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

Abstract

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 intrans 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.

Hepatitis C Virus RNA Replication Depends on Specific Cis- and Trans-Acting Activities of Viral Nonstructural Proteins

“…Our data are most consistent with a requirement for the HCV NS3 RNA binding and NTPase activities in cis. Similarly, for other Flaviviridae, the BVDV-1 NS3 NTPase active site residues are required in cis [13,118], whereas a West Nile virus NS3 helicase mutant was transcomplemented with very low efficiency [14].…”

Potyvirus P1 Proteinase

“…Enzymatically active proteases have been expressed using the T7 Vaccinia virus system [3,17], the baculovirus system [6], E. coli [5,22] and in vitro translation [31]. Fusion of GST to the N terminus of NS3 did not interfere with the activity of the protease [17].…”

“…NS3 serine protease expressed in and purified from E. coli has been used for inhibitor development [22]. To study the helicase activity of NS3 the active enzyme has been partially purified by affinity chromatography from eukaryotic cells [31].…”

Biological characteristics of dengue virus and potential targets for drug design

“…The NS3 helicase essential for in viral replication also makes it an attractive target for the design of antiviral compounds [123,124]. The 3D structure of dengue virus helicase/NTPase shows that there are three domains: domains I and II situated at the N-terminal (the NTPase site resides between these two domains); and the C-terminal domain III bound to NS5 [125].…”