Rodríguez E, Everitt E
Department of Microbiology, University of Lund, Sweden.
J Virol. 1996 Jun;70(6):3470-7. doi: 10.1128/JVI.70.6.3470-3477.1996.
We have used four established lysosomotropic agents, ammonium chloride, amantadine, chloroquine, and methylamine, to monitor the possible interference with an early low-pH-dependent step during adenovirus replication. Two concentrations of each of the different agents were selected; one was essentially nontoxic to uninfected HeLa cells, and the other resulted in some toxicity as measured by trypan blue staining and by interference with cell monolayer establishment, cell proliferation, and radioisotope labelling. It was separately determined that these concentrations displayed pH-raising effects of the same magnitude as higher concentrations previously used in similar studies. Adenovirus uncoating in vivo, normally reaching its maximum within 1 h after infection, was not affected by any of the agents. The subsequent levels of successful nuclear entry events by the parental genomes were monitored by measuring the extent of transcription of an mRNA species coding for the early 72-kDa DNA-binding protein at 10 to 12 h postinfection. In HeLa, KB, HEp-2, and A549 cells, none of the agents were able to affect the levels of early transcription after administration at the point of infection or at 3 h after infection. The cumulative synthesis of the hexon antigen was assessed late in infection, and inhibitory effects were revealed upon administration of 10, 20, and 40 mM ammonium chloride, 10 mM methylamine, and 0.5 mM amantadine, irrespective of the time point of addition. Ammonium chloride at 5 mM reduced the hexon yield by 20% at the most when added within 50 min after infection. Chloroquine at concentrations of 2.5 and 5 microM specifically reduced the hexon yields by 30 to 40% when administered within the first 50 min of infection. On the basis of the lack of effects of nontoxic concentrations of the four agents on the early virus-cell interactive event of uncoating and the early virus-specified transcription, we conclude that a low-pH-dependent step early in the adenovirus replication cycle is not mandatory for a successful infection.
我们使用了四种已确定的溶酶体促渗剂，即氯化铵、金刚烷胺、氯喹和甲胺，来监测腺病毒复制过程中对早期低pH依赖性步骤可能产生的干扰。每种不同的试剂选择了两种浓度；一种对未感染的HeLa细胞基本无毒，另一种通过台盼蓝染色以及对细胞单层形成、细胞增殖和放射性同位素标记的干扰来衡量，会产生一定毒性。分别确定这些浓度所显示的pH升高效应与之前类似研究中使用的较高浓度相同。腺病毒在体内脱壳，通常在感染后1小时内达到最大值，不受任何一种试剂的影响。通过测量感染后10至12小时编码早期72 kDa DNA结合蛋白的mRNA种类的转录程度，监测亲本基因组随后成功进入细胞核事件的水平。在HeLa、KB、HEp-2和A549细胞中，在感染时或感染后3小时给药，没有一种试剂能够影响早期转录水平。在感染后期评估六邻体抗原的累积合成，发现给予10、20和40 mM氯化铵、10 mM甲胺和0.5 mM金刚烷胺时会出现抑制作用，与添加时间点无关。感染后50分钟内添加5 mM氯化铵时，六邻体产量最多降低20%。在感染的前50分钟内给予2.5和5 microM浓度的氯喹，可使六邻体产量特异性降低30%至40%。基于这四种试剂的无毒浓度对早期病毒-细胞相互作用的脱壳事件和早期病毒特异性转录没有影响，我们得出结论，腺病毒复制周期早期的低pH依赖性步骤对于成功感染不是必需的。
