title: Generation and genetic stability of tick-borne encephalitis virus mutants dependent on processing by the foot-and-mouth disease virus 3C protease.
abstract: Mature protein C of tick-borne encephalitis virus (TBEV) is cleaved from the polyprotein precursor by the viral NS2B/3 protease (NS2B/3(pro)). We showed previously that replacement of the NS2B/3(pro) cleavage site at the C terminus of protein C by the foot-and-mouth disease virus (FMDV) 2A StopGo sequence leads to the production of infectious virions. Here, we show that infectious virions can also be produced from a TBEV mutant bearing an inactivated 2A sequence through the expression of the FMDV 3C protease (3C(pro)) either in cis or in trans (from a TBEV replicon). Cleavage at the C terminus of protein C depended on the catalytic activity of 3C(pro) as well as on the presence of an optimized 3C(pro) cleavage site. Passage of the TBEV mutants bearing a 3C(pro) cleavage site either in the absence of 3C(pro) or in the presence of a catalytically inactive 3C(pro) led to the appearance of revertants in which protein C cleavage by NS2B/3(pro) had been regained. In three different revertants, a cleavage site for NS2B/3(pro), namely RR*C, was now present, leading to an elongated protein C. Furthermore, two revertants acquired additional mutations in the C terminus of protein C, eliminating two basic residues. Although these latter mutants showed wild-type levels of early RNA synthesis, their foci were smaller and an accumulation of protein C in the cytoplasm was observed. These findings suggest a role of the positive charge of the C terminus of protein C for budding of the nucleocapsid and further support the notion that TBEV protein C is a multifunctional protein.
authors: Institute of Virology, Medical University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria.
authors: Sabrina
authors: S
authors: Schrauf
authors: Max F. Perutz Laboratories, Medical University of Vienna, Dr. Bohr-Gasse 9/3, A-1030 Vienna, Austria.
authors: Martina
authors: M
authors: Kurz
authors: Institute of Virology, Medical University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria.
authors: Christian
authors: C
authors: Taucher
authors: Institute of Virology, Medical University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria.
authors: Christian W
authors: CW
authors: Mandl
authors: Max F. Perutz Laboratories, Medical University of Vienna, Dr. Bohr-Gasse 9/3, A-1030 Vienna, Austria.
authors: Tim
authors: T
authors: Skern
mesh_terms: D000086042:3C Viral Proteases; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004669:Encephalitis Viruses, Tick-Borne / Q000235:genetics / Q000502:physiology*; D029544:Foot-and-Mouth Disease Virus / Q000201:enzymology*; D009154:Mutation; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000086042:3C Viral Proteases; D003546:Cysteine Endopeptidases; D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases