Gower Tara L, Pastey Manoj K, Peeples Mark E, Collins Peter L, McCurdy Lewis H, Hart Timothy K, Guth Alex, Johnson Teresa R, Graham Barney S
Vaccine Research Center, Building 40, Room 2502, NIAID, NIH, 40 Convent Dr., MSC 3017, Bethesda, MD 20892-3017, USA.
J Virol. 2005 May;79(9):5326-36. doi: 10.1128/JVI.79.9.5326-5336.2005.
Respiratory syncytial virus (RSV) is an important human pathogen that can cause severe and life-threatening respiratory infections in infants, the elderly, and immunocompromised adults. RSV infection of HEp-2 cells induces the activation of RhoA, a small GTPase. We therefore asked whether RhoA signaling is important for RSV replication or syncytium formation. The treatment of HEp-2 cells with Clostridium botulinum C3, an enzyme that ADP-ribosylates and specifically inactivates RhoA, inhibited RSV-induced syncytium formation and cell-to-cell fusion, although similar levels of PFU were released into the medium and viral protein expression levels were equivalent. Treatment with another inhibitor of RhoA signaling, the Rho kinase inhibitor Y-27632, yielded similar results. Scanning electron microscopy of C3-treated infected cells showed reduced numbers of single blunted filaments, in contrast to the large clumps of long filaments in untreated infected cells. These data suggest that RhoA signaling is associated with filamentous virus morphology, cell-to-cell fusion, and syncytium formation but is dispensable for the efficient infection and production of infectious virus in vitro. Next, we developed a semiquantitative method to measure spherical and filamentous virus particles by using sucrose gradient velocity sedimentation. Fluorescence and transmission electron microscopy confirmed the separation of spherical and filamentous forms of infectious virus into two identifiable peaks. The C3 treatment of RSV-infected cells resulted in a shift to relatively more spherical virions than those from untreated cells. These data suggest that viral filamentous protuberances characteristic of RSV infection are associated with RhoA signaling, are important for filamentous virion morphology, and may play a role in initiating cell-to-cell fusion.
呼吸道合胞病毒（RSV）是一种重要的人类病原体，可在婴儿、老年人和免疫功能低下的成年人中引起严重的、危及生命的呼吸道感染。RSV感染HEp-2细胞可诱导小GTP酶RhoA的激活。因此，我们询问RhoA信号传导对于RSV复制或合胞体形成是否重要。用肉毒杆菌C3（一种ADP核糖基化并特异性使RhoA失活的酶）处理HEp-2细胞，可抑制RSV诱导的合胞体形成和细胞间融合，尽管释放到培养基中的PFU水平相似且病毒蛋白表达水平相当。用另一种RhoA信号传导抑制剂Rho激酶抑制剂Y-27632处理也得到了类似的结果。对经C3处理的感染细胞进行扫描电子显微镜检查显示，单个钝端细丝数量减少，这与未处理的感染细胞中大量长细丝团块形成对比。这些数据表明，RhoA信号传导与丝状病毒形态、细胞间融合和合胞体形成有关，但对于体外有效感染和产生传染性病毒并非必需。接下来，我们开发了一种半定量方法，通过蔗糖梯度速度沉降来测量球形和丝状病毒颗粒。荧光和透射电子显微镜证实，传染性病毒的球形和丝状形式可分离为两个可识别的峰。对RSV感染细胞进行C3处理导致与未处理细胞相比，球形病毒粒子相对增多。这些数据表明，RSV感染特有的病毒丝状突起与RhoA信号传导有关，对丝状病毒粒子形态很重要，并且可能在启动细胞间融合中起作用。
