Macropinocytotic Uptake and Infection of Human Epithelial Cells with Species B2 Adenovirus Type 35
Macropinocytotic Uptake and Infection of Human Epithelial Cells with Species B2 Adenovirus Type 35
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DOI：
10.1128/jvi.02494-09
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发表时间：
2010-05-01
影响因子：
5.4
通讯作者：
Greber, Urs F.
中科院分区：
文献类型：
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作者：
Kaelin, Stefan;Amstutz, Beat;Greber, Urs F.
人类腺病毒血清型35 （HAdV-35，此处简称Ad35）可引起肾脏和尿路感染，并感染免疫功能低下个体的呼吸器官。与其他腺病毒不同，Ad35具有较低的血清阳性率，这使得基于Ad35的载体有希望成为基因治疗的候选者。Ad35利用CD46和整合素作为上皮细胞和造血细胞感染的受体。在这里，我们发现Ad35感染进入HeLa细胞、人肾HK-2细胞和正常人肺成纤维细胞强烈依赖CD46和整合素，但不依赖硫酸肝素，并且不同地需要大的GTPase动力蛋白。Ad35感染不依赖于AP180羧基末端结构域的表达，这有效地阻断了网格蛋白介导的摄取。对丝氨酸/苏氨酸激酶Pak1 （p21活化激酶）、蛋白激酶C （PKC）、钠-质子交换剂、肌动蛋白和酸性细胞器的小分子化学物质可抑制Ad35感染。值得注意的是，f -肌动蛋白抑制剂jasplakinolide、Pak1抑制剂IPA-3或钠-质子交换抑制剂5-（n -乙基- n -异丙基）amiloride （EIPA）阻断了Ad35的内吞。抑制巨噬细胞增生因子的显性阴性蛋白或小干扰rna，包括小GTPase Rac1、Pak1或Pak1效应物c端结合蛋白1 (CtBP1)，能有效抑制Ad35感染。共聚焦激光扫描显微镜、电子显微镜和活细胞成像显示，Ad35与大内吞结构中的液相标记物共定位，CD46、α - nu整合素和CtBP1均呈阳性。我们的结果扩展了先前对HAdV-3 （Ad3）的观察，并建立了巨噬细胞作用作为B种人腺病毒在上皮细胞和造血细胞中的感染途径。
Human adenovirus serotype 35 (HAdV-35; here referred to as Ad35) causes kidney and urinary tract infections and infects respiratory organs of immunocompromised individuals. Unlike other adenoviruses, Ad35 has a low seroprevalence, which makes Ad35-based vectors promising candidates for gene therapy. Ad35 utilizes CD46 and integrins as receptors for infection of epithelial and hematopoietic cells. Here we show that infectious entry of Ad35 into HeLa cells, human kidney HK-2 cells, and normal human lung fibroblasts strongly depended on CD46 and integrins but not heparan sulfate and variably required the large GTPase dynamin. Ad35 infections were independent of expression of the carboxy-terminal domain of AP180, which effectively blocks clathrin-mediated uptake. Ad35 infections were inhibited by small chemicals against serine/threonine kinase Pak1 (p21-activated kinase), protein kinase C (PKC), sodium-proton exchangers, actin, and acidic organelles. Remarkably, the F-actin inhibitor jasplakinolide, the Pak1 inhibitor IPA-3, or the sodium-proton exchange inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) blocked endocytic uptake of Ad35. Dominant-negative proteins or small interfering RNAs against factors driving macropinocytosis, including the small GTPase Rac1, Pak1, or the Pak1 effector C-terminal binding protein 1 (CtBP1), potently inhibited Ad35 infection. Confocal laser scanning microscopy, electron microscopy, and live cell imaging showed that Ad35 colocalized with fluid-phase markers in large endocytic structures that were positive for CD46, alpha nu integrins, and also CtBP1. Our results extend earlier observations with HAdV-3 (Ad3) and establish macropinocytosis as an infectious pathway for species B human adenoviruses in epithelial and hematopoietic cells.