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.