Suikkanen Sanna, Antila Mia, Jaatinen Anne, Vihinen-Ranta Maija, Vuento Matti
Department of Biological and Environmental Science, PO Box 35, FIN-40014 University of Jyväskylä, Finland.
Virology. 2003 Nov 25;316(2):267-80. doi: 10.1016/j.virol.2003.08.031.
Canine parvovirus (CPV) is a small nonenveloped virus with a single-stranded DNA genome. CPV enters cells by clathrin-mediated endocytosis and requires an acidic endosomal step for productive infection. Virion contains a potential nuclear localization signal as well as a phospholipase A(2) like domain in N-terminus of VP1. In this study we characterized the role of PLA(2) activity on CPV entry process. PLA(2) activity of CPV capsids was triggered in vitro by heat or acidic pH. PLA(2) inhibitors inhibited the viral proliferation suggesting that PLA(2) activity is needed for productive infection. The N-terminus of VP1 was exposed during the entry, suggesting that PLA(2) activity might have a role during endocytic entry. The presence of drugs modifying endocytosis (amiloride, bafilomycin A(1), brefeldin A, and monensin) caused viral proteins to remain in endosomal/lysosomal vesicles, even though the drugs were not able to inhibit the exposure of VP1 N-terminal end. These results indicate that the exposure of N-terminus of VP1 alone is not sufficient to allow CPV to proliferate. Some other pH-dependent changes are needed for productive infection. In addition to blocking endocytic entry, amiloride was able to block some postendocytic steps. The ability of CPV to permeabilize endosomal membranes was demonstrated by feeding cells with differently sized rhodamine-conjugated dextrans together with the CPV in the presence or in the absence of amiloride, bafilomycin A(1), brefeldin A, or monensin. Dextran with a molecular weight of 3000 was released from vesicles after 8 h of infection, while dextran with a molecular weight of 10,000 was mainly retained in vesicles. The results suggest that CPV infection does not cause disruption of endosomal vesicles. However, the permeability of endosomal membranes apparently changes during CPV infection, probably due to the PLA(2) activity of the virus. These results suggest that parvoviral PLA(2) activity is essential for productive infection and presumably utilized in membrane penetration process of the virus, but CPV also needs other pH-dependent changes or factors to be released to the cytoplasm from endocytic vesicles.
犬细小病毒（CPV）是一种小型无包膜病毒，具有单链DNA基因组。CPV通过网格蛋白介导的内吞作用进入细胞，并且 productive 感染需要酸性内体步骤。病毒粒子在VP1的N端含有一个潜在的核定位信号以及一个类似于磷脂酶A(2)的结构域。在本研究中，我们表征了PLA(2)活性在CPV进入过程中的作用。CPV衣壳的PLA(2)活性在体外通过加热或酸性pH触发。PLA(2)抑制剂抑制病毒增殖，表明 productive 感染需要PLA(2)活性。VP1的N端在进入过程中暴露，表明PLA(2)活性可能在内吞进入过程中起作用。改变内吞作用的药物（amiloride、bafilomycin A(1)、brefeldin A和莫能菌素）的存在导致病毒蛋白保留在内体/溶酶体囊泡中，尽管这些药物不能抑制VP1 N端的暴露。这些结果表明，仅VP1 N端的暴露不足以使CPV增殖。 productive 感染还需要其他pH依赖性变化。除了阻断内吞进入外，amiloride还能够阻断一些内吞后步骤。通过在存在或不存在amiloride、bafilomycin A(1)、brefeldin A或莫能菌素的情况下，将不同大小的罗丹明偶联葡聚糖与CPV一起喂入细胞，证明了CPV使内体膜通透的能力。感染8小时后，分子量为3000的葡聚糖从囊泡中释放出来，而分子量为10,000的葡聚糖主要保留在囊泡中。结果表明，CPV感染不会导致内体囊泡破裂。然而，在CPV感染期间，内体膜的通透性显然发生了变化，这可能是由于病毒的PLA(2)活性。这些结果表明，细小病毒PLA(2)活性对于 productive 感染至关重要，并且可能在病毒的膜穿透过程中发挥作用，但CPV还需要其他pH依赖性变化或因子才能从内吞囊泡释放到细胞质中。
