Scale bar = 10 m. Figure S2 : Dominant\bad dynamin decreases endocytosis of gB and gE. antibodies (LP2) for 16 h. Cells were then fixed, permeabilized and labeled with main antibodies to GM130 followed by isotope\specific secondary Alexa Fluor antibodies. Cells were imaged by confocal microscope. Level pub PI-3065 = 10 m. Number S2 : Dominant\bad dynamin decreases endocytosis of gB and gE. PI-3065 WT or K44A dynamin\transfected COS7 cells were infected with VP26\mTurquoise/gM\EYFP for 8 h and then fed with anti\gB (CB24) or gE (3114) antibodies for 15 min before fixing. Cells were then permeabilized, immunostained with anti\HA antibodies and LIFR then labeled with secondary antibodies. Images were acquired using epifluorescence microscope. Level pub = 10 m. Number S3 : Effect of endocytosis inhibitors on uptake of transferrin and glycoproteins into COS7 cells. A) Cells were incubated with DMSO, 15 m Dynole or Dynole bad control and 30 m PitStop2 or its bad control for 15 min. Next, transferrin conjugated to Alexa Fluor 568 was added for 5 min. Cells were then washed on snow, fixed and imaged using an epifluorescence microscope. Level pub = 20 m. B) Cells were infected with HSV\1 for 8 h. For the last 30 min, cells were incubated with DMSO, 15 m Dynole or Dynole bad control and 30 m PitStop2 or its bad control. After 15\min incubation, antibodies specific to viral glycoproteins (LP2 for gD, 3063 for gE, CB24 for gB and LP11 for gH) were added and incubated for a further 15 min. After fixing and permeabilizing, cells were stained with secondary antibodies and imaged by epifluorescence microscopy. Level pub = 20 m. Number S4 PI-3065 : Low\power images corresponding to Numbers ?44 and ?6.6. A) HFF\Tert cells were infected with VP26\mTurquoise/gM\EYFP recombinant disease for 8 h. Thirty minutes before the end of incubation cells were treated with 15 m Dynole or Dynole bad control and 30 m PitStop2 or its bad control. After 15 min antibodies specific to gD (LP2) were added and incubated for a further 15 min. Cells were then fixed, permeabilized and labeled with secondary antibodies. Images were acquired using a confocal microscope. Level pub = 10 m. B) HFF\Tert cells were infected with VP26\mTurquoise/gM\EYFP recombinant disease for 8 h. Antibodies specific to HSV\1 gD, gB, gH and gE were added in various mixtures for the last 15 min of incubation. After fixing and permeabilization, isotype\specific secondary Alexa Fluor antibodies were used and images were acquired using a confocal microscope. Level pub = 5 m. Number S5 : dSTORM images of clathrin and caveolae\dependent endocytosis of gE. HFF\Tert cells were infected with VP26\mTurquoise/gM\EYFP recombinant disease for 8 h. gE\specific antibodies were added to the cells for uptake 15 min before fixation and permeabilization. Clathrin\coated pits or caveolae PI-3065 were recognized with anti AP\2 (A) or Cav\1 (B) monoclonal antibodies and subtype\specific secondary Alexa Fluor antibodies. Wide\field images were acquired for VP26 PI-3065 (blue) and gM (yellow), and merged with dSTORM images of gE (Alexa Fluor 568 channel) and AP2/Cav1 (Alexa Fluor 647 channel). Level bars = 10 m, 500 nm and 200 nm. TRA-17-21-s001.docx (30M) GUID:?D0F1E877-1FAC-4DFF-95ED-32F055F019AC Abstract Herpes simplex virus\1 (HSV\1) is definitely a large enveloped DNA virus that belongs to the family of Herpesviridae. It has been recently shown the cytoplasmic membranes that wrap the newly put together capsids are endocytic compartments derived from the plasma membrane. Here, we display that dynamin\dependent endocytosis plays a major role in this process. Dominant\bad dynamin and clathrin adaptor AP180 significantly decrease disease production. Moreover, inhibitors focusing on dynamin and clathrin lead to a decreased transport of glycoproteins to cytoplasmic capsids, confirming that glycoproteins are delivered to assembly sites via endocytosis..