Pestivirus Virion Morphogenesis in the Absence of Uncleaved Nonstructural Protein 2-3

Abstract

The family Flaviviridae contains three genera of positive-strand RNA viruses, namely, Flavivirus, Hepacivirus (e.g., hepatitis C virus [HCV]), and Pestivirus. Pestiviruses, like bovine viral diarrhea virus (BVDV), bear a striking degree of similarity to HCV concerning polyprotein organization, processing, and function. Along this line, in both systems, release of nonstructural protein 3 (NS3) is essential for viral RNA replication. However, both viruses differ significantly with respect to processing efficiency at the NS2/3 cleavage site and abundance as well as functional relevance of uncleaved NS2-3. In BVDV-infected cells, significant amounts of NS2-3 accumulate at late time points postinfection and play an essential but ill-defined role in the production of infectious virions. In contrast, complete cleavage of the HCV NS2-3 counterpart has been reported, and unprocessed NS2-3 is not required throughout the life cycle of HCV, at least in cell culture. Here we describe the selection and characterization of the first pestiviral genome with the capability to complete productive infection in the absence of uncleaved NS2-3. Despite the insertion of a ubiquitin gene or an internal ribosomal entry site between the NS2 and NS3 coding sequences, the selected chimeric BVDV-1 genomes gave rise to infectious virus progeny. In this context, a mutation in the N-terminal third of NS2 was identified as a critical determinant for efficient production of infectious virions in the absence of uncleaved NS2-3. These findings challenge a previously accepted dogma for pestivirus replication and provide new implications for virion morphogenesis of pestiviruses and HCV.

Characterization of the Determinants of NS2-3-Independent Virion Morphogenesis of Pestiviruses

“…This virus contains the Osloss genes NS2, NS3, and NS4A and the first 48 bp of NS4B (NS4B*) in a NCP7-388 backbone. In addition, the mutation R1268Q in NS2 and the wt SL1 sequence in the 3=UTR were present, which have been shown to be critical in the context of this NCP7/Osloss chimera (34). In the present study, this chimera, termed N7/OsNS2-NS4B*, was used as a starting point (Fig.…”

“…It has been shown before that the NS2-4B* coding region of BVDV Osloss is sufficient to allow for virion morphogenesis in the absence of uncleaved NS2-3 in the context of a chimeric virus that carries either a ubiquitin gene or an encephalomyocarditis virus (EMCV)-IRES between the NS2 and NS3 coding regions (34). This virus contains the Osloss genes NS2, NS3, and NS4A and the first 48 bp of NS4B (NS4B*) in a NCP7-388 backbone.…”

“…Osloss sequences are shown in gray. 2/RQ, cell culture-adapted mutation in NS2 at position 1268 (34). Ubi, ubiquitin insertion derived from BVDV strain Osloss.…”

“…In cell culture, the processing of HCV NS2-NS3 is very efficient, and the insertion of an IRES between the NS2 and NS3 genes has no major impact on virion production (32,33). This remarkable difference between these closely related genera was challenged recently by a study of Lattwein and coworkers which demonstrated the possibility of NS2-3-independent virion morphogenesis for a pestivirus (34). This chimeric virus was based on ncp BVDV-1 strain NCP7 and contained the NS2-4B* region of cp BVDV-1 strain Osloss (4B* indicates the 16 N-terminal Osloss-specific amino acids).…”

“…The Osloss sequence deviates in the NS2-4B* part from the one of NCP7 in 48 amino acids, as well as in an Osloss-specific ubiquitin insertion located upstream of NS3. Efficient virion morphogenesis of the chimeric virus required an adaptive mutation in NS2 (R1268Q) and an authentic stem-loop 1 (SL1) in the 3=UTR (34). Titer-enhancing mutations were selected in E rns , E2, and p7.…”