Membrane Topology of Pestiviral Nonstructural Protein 2 and Determination of the Minimal Autoprotease Domain

Abstract

Pestiviruses like bovine viral diarrhea virus (BVDV) belong to the family Flaviviridae. A distinctive feature of the Flaviviridae is the importance of non-structural (NS) proteins for RNA genome replication and virus morphogenesis. For pestiviruses, the NS2 protease-mediated release of NS3 is essential for RNA replication, whereas uncleaved NS2-3 is indispensable for producing viral progeny. Accordingly, in the pestiviral life cycle the switch from RNA replication to virion morphogenesis is temporally regulated by the extent of NS2-3 cleavage, which is catalyzed by the NS2 autoprotease. A detailed knowledge of the structural and functional properties of pestiviral NS2 and NS2-3 is mandatory for a better understanding of these processes. In the present study, we experimentally determined the membrane topology of NS2 of BVDV-1 strain NCP7 by the Substituted Cysteine Accessibility Method (SCAM) assay. According to the resulting model, the N terminus of NS2 resides in the ER lumen and is followed by three transmembrane segments (TM) and a cytoplasmic C-terminal protease domain. We used the resulting model for fine mapping of the minimal autoprotease domain. Only one TM segment was found to be essential for maintaining residual autoprotease activity. While the topology of pestiviral NS2 is overall comparable to the one of hepatitis C virus (HCV) NS2, our data also reveal potentially important differences between the two molecules. The improved knowledge about structural and functional properties of this protein will support future functional and structural studies on pestiviral NS2. Importance Pestiviral NS2 is central to the regulation of RNA replication and virion morphogenesis via its autoprotease activity. This activity is temporally regulated by the cellular DNAJC14 as a cofactor: while free NS3 is required for RNA replication as a component of the viral replicase, only uncleaved NS2-3 supports virion morphogenesis. For a better understanding of the underlying molecular interactions, topological and structural data are required. The topology-based determination of the minimal NS2-protease domain in the present study will facilitate future attempts to determine the structure of this unusual protease cofactor complex. In the hepatitis C virus system, NS2 functions as a hub in virion morphogenesis by interacting with structural as well as non-structural proteins. Our knowledge of the membrane topology will significantly support future detailed interaction studies for pestiviral NS2.

A novel NS3/4A protease dependent cleavage site within pestiviral NS2

“…In summary, we demonstrated that internal cleavage in NS2 is mediated by the NS3/4A protease and occurs between L188 and G189, separating NS2 into two parts: (1) a membrane-bound, N-terminal fragment containing the complete NS2 transmembrane domain, and (2) a soluble, C-terminal part containing the NS2 protease active site (). For the latter part, we do not expect any NS2 cysteine protease activity according to the data of Walther et al [21], resulting in a large cleavage product consisting of the NS2 C-terminal domain (NS2 C-ter .) followed by NS3.…”

“…Immunoprecipitation of N-terminally H-F-tagged BVDV NS2 indicates internal NS2 processing. ( a ) Schemes of BVDV-1 NCP7- and CP7-derived expression constructs pTM7 NCP7 and CP7 p7-4A 2/H-F, respectively, encoding the p7 to NS4A region with N-terminally HA-Flag ( H- F)-tagged NS2 as described previously [21]. The BVDV-1 strain CP7-specific nine aa insertion in NS2 is displayed in a red box [25]; the H-F epitopes are indicated in green.…”

“…Scheme of BVDV NS2 membrane topology according to Walther et al [21] indicating the NS3/4A protease-mediated cleavage within NS2. The sequence of the nine aa insertion in the CP7 strain is shown in red.…”

“…To generate pTM7 CP7 p7-4A 2/H-F (H-F indicating a dual HA-Flag epitope located in NS2), first, a pCITE version (pCITE CP7 p7-4A 2/H-F) was generated from previously described pCITE CP7 E2-4A 2/H-F and pCITE p7-H-F-NS2 (1-453) [21]. A PmlI-CITE-p7-NsiI fragment was generated by PmlI/NsiI restriction of pCITE p7-H-F-NS2 (1-453) and cloned into pCITE CP7 E2-4A 2/H-F cleaved by PmlI and NsiI to generate pCITE CP7 p7-4A 2/H-F. pTM1-2 served as backbone for both pTM7 NCP7 and CP7 p7-4A 2/H-F and is a multifunctional vector expressing an ampicillin resistance gene and encoding a T7 promoter as well as an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) that is used for the transient expression of foreign proteins.…”

“…The C-terminal cytoplasmic domain contains the putative catalytic triad H311, E325 and C376 (numbering starting with the first NS2 amino acid) of the NS2 cysteine protease [12], a putative zinc coordination motif [12,19] and two independent DNAJC14-binding motifs [20]. The N-terminal portion is highly hydrophobic and has recently been proposed to fold into a membranebinding domain consisting of three transmembrane segments, with some analogy to NS2 of the related hepatitis C virus (HCV) [21].…”