A genome-wide analysis of the spatio-temporal distribution of herpesviral proteins in the course of infection
Final Report Abstract
Essential parts of the herpesviral life cycle including DNA replication, gene transcription, capsid morphogenesis, and capsid egress occur in the infected host nucleus. This requires the nucleo-cytoplasmic trafficking of numerous herpesviral proteins. The regulated and coordinated distribution of viral proteins between nucleus and cytoplasm is thus vital for the viral propagation. Transport of proteins between cytoplasm and nucleus is mediated by transport factors of the importin α- and β-families and occurs along a gradient of the small GTPase Ran. We aimed to systematically unravel the nucleo-cytoplasmic transport of herpesviral proteins. To this end, high-throughput protein interaction assays including the yeast-two-hybrid (Y2H) and LUMIER assay, followed by cell-biological assays were applied. A novel high-throughput nuclear export assay called NEX-TRAP was established for the systematic analysis of nuclear export activity. Our comprehensive Y2H analysis identified 24 protein interactions between human transport factors and Herpes simplex virus-1 (HSV1) proteins. These interactions were validated by Lumier assay confirming the interaction of seven viral proteins with transport factors of the importin α/β family. Using the newly established NEX-TRAP assay, nuclear export activity was identified in nine HSV1 proteins. Further analysis revealed actively exported tegument proteins of the gammaherpesvirus Epstein Barr Virus (EBV) that in part are conserved throughout the herpesvirus family. Mutagenesis identified a leucine-rich NES required for active nuclear export in five out of the nine HSV1 proteins. Three additional viral proteins are exported in a CRM1/Xpo1-dependent manner and thus are likely to also harbor a leucinerich NES. The NLS and NES motifs were identified, verified by mutational analysis and compared to known consensus sequences. Bioinformatical analysis was initiated to create a dynamic model of the spatio-temporal distribution of these viral proteins during herpesviral infection. Of the viral proteins with identified import and export activity, a few were selected for functional characterization of their NLS or NES in the viral context. A bipartite basic NLS within the N-terminal domain of HSV1 pUL31 is required for nuclear import of pUL31 upon isolated expression that however is redundant in the viral context. Despite that, these basic patches are essential for capsid translocation from the nucleoplasm to sites of nuclear egress and for a coordinated interaction with pUL34. Thus we have identified an NLS that performs two different roles during infection, a classical but redundant one in nuclear import of pUL31 and an unconventional one in primary envelopment of capsids. Functional analysis of the NES within the tegument protein pUL48/VP16 revealed that NES mutagenesis results in a viral mutant deficient in replication indicating that its nuclear export is an important feature of herpesviral propagation In summary, our data show that nuclear import and export are important features of the herpesviral life cycle potentially coordinating nuclear and cytoplasmic processes. Together our data considerably increase our understanding of herpesviral biology and reveal potential novel targets for viral treatment. Finally, novel assays established in the course of this funding provide invaluable tools for the automated and thus high-throughput and systematic analysis of nuclear import and export activity of proteins.
Publications
- (2009) Connecting viral with cellular interactomes. Curr Opin Microbiol. 12(4):453-9
Bailer, SM and Haas, J.
(See online at https://doi.org/10.1016/j.mib.2009.06.004) - (2011) Functional characterization of the essential tail anchor of the herpes simplex virus type 1 nuclear egress protein pUL34. J Gen Virol. 92(Pt 12):2734-45
Ott M, Tascher G, Hassdenteufel S, Zimmermann R, Haas J, Bailer SM
(See online at https://doi.org/10.1099/vir.0.032730-0) - (2012) NEX‐TRAP, a Novel Method for In Vivo Analysis of Nuclear Export of Proteins. Traffic 13(10):1326-34
Raschbichler V, Lieber D, Bailer SM
(See online at https://doi.org/10.1111/j.1600-0854.2012.01389.x) - (2013) A high-throughput yeast two hybrid protocol to determine virus-host protein interactions. Methods Mol. Biol. 1064:1-15
Striebinger, H, Kögl, M, Bailer, SM
(See online at https://doi.org/10.1007/978-1-62703-601-6_1) - (2013) Comprehensive analysis of varicella-zoster virus proteins using a new monoclonal antibody collection. J Virol. 87(12):6943-54
Bailer, S. M. , Lenac, T. , Pothineni, V. R., Ouwendijk, W. J. D., Simic, H., Babic, M., Rose, D., Endesfelder, M., Bajdaric, D., Baiker, A., Verjans, G. M. G. M., Jonjic, S., Haas, J.
(See online at https://doi.org/10.1128/JVI.00407-13) - (2013) Determination of HSV-1 infectivity using a luciferase reporter cell line. Methods Mol. Biol. 1064:171-81
Lieber, D, Bailer, SM
(See online at https://doi.org/10.1007/978-1-62703-601-6_12) - (2014) HCMV pUL135 remodels the actin cytoskeleton to impair immune recognition of infected cells. Cell Host Microbe 16(2):201-1
Stanton, RJ, Prod'homme, V, Purbhoo, MA, Moore, M, Aicheler, RJ, Heinzmann, M, Bailer, SM, Haas, J, Antrobus, R, Weekes, MP, Lehner, PJ, Vojtesek, B, Miners, KL, Man, S, Wilkie, GS, Davison, AJ, Wang, EC, Tomasec, P, Wilkinson, GW
(See online at https://doi.org/10.1016/j.chom.2014.07.005) - (2015) The Herpes simplex virus protein pUL31 escorts nucleocapsids to sites of nuclear egress, a process coordinated by its N-terminal domain. PLoS Pathog. 11(6):e1004957
Funk C , Ott M , Raschbichler V , Nagel C-H, Binz A, Sodeik B, Bauerfeind R, Bailer SM
(See online at https://doi.org/10.1371/journal.ppat.1004957) - (2016) Asna1/TRC40 that mediates membrane insertion of tail-anchored proteins is required for efficient release of Herpes simplex virus 1 virions. Virol J. 13(1):175
Ott M, Marques D, Funk C, Bailer SM
(See online at https://doi.org/10.1186/s12985-016-0638-8) - (2016) Asna1/TRC40 that mediates membrane insertion of tail-anchored proteins is required for efficient release of Herpes simplex virus 1 virions. Virol J. 13(1):175
Ott, M., Marques, D., Funk, C., Bailer, SM
(See online at https://doi.org/10.1186/s12985-016-0638-8)
