The SFV glycoproteins arrange into a hexagonal array within CPV-II while in transport to the plasma membrane. A similar array dimension between glycoproteins is observed on the plasma membrane adjacent to the budding virion and subsequently organized into the envelope of a T=4 lattice through the virion assembly to perform membrane budding. These well-defined glycoprotein arrays may help playing important roles in organizing the symmetry of the nucleocapsid in forming a mature virion.

Array of glycoproteins on budding SFV. (A) A volume rendering of the tomogram on the budding viruses on the plasma membrane of a cell infected with SFV. Several budding virions (∼70 nm in diameter) were captured before scission from plasma membrane. (B) Surface rendering of this tomogram showing the interstices of the spikes on the outer envelope of the virion. A budding virion clearly shows the hexagonal array of the glycoproteins (arrowheads), where the node-to-node distance is measured as 7 nm. PM, plasma membrane. (C) A slice section from the tomogram of the same budding virus demonstrates clearly the hexagonal density, corresponding well to the glycoprotein network in the cryo-EM density map of isolated virus. Scale bar, 5 nm.

Arrangement of viral glycoproteins at the site of budding on the plasma membrane. (A) A volume projection of SFV budding site reveals that the budding virus (arrowhead) was in proximity to the gold particles that registered the location of the E2 glycoprotein on the plasma membrane. Scale bar, 200 nm. (B) The stereo pair of projection views above plasma membrane illustrates the budding region around an exiting virion (arrowhead in panel A). This close-up view shows the exterior surface of the plasma membrane with detailed arrangement of an associated group of gold markers, with the majority of them observed with an average spacing of ∼7 nm. The conjugated gold particles are located on the plasma membrane with the brightest density as a result of the high-angle electron scattering in electron microscopic imaging.

The composition of a CPV-II tubular structure. (A) The gold particles (arrowheads) were attached to the tubular structures of the tomogram of CPV-II during immunolabeling with anti-E2 antibody. Additional short tubules were observed lying vertically to the long tubules, and one of them (asterisk) appears to make contact with the CPV-II membrane. (B) An orthoslice (0.67 nm in thickness) was taken from the tomogram of the same CPV-II as in panel A, showing two tubules that appeared to have helical turns. The gold particle on the short tube is omitted from the slice, as it was positioned on the other side of the section (see also Video S1 in the supplemental material). Note that the gold labeling was performed on both sides, and it resulted in an uneven distribution of gold particles in the z dimension. Scale bar, 100 nm. (C) Volume rendering of CPV-II tubule tomogram shows the hexagonal array (asterisks) of the density nodes. Scale bar, 15 nm. The node-to-node distance (between the pair of arrowheads) is approximately 7 nm. (D) A planar array of five adjacent hexagonal rings of SFV glycoprotein trimers indicates that the trimer-to-trimer distance is 7.2 nm. Scale bar, 7 nm.

CPV-II distribution and morphology in SFV-infected BHK-21 cells. (A) CPV-II can be found close to the plasma membrane at the late stage of SFV infection. Tubular structures were well preserved in CPV-II and attached by gold particles (white arrowheads) that registered the location of anti-E2 antibodies. The gold particle was identified by its unique profile, the darkest-contrast and sharply defined edge. A few gold particles located on the plasma membrane are not marked. (B) The plot shows the CPV-II density in the BHK-21 cells that were infected with SFV at an MOI of 2 for 30 min, an MOI of 2 for 60 min, and an MOI of 200 for 30 min. The number of type II CPVs was counted after fixation of the cells at 3, 5, and 8 h postinfection and normalized relative to the measured specimen area. The average CPV-II density (number per μm²) ± standard deviation (SD) at each time point is shown. There was no CPV-II observed in mock-infected BHK-21 cells; thus the mock infection data were omitted from the graph. ads, adsorption. (C) Five consecutive sections (95 nm thick) of the same type II CPV, showing the CPV from the top to bottom. (D) A view of a CPV-II enclosed tubular structure (white arrowhead), attached by dense circular particles on the exterior surface. The two tubules within CPV-II are approximately 50 nm in diameter. (E) A longitudinal section of CPV-II shows its dimension (exceeding 1 μm). (F) A zoomed-in image of the area enclosed in a white box in panel E, showing the dense particles attached to the external membrane layer of CPV-II and the tubules. Scale bars, 200 nm (panels A, C, and E) and 50 nm (panels D and F).