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Journal of Virology, November 1999, p. 9196-9205, Vol. 73, No. 11
0022-538X/99/$04.00+0

Assignment of the Multifunctional NS3 Protein of Bovine Viral Diarrhea Virus during RNA Replication: an In Vivo and In Vitro Study

Claus W. Grassmann, Olaf Isken, and Sven-Erik Behrens*

Institut für Virologie (FB Veterinärmedizin), Justus-Liebig-Universität Giessen, D-35392 Giessen, Germany

Received 13 May 1999/Accepted 9 August 1999

Studies on the replication of the pestivirus bovine viral diarrhea virus (BVDV) were considerably facilitated by the recent discovery of an autonomous subgenomic BVDV RNA replicon (DI9c). DI9c comprises mainly the untranslated regions of the viral genome and the coding region of the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. To assess the significance of the NS3-associated nucleoside triphosphatase/helicase activity during RNA replication and to explore other functional features of NS3, we generated a repertoire of DI9c derivatives bearing in-frame mutations in different parts of the NS3 coding unit. Most alterations resulted in deficient replicons, several of which encoded an NS3 protein with an inhibited protease function. Three lesions permitted replication, though at a lower level than that of the wild-type RNA, i.e., replacement of the third position of the DEYH helicase motif II by either T or F and an insertion of four amino acid residues in the C-terminal part of NS3. While polyprotein proteolysis was found to be almost unaffected in these latter replicons, in vitro studies with the purified mutant NS3 proteins revealed a significantly impaired helicase activity for the motif II substitutions. NS3 with a DEFH motif, moreover, showed a significantly lower ATPase activity. In contrast, the C-terminal insertion had no negative impact on the ATPase/RNA helicase activity of NS3. All three mutations affected the synthesis of both replication products---negative-strand intermediate and progeny positive-strand RNA---in a symmetric manner. Unexpectedly, various attempts to rescue or enhance the replication capability of nonfunctional or less functional DI9c NS3 derivatives, respectively, by providing intact NS3 in trans failed. Our experimental data thus demonstrate that the diverse enzymatic activities of the NS3 protein---in particular the ATPase/RNA helicase---play a pivotal role even during early steps of the viral replication pathway. They may further indicate the C-terminal part of NS3 to be an important functional determinant of the RNA replication process.

* Corresponding author. Mailing address: Institut für Virologie (FB Veterinärmedizin), Justus-Liebig-Universität Giessen, Frankfurter Str. 107, D-35392 Giessen, Germany. Phone: 496419938373. Fax: 496419938359. E-mail: Sven-Erik.Behrens{at}vetmed.uni-giessen.de.

Journal of Virology, November 1999, p. 9196-9205, Vol. 73, No. 11
0022-538X/99/$04.00+0


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