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Journal of Virology, March 2004, p. 2414-2425, Vol. 78, No. 5
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.5.2414-2425.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Uncleaved NS2-3 Is Required for Production of Infectious Bovine Viral Diarrhea Virus

Eugene V. Agapov,1,{dagger} Catherine L. Murray,2,{dagger} Ilya Frolov,1,§ Lin Qu,1,|| Tina M. Myers,1,# and Charles M. Rice1,2*

Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110,1 Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, New York 100212

Received 29 August 2003/ Accepted 30 October 2003

Despite increasing characterization of pestivirus-encoded proteins, functions for nonstructural (NS) proteins NS2, NS2-3, NS4B, and NS5A have not yet been reported. Here we investigated the function of bovine viral diarrhea virus (BVDV) uncleaved NS2-3. To test whether NS2-3 has a discrete function, the uncleaved protein was specifically abolished in two ways: first by inserting a ubiquitin monomer between NS2 and NS3, and second by placing an internal ribosome entry site between the two proteins (a bicistronic genome). In both cases, complete processing of NS2-3 prevented infectious virion formation without affecting RNA replication. We tested the hypothesis that uncleaved NS2-3 was involved in morphogenesis by creating a bicistronic genome in which NS2-3 was restored in the second cistron. With this genome, both uncleaved NS2-3 expression and particle production returned. We then investigated the minimal regions of the polyprotein that could rescue an NS2-3 defect by developing a trans-complementation assay. We determined that the expression of NS4A in cis with NS2-3 markedly increased its activity, while p7 could be supplied in trans. Based on these data, we propose a model for NS2-3 action in virion morphogenesis.

* Corresponding author. Mailing address: Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, 1230 York Ave., New York, NY 10021. Phone: (212) 327-7046. Fax: (212) 327-7048. E-mail: ricec{at}rockefeller.edu.

{dagger} These authors contributed equally to this work.

{ddagger} Present address: Department of Medicine, Division of Pulmonary and Critical Care Medicine, Washington University School of Medicine, St. Louis, MO 63110.

§ Present address: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555-1019.

|| Present address: Idenix Pharmaceuticals, Inc., Cambridge, MA 02140.

# Present address: Eli Lilly & Co., Indianapolis, IN 46225.

Journal of Virology, March 2004, p. 2414-2425, Vol. 78, No. 5
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.5.2414-2425.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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