Actin dynamics regulate multiple endosomal steps during Kaposi's sarcoma-associated herpesvirus entry and trafficking in endothelial cells.
Actin dynamics regulate multiple endosomal steps during Kaposi's sarcoma-associated herpesvirus entry and trafficking in endothelial cells.
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DOI：
10.1371/journal.ppat.1000512
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发表时间：
2009-07
期刊：
影响因子：
6.7
通讯作者：
Gao SJ
中科院分区：
文献类型：
--
作者：
Greene W;Gao SJ
肌动蛋白动力学在哺乳动物细胞中网格蛋白介导的内吞作用中的作用尚不清楚。在这项研究中，我们使用基于免疫荧光的方法来可视化病毒衣壳和相关细胞成分，确定了肌动蛋白细胞骨架在卡波西肉瘤相关疱疹病毒（KSHV）进入内皮细胞和运输中的作用。与感染性或报告基因试验相比，这种方法不依赖于任何病毒和报告基因的表达，而是直接跟踪单个病毒颗粒在核膜上的积累，作为病毒成功进入和运输细胞的指标。内体酸化抑制剂降低了含有病毒颗粒的细胞核的百分比和在核周区域停靠的病毒颗粒的总数，表明内吞作用，而不是质膜融合，是KSHV进入内皮细胞的主要途径。因此，病毒包膜蛋白仅在感染早期内化的KSHV颗粒上检测到，而在感染后期未检测到。网格蛋白而非小泡/脂质筏介导的内吞作用抑制了KSHV的进入，表明网格蛋白介导的内吞作用是KSHV进入内皮细胞的主要途径。KSHV颗粒不仅与早期和再循环核内体和溶酶体标记物共定位，而且与感染早期的肌动蛋白丝共定位。与这些观察结果一致的是，通过网格蛋白介导的内吞作用进入细胞的转铁蛋白被发现与肌动蛋白丝以及早期和再循环核内体有关，并且在较小程度上与晚期核内体和溶酶体有关。KSHV感染诱导动态肌动蛋白细胞骨架重排。破坏肌动蛋白细胞骨架和抑制肌动蛋白成核调节因子，如Rho GTPases和Arp2/3复合物，会严重阻断KSHV的进入和运输。总之，这些结果表明肌动蛋白动力学在内皮细胞中KSHV的内化和内体分选/运输以及网格蛋白介导的内吞作用中起重要作用。内吞作用是介导细胞内化事件的重要生物过程，病原体经常利用内吞作用进入靶细胞。肌动蛋白细胞骨架在哺乳动物细胞中网格蛋白介导的内吞作用中的作用尚不清楚。卡波西肉瘤相关疱疹病毒（KSHV）是一种伽玛疱疹病毒，与卡波西肉瘤（一种常见于艾滋病患者的内皮恶性肿瘤）和其他几种恶性肿瘤的发展有关。在本研究中，我们发现KSHV通过网格蛋白介导的内吞途径进入内皮细胞，这一过程受肌动蛋白动力学调节。我们发现KSHV颗粒存在于早期和循环的核内体和溶酶体中，它们在病毒感染的早期时间点停靠在肌动蛋白丝上。同样，通过网格蛋白介导的内吞作用进入细胞的转铁蛋白与肌动蛋白丝以及早期和循环核内体相关，并且在较小程度上与晚期核内体和溶酶体相关。破坏肌动蛋白细胞骨架和抑制肌动蛋白成核调节因子，如Rho GTPases和Arp2/3复合物，严重阻断了KSHV在内皮细胞中的进入和运输。总之，这些结果确定了肌动蛋白动力学在KSHV感染和网格蛋白介导的内皮细胞内吞过程中的多个内体步骤中的重要作用。
The role of actin dynamics in clathrin-mediated endocytosis in mammalian cells is unclear. In this study, we define the role of actin cytoskeleton in Kaposi's sarcoma-associated herpesvirus (KSHV) entry and trafficking in endothelial cells using an immunofluorescence-based assay to visualize viral capsids and the associated cellular components. In contrast to infectivity or reporter assays, this method does not rely on the expression of any viral and reporter genes, but instead directly tracks the accumulation of individual viral particles at the nuclear membrane as an indicator of successful viral entry and trafficking in cells. Inhibitors of endosomal acidification reduced both the percentage of nuclei with viral particles and the total number of viral particles docking at the perinuclear region, indicating endocytosis, rather than plasma membrane fusion, as the primary route for KSHV entry into endothelial cells. Accordingly, a viral envelope protein was only detected on internalized KSHV particles at the early but not late stage of infection. Inhibitors of clathrin- but not caveolae/lipid raft-mediated endocytosis blocked KSHV entry, indicating that clathrin-mediated endocytosis is the major route of KSHV entry into endothelial cells. KSHV particles were colocalized not only with markers of early and recycling endosomes, and lysosomes, but also with actin filaments at the early time points of infection. Consistent with these observations, transferrin, which enters cells by clathrin-mediated endocytosis, was found to be associated with actin filaments together with early and recycling endosomes, and to a lesser degree, with late endosomes and lysosomes. KSHV infection induced dynamic actin cytoskeleton rearrangements. Disruption of the actin cytoskeleton and inhibition of regulators of actin nucleation such as Rho GTPases and Arp2/3 complex profoundly blocked KSHV entry and trafficking. Together, these results indicate an important role for actin dynamics in the internalization and endosomal sorting/trafficking of KSHV and clathrin-mediated endocytosis in endothelial cells. Endocytosis, an essential biological process mediating cellular internalization events, is often exploited by pathogens for their entry into target cells. The role of actin cytoskeleton in clathrin-mediated endocytosis in mammalian cells remains unclear. Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus linked to the development of Kaposi's sarcoma, an endothelial malignancy commonly found in AIDS patients, and several other malignancies. In this study, we found that KSHV uses the clathrin-mediated endocytosis pathway to enter endothelial cells, and this process is regulated by actin dynamics. We found KSHV particles in early and recycling endosomes, and lysosomes, which are docked on actin filaments at the early time points of viral infection. Similarly, transferrin, which enters cells by clathrin-mediated endocytosis, is associated with actin filaments together with early and recycling endosomes, and, to a lesser degree, with late endosomes and lysosomes. Disruption of the actin cytoskeleton and inhibition of regulators of actin nucleation such as Rho GTPases and Arp2/3 complex profoundly blocked KSHV entry and trafficking in endothelial cells. Together, these results define an important role for actin dynamics in multiple endosomal steps during KSHV infection and clathrin-mediated endocytosis in endothelial cells.
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Formin MDIA2独立于其肌动蛋白成核活性稳定微管。
影响因子：
7.8
作者：
Bartolini, Francesca;Moseley, James B.;Schmoranzer, Jan;Cassimeris, Lynne;Goode, Bruce L.;Gundersen, Gregg G.
通讯作者：
Gundersen, Gregg G.
DOI：
10.1083/jcb.131.1.69
发表时间：
1995-10
期刊：
The Journal of cell biology
影响因子：
--
作者：
Damke H;Baba T;van der Bliek AM;Schmid SL
通讯作者：
Schmid SL
影响因子：
3.7
作者：
Foerg, Christina;Ziegler, Urs;Merkle, Hans P.
通讯作者：
Merkle, Hans P.
DOI：
10.1101/sqb.1995.060.01.060
发表时间：
1995-01-01
期刊：
COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY
影响因子：
--
作者：
Dai, J;Sheetz, MP
通讯作者：
Sheetz, MP
影响因子：
3.3
作者：
Johnson, Martha B.;Chen, Juxing;Enns, Caroline A.
通讯作者：
Enns, Caroline A.