Bovine viral diarrhea virus NS4B protein is an integral membrane protein associated with Golgi markers and rearranged host membranes

Background: Very little is known about BVDV NS4B, a protein of approximately 38 kDa. However, a missense mutation in NS4B has been implicated in changing BVDV from a cytopathic to noncytopathic virus, suggesting that NS4B might play a role in BVDV pathogenesis. Though this is one possible function, it is also likely that NS4B plays a role in BVDV genome replication. For example, BVDV NS4B interacts with NS3 and NS5A, implying that NS4B is part of a complex, which contains BVDV replicase proteins. Other possible BVDV NS4B functions can be inferred by analogy to hepatitis C virus (HCV) NS4B protein. For instance, HCV NS4B remodels host membranes to form the so-called membranous web, the site for HCV genome replication. Finally, HCV NS4B is membrane-associated, implying that HCV NS4B may anchor the virus replication complex to the membranous web structure. Unlike its HCV counterpart, we know little about the subcellular distribution of BVDV NS4B protein. Further, it is not clear whether NS4B is localized to host membrane alterations associated with BVDV infection. Results: We show first that release of infectious BVDV correlates with the kinetics of BVDV genome replication in infected cells. Secondly, we found that NS4B subcellular distribution changes over the course of BVDV infection. Further, BVDV NS4B is an integral membrane protein, which colocalizes mainly with the Golgi compartment when expressed alone or in the context of BVDV infection. Additionally, BVDV induces host membrane rearrangement and these membranes contain BVDV NS4B protein. Finally, NS4B colocalizes with replicase proteins NS5A and NS5B proteins, raising the possibility that NS4B is a component of the BVDV replication complex. Interestingly, NS4B was found to colocalize with mitochondria suggesting that this organelle might play a role in BVDV genome replication or cytopathogenicity. Conclusion: These results show that BVDV NS4B is an integral membrane protein associated with the Golgi apparatus and virus-induced membranes, the putative site for BVDV genome replication. On the basis of NS4B Colocalization with NS5A and NS5B, we conclude that NS4B protein is an integral component of the BVDV replication complex.