CENTRIPETAL TRANSPORT OF HERPES-SIMPLEX VIRUS IN HUMAN RETINAL-PIGMENT EPITHELIAL-CELLS IN-VITRO

Herpes simplex virus displays tropism for neurons and other polarized epithelial cells. We have grown human retinal pigment epithelial cells in culture to study potential mechanisms whereby herpes simplex virus (type I) is transported from the plasma membrane of the cell to the n ucleus. The cells were highly polarized as determined by a variety of criteria. They were tightly coupled by junctional complexes, as determ ined by electron microscopy, immunofluorescent staining of tight junct ions and measurements of transepithelial electrical resistances > 200 Omega . cm(2). Immunofluorescence and confocal microscopy were used to visualize microtubule orientation. The microtubules were arranged (i) in a single apical cilium, (ii) in a meshwork beneath the apical memb rane and (iii) in longitudinally arranged bundles near the lateral mem branes and nucleus. The latter microtubules were primarily oriented wi th their plus ends directed toward the basal surface of the cells. We infected retinal pigment epithelial cells at the apical surface with v irus and assayed the uptake and transport of virus to the nucleus by q uantitative immunoblot and immunocytochemical staining for the viral i mmediate early gene product, infected cell protein 4. The antigen firs t appeared in retinal pigment epithelial cells 2 h after infection. Tr eatment of the cells with 33 mu M nocodazole, a microtubule-destabiliz ing drug, delayed the appearance of the viral antigen by 1 h. The effe ct of nocodazole treatment on microtubule integrity was confirmed by i mmunofluorescent staining and immunoblots of tubulin. Both cytoplasmic dynein and the ubiquitous form of kinesin were identified in the cell s using immunoblots. These novel data indicate that human retinal pigm ent epithelial cells, like neurons, are susceptible to infection by he rpes simplex virus and that the centripetal transport of virus to the nucleus in both cell types is facilitated by microtubules. The orienta tion of microtubules in retinal pigment epithelial cells suggests that the transport of herpes simplex virus from the apical surface is medi ated by a microtubule-activated motor enzyme, possibly kinesin.