Project C06.2 (associated)

Principal Investigator

Dr. Olaf Isken & Prof. Dr. Norbert Tautz

Universität zu Lübeck

C6.2

PhD candidate

Yasmin Roeder

C6.23

Project Summary

Dissection of the pestiviral replicase and packaging complexes by proximity labelling and biorthogonal crosslinking approaches

The bovine viral diarrhea virus (BVDV) is a critical pathogen of livestock and belongs to the genus Pestivirus. Like in all members of the Flaviviridae family, BVDV non-structural (NS) proteins serve multiple roles in genome replication and virion morphogenesis [1]. In this context, the NS2-3 protein is of particular importance. In classical pestiviruses, uncleaved NS2-3 in complex with NS4A (NS2-3/4A) is essential for virion morphogenesis (VM) [2]. Importantly, it cannot functionally replace cleaved NS3/4A in the minimal viral replicase comprised of NS3-NS5B. This strict dependence on two different NS3/4A-containing protein complexes for RNA replication and VM is in contrast to the closely related Hepatitis C Virus, however pestiviruses can also be adapted to NS2-3-independent virion morphogenesis. It was shown, that the strength of surface interactions between the NS3 protease domain and the C-terminal domain of NS4A represent a molecular switch between RNA replication and VM [3]. Our hypothesis is, that the NS3/4A complex most likely exists in several different conformations to accommodate cis- as well as trans-cleavages of the polyprotein. Furthermore, the NS2-3/4A complex most likely differs from the NS3/4A complex in its conformation and/or its composition of accessory factors to support packaging instead of replication. To date, structural information on these multiprotein complexes is limited. Additional insight is necessary to understand how these complexes support the different steps in the pestiviral life cycle.

The aims of this projects are:

  1. Characterization of these interactions by bioorthogonal labeling: TAG stop-codon mutagenesis at critical surface residues of NS3 or NS4A in the context of different viral complexes will be applied. Via amber stop-codon suppression, a reactive diazirine-containing orthogonal amino acid is introduced at these positions [4-7]. UV-crosslinking followed by Western blotting enables the detection of site-specific protein interactions, thereby allowing the identification of critical NS3/NS4A residues as well as the identification of important host factors. For the identification of host factors, we will combine the information from bioorthogonal labelling with proximity labelling efforts using viral protein fusions with biotin ligases.
  2. The identified NS3/NS4A residues will be investigated by a reverse genetics approach to obtain a better understanding of the NS3/4A interaction on molecular level. 
  3. The identified potential host factors will be characterized for their participation in either replication or virion morphogenesis. To accomplish this, we will use different methods: Co-Immunoprecipitation to identify proteins directly/strongly interacting with viral protein complexes; Knock-out/Knock-down and overexpression cell lines for functional assays as well as co-localization studies in infected living cells. 

These experiments are expected to provide a better understanding of the pestiviral life cycle.

References

  1. Tautz N, Tews BA, Meyers G. The Molecular Biology of Pestiviruses. Adv Virus Res. 2015;93:47-160. doi: 10.1016/bs.aivir.2015.03.002.
  2. Agapov EV, Murray CL, Frolov I, Qu L, Myers TM, Rice CM. Uncleaved NS2-3 is required for production of infectious bovine viral diarrhea virus. J Virol. 2004 Mar;78(5):2414-25. doi: 10.1128/jvi.78.5.2414-2425.2004.
  3. Dubrau D, Tortorici MA, Rey FA, Tautz N. A positive-strand RNA virus uses alternative protein-protein interactions within a viral protease/cofactor complex to switch between RNA replication and virion morphogenesis. PLoS Pathog. 2017 Feb 2;13(2):e1006134. doi: 10.1371/journal.ppat.1006134.
  4. Liu CC, Schultz PG. Adding new chemistries to the genetic code. Annu Rev Biochem. 2010;79:413-44. doi: 10.1146/annurev.biochem.052308.105824. 
  5. Chin JW. Expanding and reprogramming the genetic code of cells and animals. Annu Rev Biochem. 2014;83:379-408. doi: 10.1146/annurev-biochem-060713-035737.
  6. Chin JW. Expanding and reprogramming the genetic code. Nature. 2017 Oct 4;550(7674):53-60. doi: 10.1038/nature24031.
  7. E. Oluwabusola. Development of Diazirine-based crosslinking agents for covalently linking protein. 2015. doi: 10.13140/RG.2.1.4493.0647.