Dynein and kinesin receptors of Herpes Simplex Virus Type 1

单纯疱疹病毒1型（HSV1）是一种包膜双链DNA病毒，通过与质膜融合进入细胞。进入的病毒衣壳通过细胞质动力蛋白（一种负端定向运动atp酶）沿着微管（MT）运输到宿主细胞核。衣壳的最终目标是核孔，病毒基因组在病毒转录和复制之前被注射到核质中。在病毒组装和输出过程中，外向的细胞质衣壳沿着MTs顺行运输，但这次可能使用视频显微镜，我们开发了一种体外运动性试验，包括纯化的衣壳，其中包含与荧光蛋白融合的衣壳蛋白（GFP/VP26或CFP/VP26）， cy3标记的MTs，细胞质和ATP再生系统。利用这个系统以及我们的体内实验，我们将确定MT运输所需的病毒衣壳的分子决定因素。利用编码HSV1全基因组的重组bac载体（细菌人工染色体），我们将产生具有系统特异性的HSV1突变体。这些突变体和现有的结构病毒蛋白缺失突变体将被测试它们沿着mt移动的能力以及它们的方向性。与沿着MTs运输的其他货物相比，这个蛋白质的组成是已知的（大约有20个候选）。因此，它作为一个极好的模型，以最小的功能蛋白质组学方法鉴定假定的运动受体。此外，根据病毒生命周期的阶段和细胞环境，病毒衣壳可以运输到MT的负端或正端。我们将使用该系统来分析不同MT马达的结合和激活要求以及运输方向的控制。

Herpes Simplex Virus Type 1 (HSV1) is an enveloped double-stranded DNA virus which enters cells by fusion with the plasma membrane. The incoming viral capsids are transported along microtubules (MT) to the host nucleus using cytosolic dynein, a minus-end directed motor ATPase. The capsids are ultimately targeted to the nuclear pores where the viral genome is injected into the nucleoplasm prior to viral transcription and replication. During virus assembly and egress, outgoing cytosolic capsids are anterogradely transported along MTs but this time possibly Using videomicroscopy, we have developed an in vitro motility assay consisting of purified capsids containing a capsid protein fused to fluorescent proteins (GFP/VP26 or CFP/VP26), Cy3-labeled MTs, cytosol and an ATP regenerating system. Exploiting this system as well as our in vivo assays, we will determine the molecular determinants of the viral capsids required for MT transport. Using a recombinant BAC-vector (bacterial artificial chromosome) encoding the entire HSV1 genome, we will generate stystematicaly specific mutants of HSV1. These mutants and existing deletion mutants of structural viral proteins will be tested for their ability to move along MTs as well as for the directionality of. In contrast to other cargo transported along MTs, the protein composition of this one is known (about 20 candidates). It therefore serves as an excellent model to identify in a minimal functional proteomics approach putative motor receptors. Moreover, dependent of the stage of the viral life cycle and the cellular context, the viral capsid can be transported to the minus-end or to the plus-end of MTs. We will use this system to analyse the requirements for binding and activation of different MT motors as well as the control of transport direction.