Project C03.2

Principal Investigator

Prof. Dr. Norbert Tautz & Prof. Dr. Arwen Pearson

Universität zu Lübeck &
Universität Hamburg

C3.2

PhD candidate

Malte Morische

C3.2

Project Summary

Structural and functional characterization of the pestiviral NS2pro-NS3pro polyprotein region

Pestiviruses, such as bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV), possess a single-stranded RNA genome of positive polarity, which is translated into one polyprotein and processed by cellular and viral proteases to produce structural as well as non-structural (NS) proteins [1]. These proteins are forming the packaging or replication complexes. The switch from replication to virion morphogenesis is temporally controlled by the extent of NS2-NS3 cleavage mediated by the NS2 autoprotease [2,3]. For efficient NS2-NS3 cleavage, the NS2 autoprotease needs to be activated by the cellular cofactor DNAJC-14 [4,5]. 

The generated free NS3 together with other non-structural and cellular proteins forms the replication complex, replicating the viral genome. In non-cytopathogenic Pestiviruses (such as BVDV NCP7), the available cellular supply of DNAJC-14 is decreased at late stages of infection, leading to accumulation of uncleaved NS2-3, participating in virion morphogenesis. In contrast, in cytopathogenic strains, like BVDV CP7 and NADL, alterations of the pestiviral genome deregulate NS2-NS3 cleavage, making them DNAJC-14 independent [4,6].

Pestiviral NS2 is a membrane protein with three transmembrane segments followed by a cytoplasmic domain [7]. While structural information on the NS3/4A serine protease is available [2], no crystal structures of uncleaved NS2-3 as well as NS2 exist. Due to this missing information, the mechanism leading to the NS2 autoprotease activation by DNAJC-14 is poorly understood. Therefore, we aim to determine the structures of pestiviral NS2 protease domain (NS2pro) and its uncleaved precursor fragment (NS2pro-NS3pro). In addition, structural investigations of the NS2pro-NS3pro variants of cytopathic strains are expected to provide additional information to understand ways to activate the NS2 autoprotease.

In this project we aim to: 

  1. Design and establish bacterial expression systems and protein isolation protocols for NS2pro and NS2pro-NS3pro from BVDV strains NCP7, CP7 and NADL, as well as CSFV.
  2. Design and establish bacterial expression systems and protein isolation protocols for its DNAJC-14 co-factor domain Jiv90. This co-factor domain will be used in co-crystallization efforts with NS2pro-NS3pro of BVDV NCP7 and CSFV NS2pro-NS3pro.
  3. Purified proteins will be used for crystallization trials and further structural investigation. 
  4. A long-term goal of this project is the time-resolved investigation of the NS2pro-NS3pro cleavage process. 

These efforts are expected to lead to structural information that will help to understand pestiviral NS2pro activation on a molecular level.

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. 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. Cherry S, editor. PLOS Pathog. 2017;13: e1006134. doi:10.1371/journal.ppat.1006134
  3. 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;78: 2414–2425. doi:10.1128/JVI.78.5.2414-2425.2004
  4. Isken O, Postel A, Bruhn B, Lattwein E, Becher P, Tautz N. CRISPR/Cas9-Mediated Knockout of DNAJC14 Verifies This Chaperone as a Pivotal Host Factor for RNA Replication of Pestiviruses. Pfeiffer JK, editor. J Virol. 2019;93: e01714-18. doi:10.1128/JVI.01714-18
  5. Rinck G, Birghan C, Harada T, Meyers G, Thiel H-J, Tautz N. A Cellular J-Domain Protein Modulates Polyprotein Processing and Cytopathogenicity of a Pestivirus. J Virol. 2001;75: 9470–9482. doi:10.1128/JVI.75.19.9470-9482.2001
  6. Becher P, Tautz N. RNA recombination in pestiviruses: Cellular RNA sequences in viral genomes highlight the role of host factors for viral persistence and lethal disease. RNA Biol. 2011;8: 216–224. doi:10.4161/rna.8.2.14514
  7. Walther T, Fellenberg J, Klemens O, Isken O, Tautz N. Membrane Topology of Pestiviral Nonstructural Protein 2 and Determination of the Minimal Autoprotease Domain. J Virol. 2021 May 10;95(11):e00154-21. Epub 2021 Mar 17. doi: 10.1128/JVI.00154-21
  8. Created in BioRender. M, M. (2026) https://BioRender.com/ilsztto