmetric unit). The ribbon coloring scheme is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049607#pone-0049607-g002" target="_blank">Figure 2</a>.</p

Alternate model conformations for MPER next to penton base.

<p>(A) Simulated density for hexon (blue) shown with a ribbon representation of an MDFF model of the MPER in an α-helical conformation or (B) extended conformation. The 14 amino acid 2F5 epitope is shown with a thicker ribbon in the extended model. The ribbons are colored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049607#pone-0049607-g002" target="_blank">Figure 2</a>.</p

Proposed vector modifications for optimizing MPER presentation at the Ad hexon HVR2 site.

<p>(A) Schematic representation of the MPER insertion incorporated within the Ad-HVR2-GP41-L15 vector. The hexon capsid protein (gray) is shown together with the N-terminal 3aa linker (blue), the MPER peptide (green), and the C-terminal 10aa linker (red). The protein-protein interface between hexons in the Ad capsid is represented by a hashed region. (B-E) Based on the structural analysis of the Ad-HVR2-GP41-L15 vector, four possible modifications are proposed which include (B) extending the N-terminal linker by 3aa, (C) extending the C-terminal linker by 4aa, (D) extending both the N- and C-terminal linkers by 2aa, and (E) swapping the N- and C- terminal linkers.</p

Characterization of XMRV pseudovirus and single-round neutralization assay.

<p>(A) Comparison of XMRV and control HIV-1 pseudoviruses in yield (p24 accumulation) and infectivity (IU/ml on TZM-bl cells). (B) Detection of antibody specificity to XMRV and HIV-1 pseudoviruses. Pseudoviruses were tested in the neutralization assay with mAb 83A25 that recognizes a shared epitope of MLV Env glycoprotein and with mAb b12 that recognizes HIV-1 Env glycoprotein. (C) Neutralization of the XMRV and HIV-1 pseudoviruses showing a broad range of sensitivity and specificity of the assay using polyclonal antibodies (anti Friend-MuLV).</p

Detection of XMRV-specific antibody production in mouse sera.

<p>Time course of the production of (A) ELISA-binding antibodies and (B) NAb in Balb/C mice (10 animals in each group) immunized with pDP1-XMRV<i>envgag</i> (first arrow; P), Ad5-XMRV (second and third arrows; A) and XMRV VLP (fourth arrow; V). Determination of (C) endpoint dilution and (D) serum neutralizing titers at the peak time point indicated by asterisks in Panels A and B, respectively. The arrow indicates endpoint dilution. (E) The specificity of the serum neutralizing activity was determined by comparing XMRV and HIV-1 pseudoviruses and showed that the primary target for neutralization is the XMRV Env.</p

Expression of XMRV Env, Gag and VLP.

<p>(A) Western blot analysis of XMRV gag expression. HeLa cells were infected with Ad5-XMRV (10 MOI) for 24 h and then whole cell lysate (Lane 1) and cell culture media concentrated 100-fold by centrifugation through a 20% sucrose cushion (Lane 2) were subjected to 10% SDS-PAGE and then transferred to PVDF. The blots were probed with anti-Gag mAb R187 and HRP-conjugated goat anti-rat immunoglobulin G antiserum (Southern Biotechnology Associates, Inc.). The masses (kDa) of the molecular weight standards (Std) are shown on the left. The arrows (←) indicate the positions of the Gag precursor at ∼65 kDa (top arrow) and a cleaved, lower molecular mass Gag protein (bottom arrow). (B) Detection of XMRV envelope expression by flow cytometric (left) and Western blot (right) analyses. For flow cytometry, HeLa cells infected as in (A) were stained with mAb 83A25 and fluorescein isothiocyanate-conjugated goat anti-rat immunoglobulin G antiserum. For Western blot analysis, VLP produced by those cells were purified from culture media and probed with mAb 83A25. MAb 83A25 recognizes an epitope located near the carboxyl terminus of Env that common for many MuLVs. (C) Electron microscopy showing VLP production in HeLa cells after 48 hours of infection with Ad5-XMRV (Panels I and II). An infectious XMRV virus is shown budding (arrows) from Du145-C7 cells, a prostate cancer cell line that constitutively produces XMRV (Panels III and IV). The similarities in morphology and size between the VLP and live XMRV particles are in the insets of Panels II and IV.</p