Gilbert J M, Mason D, White J M
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0450.
J Virol. 1990 Oct;64(10):5106-13. doi: 10.1128/JVI.64.10.5106-5113.1990.
We investigated whether Rous sarcoma virus (RSV) infects cells through a pH-independent or a low-pH-dependent pathway. To do this, the effects of lysosomotropic agents and acid pretreatment on RSV infectivity of, and fusion with, chicken embryo fibroblasts (CEFs) were studied. High concentrations of lysosomotropic agents (ammonium chloride and monensin) did not inhibit virus infectivity: equal titers of RSV were produced in the presence and absence of these agents. Similarly, low-pH pretreatment did not inhibit RSV infectivity. In parallel experiments, lysosomotropic agents and acid pretreatment completely abolished the ability of influenza virus to infect CEFs. To monitor the fusion activity of RSV directly, the viral membrane was labeled with the fluorescent lipid probe octadecyl rhodamine at a self-quenching concentration. Upon fusion with a host cell, the probe is diluted in the cell membrane, resulting in fluorescence dequenching (D. Hoekstra, T. de Boer, K. Klappe, and J. Wilschut, Biochemistry 23:5675-5681, 1984). In this assay, fusion of RSV with CEFs was found to occur in both a time-dependent and a strictly temperature-dependent fashion. No fusion occurred unless cells with prebound virus were warmed to temperatures greater than 20 degrees C. Fusion, but not binding, was abolished if virus was pretreated with low concentrations of glutaraldehyde. High concentrations of ammonium chloride had no effect on fusion of RSV with CEFs but greatly diminished the ability of influenza virus and Semliki Forest virus to fuse with CEFs. Similarly, acid pretreatment of RSV had no effect on fusion with CEFs while markedly inhibiting fusion of both influenza and Semliki Forest viruses. Collectively, our results show that RSV fusion with and hence infection of CEFs does not require exposure of the virus to low pH. In this respect, RSV resembles another retrovirus, human immunodeficiency virus.
我们研究了劳氏肉瘤病毒（RSV）是通过不依赖pH值的途径还是低pH值依赖的途径感染细胞。为此，我们研究了溶酶体促渗剂和酸预处理对RSV感染鸡胚成纤维细胞（CEF）以及与CEF融合的影响。高浓度的溶酶体促渗剂（氯化铵和莫能菌素）并不抑制病毒感染性：在有或没有这些试剂的情况下产生的RSV滴度相同。同样，低pH预处理也不抑制RSV感染性。在平行实验中，溶酶体促渗剂和酸预处理完全消除了流感病毒感染CEF的能力。为了直接监测RSV的融合活性，用自猝灭浓度的荧光脂质探针十八烷基罗丹明标记病毒膜。与宿主细胞融合时，探针在细胞膜中被稀释，导致荧光去猝灭（D. Hoekstra、T. de Boer、K. Klappe和J. Wilschut，《生物化学》23:5675 - 5681，1984）。在该检测中，发现RSV与CEF的融合以时间依赖性和严格温度依赖性方式发生。除非预先结合病毒的细胞被加热到高于20摄氏度的温度，否则不会发生融合。如果用低浓度戊二醛预处理病毒，融合（而非结合）会被消除。高浓度氯化铵对RSV与CEF的融合没有影响，但大大降低了流感病毒和Semliki森林病毒与CEF融合的能力。同样，RSV的酸预处理对其与CEF的融合没有影响，而显著抑制了流感病毒和Semliki森林病毒的融合。总体而言，我们的结果表明，RSV与CEF的融合以及由此导致的感染并不需要病毒暴露于低pH值环境。在这方面，RSV类似于另一种逆转录病毒——人类免疫缺陷病毒。
