Core protein of Flaviviridae is regarded as essential factor for nucleocapsid formation. Yet, core protein is not encoded by all isolates (GBV- A and GBV- C). Pestiviruses are a genus within the family Flaviviridae that affect cloven-hoofed animals, causing economically important diseases like classical swine fever (CSF) and bovine viral diarrhea (BVD). Recent findings describe the ability of NS3 of classical swine fever virus (CSFV) to compensate for disabling size increase of core protein (Riedel et al., 2010). NS3 is a nonstructural protein possessing protease, helicase and NTPase activity and a key player in virus replication. A role of NS3 in particle morphogenesis has also been described for other members of the Flaviviridae (Patkar et al., 2008; Ma et al., 2008). These findings raise questions about the necessity and function of core protein and the role of NS3 in particle assembly. A reverse genetic system for CSFV was employed to generate poorly growing CSFVs by modification of the core gene. After passaging, rescued viruses had acquired single amino acid substitutions (SAAS) within NS3 helicase subdomain 3. Upon introduction of these SAAS in a nonviable CSFV with deletion of almost the entire core gene (Vp447Δc), virus could be rescued. Further characterization of this virus with regard to its physical properties, morphology and behavior in cell culture did not reveal major differences between wildtype (Vp447) and Vp447Δc. Upon infection of the natural host, Vp447Δc was attenuated. Hence we conclude that core protein is not essential for particle assembly of a core-encoding member of the Flaviviridae, but important for its virulence. This raises questions about capsid structure and necessity, the role of NS3 in particle assembly and the function of core protein in general.
Virus particles of members of the Flaviviridae consist of an inner complex of viral RNA genome and core protein that together form the nucleocapsid, and an outer lipid layer containing the viral glycoproteins. Functional analyses of core protein of the classical swine fever virus (CSFV), a pestivirus related to hepatitis C virus (HCV), led to the observation that crippling mutations or even complete deletion of the core gene were compensated by single amino acid substitutions in the helicase domain of non-structural protein 3 (NS3). NS3 is well conserved among the Flaviviridae and acts as protease and helicase. In addition to its essential role in RNA replication, NS3 apparently organizes the incorporation of RNA into budding virus particles. Characterization of core deficient CSFV particles (Vp447Δc) revealed that the lack of core had no effect with regard to thermostability, size, density, and morphology. Vp447Δc was fully attenuated in the natural host. Our results provide evidence that core protein is not essential for virus assembly. Hence, Vp447Δc might help to explain the enigmatic existence of GB viruses -A and -C, close relatives of HCV that do not encode an apparent core protein.