Dynein and kinesin receptors of Herpes Simplex Virus Type 1

点击翻译按钮获取中文摘要

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.