Essential role of <i>cis</i> -encoded mature NS3 in the genome packaging of classical swine fever virus

Pestiviruses are economically significant pathogens in livestock. Although genome organization and non-structural protein functions resemble those of other <i>Flaviviridae</i> genera, distinct differences can be observed. Previous studies showed that coreless CSFV strains can produce coreless virions mediated by single compensatory mutations in NS3. In this study, we could show that only RNA molecules encoding these mutations in the mature NS3 are packaged in the absence of the core protein. Unlike this selectivity, a pool of structural proteins in the host cell was readily available for packaging all CSFV genomes. Similarly, the NS2-3-4A precursor molecules required for packaging could also be provided in <i>trans</i>. Consequently, genome packaging in pestiviruses is governed by <i>cis</i>-encoded mature NS3. Reliance on <i>cis</i>-acting proteins restricts the acceptance of defective genomes and establishes packaging specificity regardless of RNA sequence-specific packaging signals. Understanding the role of NS3 in pestiviral genome packaging might uncover new targets for antiviral therapies.