Characterization of an Autonomous Subgenomic Pestivirus RNA Replicon

Abstract

As an initial approach to define the requirements for the replication of bovine viral diarrhea virus (BVDV), a member of theFlaviviridae family with a positive-strand RNA genome, full-length genomic and subgenomic RNAs were originated by in vitro transcription of diverse BVDV cDNA constructs and transfected into eucaryotic host cells. RNA replication was measured either directly by an RNase protection method or by monitoring the synthesis of viral protein. When full-length BVDV cRNA was initially applied, the synthesis of negative-strand RNA intermediates as well as progeny positive-strand RNA was detected posttransfection in the cytoplasm of the host cells. Compared to the negative-strand RNA intermediate, an excess of positive-strand RNA was synthesized. Surprisingly, a subgenomic RNA molecule, DI9c, corresponding to a previously characterized defective interfering particle, was found to support both steps of RNA replication in the absence of a helper virus as well, thus functioning as an autonomous replicon. DI9c comprises the 5′ and 3′ untranslated regions of the BVDV genome and the coding regions of the autoprotease Npro and the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. Most interestingly, the NS2 polypeptide was thus determined to be nonessential for RNA replication. As expected, deletion of the genomic 3′ end as well as abolition of the catalytic function of the virus-encoded serine protease resulted in DI9c molecules that were unable to replicate. Deletion of the entire Npro gene also destroyed the ability of DI9c molecules to replicate. On the other hand, DI9c derivatives in which the 5′ third of the Npro gene was fused to a ubiquitin gene, allowing the proteolytic release of NS3 in trans, turned out to be replication competent. These results suggest that the RNA sequence located at the 5′ end of the open reading frame exerts an essential role during BVDV replication. Replication of DI9c and DI9c derivatives was found not to be limited to host cells of bovine origin, indicating that cellular factors functioning as potential parts of the viral replication machinery are well conserved between different mammalian cells. Our data provide an important step toward the ready identification and characterization of viral factors and genomic elements involved in the life cycle of pestiviruses. The implications for other Flaviviridae and, in particular, the BVDV-related human hepatitis C virus are discussed.

Derivation of a novel SARS–coronavirus replicon cell line and its application for anti-SARS drug screening

“…To this aim, partial viral RNA genomes have been constructed such that they replicate and persist in dividing cells without producing viral particles (Kaplan and Racaniello, 1988;Liljestrom and Garoff, 1991;Khromykh and Westaway, 1997;Behrens et al, 1998;Lohmann et al, 1999;Pang et al, 2001;Shi et al, 2002;Thumfart and Meyers, 2002;Hertzig et al, 2004). These viral replicons were derived from viral genomes through the deletion of all or some structural genes.…”

Architects of assembly: roles of Flaviviridae non-structural proteins in virion morphogenesis

“…Replication requires the protease and helicase/NTPase functions of NS3. HCV and pestivirus NS2 proteins are not involved in RNA accumulation after release of NS3 7,8,32 ; flavivirus NS2A and NS2B are essential 65 . Infectious virus production by HCV and pestivirus genomes requires the p7 protein 34,39,40 .…”

“…Most nonstructural proteins have essential roles in replication, and it was long thought that these replicase proteins were responsible solely for catalyzing the accumulation of genomic RNA molecules that could subsequently be packaged by the structural proteins. This canonical division of labour was initially supported by the ability of subgenomes that lack the structural region to efficiently undergo RNA synthesis 7,8,32,33 . Whereas replication is independent of the structural proteins, however, packaging of genomes into infectious particles seems to require more than just the physical components of the virion.…”

“…Homologues of this enzyme are present in hepaciviruses and pestiviruses, but not flaviviruses. After liberation of NS3, NS2 is dispensable for RNA replication 7,8,32,53,56 , but is essential for progeny particle production ( Fig 4) 39,57,58 . HCV NS2 functions early in morphogenesis, before the assembly of an infectious intracellular virus 39 .…”

Self-Replicating RNA

“…4). Such replicons were important tools for research on RNA replication of for example pestiviruses and hepaciviruses [73,74]. For pestiviruses, replicons have also been tested in vaccination approaches [75].…”