Membrane budding by rabies virus matrix- and phosphoprotein

狂犬病毒（RV）和相关的横纹肌病毒的组装和出芽发生在质膜上。包膜和膜出芽的主要驱动力是结合膜、病毒核和糖蛋白的病毒基质蛋白M，它活跃于膜弯曲和挤压膜囊泡。胞外分泌的活跃过程涉及细胞膜相关因子与各种包膜病毒的M或M样蛋白中鉴定的后期结构域结合。然而，在RV M中发现的2个经典晚结构域基序中，没有一个是必需的，并且RV M出芽不依赖于ESCRT下游的VPS4因子，这与从内部膜出芽的病毒（如埃博拉病毒或HIV）不同。这表明在RV M中存在到目前为止尚未确定的晚期结构域，或者使用替代的，到目前为止未知的质膜病毒或囊泡胞外分泌机制。在拟议的项目中，我们将通过筛选一个全面的M ala扫描突变体库来剖析RV M的多种功能，以补充由反向遗传学设计的缺陷RV生命周期的定义步骤。在分级系统中，突变体将被确定为能够调节病毒RNA合成、病毒核心和糖蛋白的结合和凝聚，但在(i)膜结合（ii）¿分选（iii）弯曲和（iv）囊泡夹断中具有离散缺陷。与wtM相比，这些突变体将揭示M结构域和细胞相互作用因子的特性以及病毒在质膜上胞外分泌的分子机制。

Assembly and budding of Rabies virus (RV) and related Rhabdoviruses takes place at the plasma membrane. The major driving force of envelopment and membrane budding is the viral matrix protein M which binds membranes, virus cores and glycoproteins, and which is active in membrane bending and pinching off membrane vesicles. The active process of ¿exocytosis¿ involves cellular membrane-associated factors binding to ¿late domains¿ iden-tified in M or M-like proteins of various enveloped viruses. Of the 2 classical late domain motifs identified in RV M, however, none is essential, and RV M budding is independent of the factor VPS4 downstream of ESCRT, in contrast to viruses budding from internal membranes, such as Ebola virus or HIV. This suggests the presence of so far unidentified late domains in RV M, or the use of alternative, so far unknown mechanisms for viral or vesicle exocytosis at the plasma membrane. In the proposed project, we will dissect the multiple functions of RV M by screening a comprehensive bank of M Ala-scan mutants for complementation of defined steps of the life cycle of defective RVs designed by reverse genetics. In a hierarchical system mutants will be identified that are competent in regulating viral RNA synthesis, binding and con-densation of viral cores and glycoproteins, but have discrete defects in (i) membrane bind-ing (ii) ¿sorting, (iii) -bending and (iv) vesicle pinching-off. In comparison with wtM, the mutants will shed light on the identity of M domains and cellular interacting factors and the molecular mechanisms of viral exocytosis at the plasma membrane.