Function and transport of a herpesvirus encoded Ubiquitin-specific protease in virus entry and assembly

摘要

Herpes simplex virus type I (HSV-1), the prototype α-herpesvirus (HV), is a double stranded DNA virus that replicates in the nucleus of infected cells. The nuclear pore represents a gateway that must be engaged and navigated immediately after cell entry for successful infection by many classes of human viruses. For herpesviruses, capsid-tegument assemblies must be targeted to the pore where the viral genome exits and transport into the nucleus occurs. We currently have little mechanistic knowledge of this fundamental step of infection.udA swath of evidence indicates that the conserved tegument protein VP1-2 is essential for early capsid transport and pore binding, and that it contains a conserved nuclear localisation signal (NLS) required for pore docking. In this thesis I undertook a detailed analysis to dissect functional determinants within the NLS from herpes simplex virus, to examine putative NLSs in VP1-2 homologues from representatives of all sub-families, to characterise protein interactions with VP1-2 and finally to construct a GFP expressing entry defective recombinant virus to study the consequences of infection.udI show that the HSV NLS can function as a mono-or bipartite motif and has a particular organisation conserved in the a-herpesvirus homologues but distinct from those in the eta- and gamma-herpesviruses. The representatives of all 3 classes contain a functional NLS at approximately the same position. All bi-partite motifs were able to rescue the HSV VP1-2ΔNLS virus defect albeit to varying extent whereas the mono-partite HHV-8 motif did not. I constructed and purified chimeric recombinant viruses for the VZV, HCMV and EBV motifs and show distinct differences in their ability to replicate in non-complementing cells. In HSV, NLS function in the context of protein nuclear import or of viable virus replication, was dependent on lysine 428 and the integrity of the full bi-partite motif. Mutations which reduced NLS activity generally caused reduced fitness of recombinant viruses.udFor the analysis of interaction partners of VP1-2 and the NLS, I developed a one-step approach to analyse the capsid interactome during entry. Additionally, I constructed mammalian GST-VP1-2.NLS fusion proteins and cell lines which inducibly express the N-terminal region of VP1-2 for analysis of NLS interacting proteins. Using mass spectrometry (MS) I identified a number of VP1-2-interacting, cellular proteins including DTX3L, an important regulator of the DNA damage response.