Fig 2: Cellular localization of Rab6a and HSV-1 proteins. (A) TauSTED confocal imaging shows that HSV-1 mRFP-VP26 puncta and EmGFP-Rab6a colocalize with trans-Golgi marker Golgin97 in Vero cells. Samples were infected with HSV-1 amplicon vector expressing EmGFP-Rab6a and HSV-1 OK14 helper virus and fixed and stained at 6 hpi. Scale bar represents 5 µm. (B) Confocal microscopy shows that endogenous Rab6a (blue), exogenous mCherry-Rab6a (red), and HSV-1 gM-pHluorin (green) colocalize at the Golgi (arrows) and at the cell periphery (arrowhead) in Vero cells. Cells were transduced to express mCherry-Rab6a, infected with HSV-1 IH01, and fixed and stained at 7 hpi. Scale bar represents 10 µm. (C) Confocal microscopy shows that endogenous Rab6a (blue) and HSV-1 gM-pHluorin protein (green) colocalize similarly in Vero cells that are not transduced and do not express exogenous Rab6a. Cells were infected with HSV-1 IH01 and fixed and stained at 7 hpi. Scale bar represents 10 µm.

Fig 3: A non-functional GDP-locked Rab6a mutant affects gM-pHluorin localization, but does not affect HSV-1 replication. (A–C) Vero cells were transduced with adenovirus vectors expressing mCherry-Rab6a or (T27N) mutant, infected with HSV-1 IH01, and imaged by widefield fluorescence microscopy at 7 hpi. gM-pHluorin localizes to ER membranes, visible as a distinct ring around the nucleus (N), juxtanuclear Golgi, and accumulates forming clusters at the cell periphery (arrowheads). Scale bars = 10 µm. (A) mCherry-Rab6a localizes to the juxtanuclear Golgi region and peripheral accumulations. (B) GDP-locked mCherry-Rab6a(T27N) does not form discrete puncta and is diffuse throughout the cell. (C) Cells (n = 50) were scored based on whether gM-pHluorin localizes predominantly to ER or the juxtanuclear Golgi region and whether gM-pHluorin accumulates to form peripheral clusters. Expression of Rab6a(T27N) caused a significant increase (P < 0.001) in ER localization but no significant difference in peripheral accumulations (ns; P > 0.05) by Fisher’s exact test. (D) Single-step replication kinetics of HSV-1 in Vero cells transduced with adenovirus vectors expressing mCherry-Rab6a, mCherry-Rab6a(T27N), mCherry-Rab27a, or mCherry-Fyn(10). Data represent geometric means of three experimental replicates.

Fig 4: TIRF microscopy at 6 hpi of Vero cells coinfected with HSV-1 IH01 expressing gM-pHluorin (green) and an adenovirus vector expressing mCherry-Rab6a (red). Representative cell shown from 82 cells imaged across 12 experimental replicates. Scale bars = 10 µm. (A) Schematic of mCherry-Rab6a secretory vesicles transporting virus particles to the plasma membrane. gM-pHluorin becomes fluorescent with the pH change at the moment of exocytosis. TIRF microscopy excites fluorescent molecules at the plasma membrane (Movie S4). (B) HSV-1 gM accumulates in the cell periphery in cells that are not infected (1) and are infected (3). Rab6a accumulates in the cell periphery in cells that are not transduced (2) and are transduced (3). (C) HSV-1 particles undergo exocytosis at the cell periphery where and HSV-1 particles and Rab6a accumulate.

Fig 5: Quantification of HSV-1 and Rab6a peripheral accumulations. Percentage of cells with peripheral accumulations of Rab6a, comparing cells that are coinfected vs not productively infected with HSV-1. ns = no significant difference, P > 0.05 by Fisher’s exact test. (A) Data correspond to experiments shown in Fig. 2 and are representative of 27 cells across eight experimental replicates. (B) Data correspond to experiments shown in Fig. 5 and are representative of 82 cells imaged across 12 experimental replicates.