Xie Jiansong, Chiang Lilian, Contreras Janette, Wu Kaijin, Garner Judy A, Medina-Kauwe Lali, Hamm-Alvarez Sarah F
Department of Pharmacology and Pharmaceutical Sciences, USC School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90033, USA.
J Virol. 2006 Dec;80(23):11833-51. doi: 10.1128/JVI.00857-06. Epub 2006 Sep 20.
The established mechanism for infection of most cells with adenovirus serotype 5 (Ad5) involves fiber capsid protein binding to coxsackievirus-adenovirus receptor (CAR) at the cell surface, followed by penton base capsid protein binding to alpha(v) integrins, which triggers clathrin-mediated endocytosis of the virus. Here we determined the identity of the capsid proteins responsible for mediating Ad5 entry into the acinar epithelial cells of the lacrimal gland. Ad5 transduction of primary rabbit lacrimal acinar cells was inhibited by excess Ad5 fiber or knob (terminal region of the fiber) but not excess penton base. Investigation of the interactions of recombinant Ad5 penton base, fiber, and knob with lacrimal acini revealed that the penton base capsid protein remained surface associated, while the knob domain of the fiber capsid protein was rapidly internalized. Introduction of rabbit CAR-specific small interfering RNA (siRNA) into lacrimal acini under conditions that reduced intracellular CAR mRNA significantly inhibited Ad5 transduction, in contrast to a control (nonspecific) siRNA. Preincubation of Ad5 with excess heparin or pretreatment of acini with a heparinase cocktail each inhibited Ad5 transduction by a separate and apparently additive mechanism. Functional and imaging studies revealed that Ad5, fiber, and knob, but not penton base, stimulated macropinocytosis in acini and that inhibition of macropinocytosis significantly reduced Ad5 transduction of acini. However, inhibition of macropinocytosis did not reduce Ad5 uptake. We propose that internalization of Ad5 into lacrimal acini is through a novel fiber-dependent mechanism that includes CAR and heparan sulfate glycosaminoglycans and that the subsequent intracellular trafficking of Ad5 is enhanced by fiber-induced macropinocytosis.
腺病毒血清型5（Ad5）感染大多数细胞的既定机制涉及纤维衣壳蛋白在细胞表面与柯萨奇病毒-腺病毒受体（CAR）结合，随后五邻体基底衣壳蛋白与α(v)整合素结合，从而触发病毒的网格蛋白介导的内吞作用。在此，我们确定了介导Ad5进入泪腺腺泡上皮细胞的衣壳蛋白的身份。过量的Ad5纤维或纤维的末端区域（钮）可抑制原代兔泪腺腺泡细胞的Ad5转导，但过量的五邻体基底则无此作用。对重组Ad5五邻体基底、纤维和钮与泪腺腺泡相互作用的研究表明，五邻体基底衣壳蛋白仍与表面相关，而纤维衣壳蛋白的钮结构域则迅速内化。在显著降低细胞内CAR mRNA的条件下，将兔CAR特异性小干扰RNA（siRNA）导入泪腺腺泡，与对照（非特异性）siRNA相比，显著抑制了Ad5转导。用过量肝素预孵育Ad5或用肝素酶混合物预处理腺泡，每种方法都通过一种单独且明显相加的机制抑制Ad5转导。功能和成像研究表明，Ad5、纤维和钮而非五邻体基底可刺激腺泡中的巨胞饮作用，且抑制巨胞饮作用可显著降低腺泡的Ad5转导。然而，抑制巨胞饮作用并未减少Ad5的摄取。我们提出，Ad5进入泪腺腺泡是通过一种新的纤维依赖性机制，该机制包括CAR和硫酸乙酰肝素糖胺聚糖，并且纤维诱导的巨胞饮作用增强了Ad5随后的细胞内运输。
