FIG. 6.

The early gM nuclear pool does not transit through the TGN. 143B cells were infected with wild-type HSV-1 strain 17⁺ (HSVwt) at an MOI of 2 for 4 h at 37°C, followed by an additional 4 h at 37°C or 15°C. The cells were fixed and stained against the ERGIC (red) and gM (A to D) or gB (E and F) (green). gD and gH were also analyzed, with results similar to those for gB (data not shown). (G) Quantification of the distribution of the glycoproteins in either the nucleus (NM [nuclear membrane]) or ERGIC in panels A to F was done for 10 random microscopy fields. The total cell number was between 34 and 147 for each condition and was normalized to 100%. (H) HeLa cells were mock infected or infected for 8 h with wild-type HSV-1 strain 17⁺. At that point, the nuclei were isolated as described in Materials and Methods and treated with endo H (+) or incubated in the absence of the enzyme (−) and loaded on an SDS-PAGE gel. The separated proteins were transferred onto a polyvinylidene difluoride membrane and probed for gM or gD by Western blotting. Note the various forms of the glycoproteins (nonglycosylated precursor, immature high-mannose form, and mature protein). (I) Quantification of endo H resistance by Image J. gM and gD on nuclei (H), in 24-h p.i. cell lysates, or in extracellular virions were resolved by SDS-PAGE, digested with endo H, probed by Western blotting, and quantified (see Materials and Methods). The error bars depict the standard deviations of the means.