Topp K S, Rothman A L, Lavail J H
Department of Anatomy, University of California, San Francisco 94143, USA.
Exp Eye Res. 1997 Mar;64(3):343-54. doi: 10.1006/exer.1996.0209.
Our objective was to determine quantitatively whether herpes simplex virus infects preferentially the apical or basolateral surfaces of two well-differentiated cell types, human retinal pigment epithelial cell and Madin-Darby canine kidney epithelial cells. Secondarily, we sought to localise the mannose 6-phosphate/insulin-like growth factor II receptor, a putative receptor for herpes simplex virus, in the membrane domains of the retinal pigment epithelial cells. Although it has been suggested that receptors utilized by the herpesviruses are heterogeneously distributed on epithelial cells, no quantitative evidence of preferential polarized uptake of wild-type herpes simplex virus into an epithelial cell has yet appeared. Moreover, no evidence has appeared of the distribution of mannose-6-phosphate/insulin-like growth factor II receptor in human retinal pigment epithelial cells. We hypothesized that the preferred pole of uptake and infection by HSV would correlate with the distribution of the receptor. Understanding the preferred site of entry in these cells may shed light on the mechanism of pathological infection and spread of this and related viruses, such as cytomegalovirus, in acute retinal necrosis and herpetic encephalitis. The efficiency of viral infection was assayed two ways. First, using permeable filters on which the monolayer of polarized epithelial cells was grown, we compared the number of foci of infected cells that resulted from an apical infection with that resulting from application of virus to the underside of the filter from which the virus could reach the basolateral surface of the cells. Second, we compared the number of infected cell foci that resulted from an apical infection to the number formed following infection at both the apical and basolateral surfaces of the cells. Both surfaces were exposed to virus following disruption of the tight junctions between cells with a Ca2+ chelator. After the efficiency of infection was normalized for relative surface areas, we found that both cell types were equally infectable with the F strain of the virus. However, there was a difference in the degree of polarized uptake of virus by the two cell types. Virus infected the basolateral surface of the retinal cells only about 6.5 times as effectively as it infected the apical surface of those cells, whereas virus infected the basolateral surface of the kidney epithelial cells about 435 times as effectively as it infected the apical surface of the same cells. These data suggest that herpes simplex virus can efficiently enter either the apical or basolateral surface of retinal pigment epithelial cells, unlike its more polarized preference for the basolateral surface of the kidney epithelial cell type. The mannose 6-phosphate/insulin-like growth factor II receptor was present in human retinal pigment epithelial cells, as determined by Western blotting. Surface biotinylation experiments revealed the presence of the receptor in both the apical and basolateral membranes of the retinal epithelial cells. Our evidence is consistent with the hypothesis that the virus may utilize the mannose 6-phosphate/insulin-like growth factor II receptor to facilitate entry.
我们的目标是定量确定单纯疱疹病毒是否优先感染两种高度分化的细胞类型，即人视网膜色素上皮细胞和马-达二氏犬肾上皮细胞的顶端或基底外侧表面。其次，我们试图在视网膜色素上皮细胞的膜结构域中定位甘露糖6-磷酸/胰岛素样生长因子II受体，这是一种推测的单纯疱疹病毒受体。虽然有人提出疱疹病毒利用的受体在上皮细胞上分布不均，但尚未出现野生型单纯疱疹病毒优先极化摄取进入上皮细胞的定量证据。此外，也没有关于甘露糖-6-磷酸/胰岛素样生长因子II受体在人视网膜色素上皮细胞中分布的证据。我们假设HSV摄取和感染的偏好极与受体的分布相关。了解这些细胞中病毒的优先进入位点可能有助于揭示这种及相关病毒（如巨细胞病毒）在急性视网膜坏死和疱疹性脑炎中的病理感染和传播机制。病毒感染效率通过两种方法进行测定。首先，使用生长有极化上皮细胞单层的可渗透滤器，我们比较了顶端感染产生的感染细胞灶数量与将病毒应用于滤器底部（病毒可由此到达细胞基底外侧表面）产生的感染细胞灶数量。其次，我们比较了顶端感染产生的感染细胞灶数量与细胞顶端和基底外侧表面均感染后形成的感染细胞灶数量。在用Ca2+螯合剂破坏细胞间紧密连接后，两个表面均暴露于病毒。在将感染效率按相对表面积进行归一化后，我们发现两种细胞类型对病毒F株的感染能力相同。然而，两种细胞类型对病毒的极化摄取程度存在差异。病毒感染视网膜细胞基底外侧表面的效率仅约为感染顶端表面的6.5倍，而病毒感染肾上皮细胞基底外侧表面的效率约为感染同一细胞顶端表面的4
