Activities of TBC1D20 and RN-Tre are important for production of infectious HSV-1. (A) COS7 cells were cotransfected with a plasmid expressing the essential virus protein pUL36 and plasmids expressing TBC domain proteins as described in Materials and Methods. The positive control was transfected with the UL36 plasmid and pcDNA3, while the negative control was transfected with only pcDNA3. At 24 h posttransfection cells were infected with HSV-1ΔUL36 at 5 PFU/cell, progeny virus was collected 16 hpi, and infectious virus levels were determined by titration on HS30 cells. Titers were normalized to the positive control, and bars represent the means of at least three independent experiments. Error bars are standard errors of the means. The solid line represents the median HSV-1 titer across all samples, and dashed lines represent ±1 standard deviation from the median. (B) Rab GAPs of interest (gray bars) were expressed alongside their inactive controls (white bars) and infected as described above. Titers were normalized to the positive control, and bars represent the means of at least two independent experiments. Error bars are standard errors of the means. The dashed line represents a 5-fold reduction in titer compared to the positive control.

Rab1a/b is required for transport of glycoproteins from the ER. (A) COS7 cells were cotransfected with a plasmid expressing TBC1D20-myc or TBC1D20RA-myc and HSV-1 gM-GFP (green) or gB (green). Eight hours posttransfection cells were fixed and stained with anti-myc (blue) and anti-calnexin (red). (B and C) HeLa cells were transfected with siRNA duplexes as described in the text. (B) Cells were infected with HSV-1ΔgE (MOI, 3), fixed at 8 hpi, and labeled with TGN46 (red) or calnexin (red) and gD (green). Bars, 10 μm. (C) Cells were infected with HSV-1 (MOI, 5), and 20 hpi cell pellets were lysed, separated by SDS-PAGE, and analyzed by Western blotting with antibodies to gD, gB, and gH.

Rab43 causes Golgi complex fragmentation but is not required for delivery of glycoproteins to the plasma membrane. (A and B) Cos7 cells were transfected with plasmids expressing RN-treRA-myc or RN-tre-myc and gM-GFP (A, upper panels) or gB (A, green, lower panels) or gD (B, all green), fixed 24 h posttransfection, and stained with antibodies to myc (blue) and TGN46 (red) or GM130 (red). (C) HeLa cells were transfected with siRNA and infected (MOI, 3) as detailed in the text. At 8 hpi cells were fixed and labeled with antibodies to gD (green, left panels) or gB (green, right panels) and TGN46 (red). Bars, 10 μm.

Functional Rab43 is required for HSV-1 assembly. (A) A549 cells were transfected with Rab43 or control siRNA and infected with HSV-1 (MOI, 5). At 18 hpi supernatant virions were collected as described in Materials and Methods. The virus samples as well as parallel cell lysates were separated by SDS-PAGE and blotted for pUL48 (upper blot) or tubulin (lower blot). Blots were quantified using the LI-COR Odyssey infrared imager, and the Odyssey v3.0 software was used to calculate the integrated intensity for quantification. The graph represents the amount of pUL48 in the supernatant normalized to the pUL48 signal in the cell lysate. (B) A549 cells were grown on coverslips, transfected, and infected (MOI, 3) as described in Materials and Methods. At 16 hpi cells were fixed, processed, and analyzed by transmission EM. Arrows indicate enveloped capsids, and arrowheads indicate unenveloped capsid. Bars, 500 nm.

Rab1a/b and Rab43 are required for infectious virus production. HeLa cells were transfected once (Rab1a/b) or twice (Rab43) with siRNA duplexes as detailed in Materials and Methods. At 48 h after the final transfection, cells were infected with HSV-1 (MOI, 5), and cell-associated virus was collected 20 hpi. Titers were established on Vero cells, and bars represent the means of three independent experiments, normalized to the control siRNA (A). Parallel samples of infected cells were collected 20 hpi, lysed, separated by SDS-PAGE, and analyzed by Western blotting (B and C). For panel C, cells were additionally transfected with a plasmid expressing Rab43-GFP.

Functional Rab1a/b is required for HSV-1 assembly. (A) A549 cells were transfected with Rab1a/b or control siRNA and infected with HSV-1 (MOI, 5). At 18 hpi, supernatant virions were collected as described in Materials and Methods. Virus samples treated with trypsin, as well as parallel cell lysates, were separated by SDS-PAGE and blotted for pUL48 (upper blots) or tubulin (lower blots). Blots were quantified using the LI-COR Odyssey infrared imager, and the Odyssey v3.0 software was used to calculate the integrated intensity for quantification. The graph represents the amount of pUL48 in the supernatant normalized to the pUL48 signal in the cell lysate. (B) A549 cells were grown on coverslips, transfected, and infected (MOI, 3) as described in Materials and Methods. At 16 hpi cells were fixed, processed, and analyzed by transmission EM. Arrows indicate enveloped capsids, and arrowheads indicate unenveloped capsid. Bars, 500 nm.

Analysis of glycoprotein localization after internalization from the plasma membrane in infected cells. (A to C) HeLa cells were cotransfected with plasmids expressing RN-tre or RN-treRA-myc (blue) (A) or RN-tre or RN-treRA-GFP (green) (B and C). At 24 h posttransfection cells were infected with HSV-1ΔgE (MOI, 3) for 8 h, whereupon anti-gD antibody was bound at 4°C for 1 h and subsequently incubated at 37°C for 60 min (A), 15 min (B), or 30 min (C). The cells were then fixed and labeled with appropriate secondary antibodies as well as anti-TGN46 (red) or anti-EEA1 (blue). For panel C, anti-gB antibody (blue) was used alongside anti-gD antibody.