Association of Orf65+ KSHV capsids with actin filaments at different stages of endocytosis during KSHV entry and trafficking in endothelial cells.

(A) HUVEC were infected with KSHV, fixed at 30 mpi, stained for Orf65+ viral capsids (red), actin filaments (green), and microtubules (white). The area highlighted by the square is enlarged and projected as 3D-projection images in the lower panels, which reveal the association of Orf65+ viral particles with actin filaments (middle), microtubules (right), and the merged image (left). (B) Time course analysis of the association Orf65+ viral particles with actin filaments. HUVEC were infected with KSHV, fixed at indicated time points post-infection, and stained for actin filaments (green), Orf65+ viral particles (red), and nuclei (blue). Highlighted sections in the upper panels were enlarged and shown as 3D-projection images in the bottom panels, which demonstrate the association of viral capsids with actin filaments at different time points of infection. (C) Quantification of the kinetics of colocalization of Orf65+ particles with actin filaments. (D–F) Association of Orf65+ viral particles shown in white with the endosomal protein EEA1 (D), Rab11 (E), or LAMP-1 (F), all in red, and actin filaments in green during KSHV entry of endothelial cells. HUVEC were prepared as described in (A) and labeled to visualize endosomal proteins, Orf65+ viral capsids and actin filaments. The sections highlighted by the squares are enlarged in the lower panels. For corresponding 3D projection of colocalization of Orf65+ particles with actin filaments and EEA1, Rab11 and LAMP-1, see Videos S13, S14 and S15.