Herpes simplex virus gE/gI sorts nascent virions to epithelial cell junctions, promoting virus spread

Abstract

Alphaherpesviruses spread rapidly through dermal tissues and within synaptically connected neuronal circuitry. Spread of virus particles in epithelial tissues involves movement across cell junctions. Herpes simplex virus (HSV), varicella-zoster virus (VZV), and pseudorabies virus (PRV) all utilize a complex of two glycoproteins, gE and gI, to move from cell to cell. HSV gE/gI appears to function primarily, if not exclusively, in polarized cells such as epithelial cells and neurons and not in nonpolarized cells or cells that form less extensive cell junctions. Here, we show that HSV particles are specifically sorted to cell junctions and few virions reach the apical surfaces of polarized epithelial cells. gE/gI participates in this sorting. Mutant HSV virions lacking gE or just the cytoplasmic domain of gE were rarely found at cell junctions; instead, they were found on apical surfaces and in cell culture fluids and accumulated in the cytoplasm. A component of the AP-1 clathrin adapter complexes, mu1B, that is involved in sorting of proteins to basolateral surfaces was involved in targeting of PRV particles to lateral surfaces. These results are related to recent observations that (i) HSV gE/gI localizes specifically to the trans-Golgi network (TGN) during early phases of infection but moves out to cell junctions at intermediate to late times (T. McMillan and D. C. Johnson, J. Virol., in press) and (ii) PRV gE/gI participates in envelopment of nucleocapsids into cytoplasmic membrane vesicles (A. R. Brack, B. G. Klupp, H. Granzow, R. Tirabassi, L. W. Enquist, and T. C. Mettenleiter, J. Virol. 74:4004-4016, 2000). Therefore, interactions between the cytoplasmic domains of gE/gI and the AP-1 cellular sorting machinery cause glycoprotein accumulation and envelopment into specific TGN compartments that are sorted to lateral cell surfaces. Delivery of virus particles to cell junctions would be expected to enhance virus spread and enable viruses to avoid host immune defenses.

FIG. 1

Electron micrographs of HEC-1A cells infected with wild-type HSV-1. HEC-1A human epithelial cells were grown to confluence on plastic coverslips and then infected with wild-type HSV-1. After 16 to 18 h, the cells were fixed and processed for electron microscopy. The basal surface is along the bottom of each image.

FIG. 2

Electron micrographs of HEC-1A cells infected with wild-type HSV-1 as described in the legend to Fig. 1.

FIG. 3

Electron micrographs of HEC-1A cells infected with F-gEβ. HEC-1A cells were grown as described for Fig. 1 and then infected with F-gEβ, a gE⁻ HSV-1 mutant, for 16 to 18 h before the cells were fixed and processed for electron microscopy.

FIG. 4

Electron micrographs of HEC-1A cells infected with F-gEβ, as described for Fig. 3. N, nucleus.

FIG. 5

Electron micrographs of HEp-2 cells infected with wild type (W.t.) or F-gEβ. HEp-2 (nonpolarized human cells) were grown to confluence on plastic coverslips, infected with either wild-type HSV-1 (A) or F-gEβ (B), and processed for electron microscopy after 17 h.

FIG. 6

Infectious HSV found in cell culture supernatants of epithelial cells. HEC-1A cells (A and B) and MDBK cells (C) were infected with wild-type HSV-1 (filled symbols and bars) or with F-gEβ (open symbols and bars). After various times, the cell culture supernatants (and cells [data not shown]) were harvested, and titers of infectious virus were determined on Vero cell monolayers. The value at each time point is the mean for five independent dishes, and standard deviations are shown. Panel A is a logarithmic scale, whereas panels B and C are linear scales.

FIG. 7

Electron micrographs of MDBK cells infected with wild-type (W.t.) HSV. Cells were fixed and processed for electron microscopy after 17 h.

FIG. 8

Electron micrographs of MDBK cells infected with F-gEβ (A) or F-gEΔCT (B and C). Cells were fixed and processed for electron microscopy after 17 h. N, nucleus.