Isolation and characterization of noncytopathic pestivirus mutants revealsa role for nonstructural protein NS4B in viral cytopathogenicity

Isolates of bovine viral diarrhea virus (BVDV), the prototype pestivirus, a re divided into cytopathic (cp) and noncytopathic (ncp) biotypes according to their effect on cultured cells. The cp viruses also differ from ncp viru ses by the production of viral nonstructural protein NS3. However, the mech anism by which cp viruses induce cytopathic effect in cell culture remains unknown. Here we used a genetic approach to isolate ncp variants that arose from a cp virus at low frequency. A bicistronic BVDV (cp strain NADL) was created that expressed puromycin acetyltransferase as a dominant selectable marker. This bicistronic virus exhibited slightly slower growth kinetics a nd smaller plaques than NADL but remained cp. A number of independent ncp v ariants were isolated by puromycin selection. Remarkably, these ncp variant s produced NS3 and viral RNA at levels comparable to those of the cp parent . Sequence analyses uncovered no change in NS3, but for all ncp variants a Y2441C substitution at residue 15 of NS4B was found. Introduction of the Y2 441C substitution into the NADL or bicistronic cp viruses reconstituted the ncp phenotype. Y2441 is highly conserved among pestiviruses and is located in a region of NS4B predicted to be on the cytosolic side of the endoplasm ic reticulum membrane. Other engineered substitutions for Y2441 also affect ed viral cytopathogenicity and viability, with Y2441V being cp, Y2441A bein g ncp, and Y2441D rendering the virus unable to replicate. The ncp substitu tions for Y2441 resulted in slightly increased levels of NS2-3 relative to NS3. We also showed that NS3, NS4B, and NS5A could be chemically cross-link ed in NADL-infected cells, indicating that they are associated as component s of a multiprotein complex. Although the mechanism remains to be elucidate d, these results demonstrate that mutations in NS413 can attenuate BVDV cyt opathogenicity despite NS3 production.