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Journal of Virology, June 2004, p. 6610-6620, Vol. 78, No. 12
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.12.6610-6620.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Murine Gammaherpesvirus 68 Open Reading Frame 31 Is Required for Viral Replication

Qingmei Jia, Ting-Ting Wu, Hsiang-I Liao, Vasili Chernishof, and Ren Sun*

Department of Molecular and Medical Pharmacology, AIDS Institute, Jonsson Comprehensive Cancer Center, Dental Research Institute, and Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90095

Received 30 October 2003/ Accepted 22 January 2004

Murine gammaherpesvirus 68 (MHV-68) is genetically related to the human gammaherpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) and Epstein-Barr virus (EBV). It has been proposed as a model for gammaherpesvirus infection and pathogenesis. Open reading frame 31 (ORF31) is conserved among the Beta- and Gammaherpesvirinae subfamily, and there is no known mammalian homologue of this protein. The function of MHV-68 ORF31 and its viral homologues has not yet been determined. We described here a primary characterization of this protein and its requirement for lytic replication. The native MHV-68 ORF31 was detected at peak levels by 24 h postinfection, and the FLAG-tagged and green fluorescent protein fusion ORF31 were localized in the cytoplasm and nucleus in a diffuse pattern. Two independent experimental approaches were then utilized to demonstrate that ORF31 was required for lytic replication. First, small interfering RNA generated against ORF31 expression blocked protein expression and virus production in transfected cells. Then, two-independent bacterial artificial chromosome-derived ORF31-null MHV-68 mutants (31STOP) were generated and found to be defective in virus production in fibroblast cells. This defect can be rescued in trans by MHV-68 ORF31 and importantly by its KSHV homologue. A repair virus of 31STOP was also generated by homologous recombination in fibroblast cells. Finally, we showed that the defect in ORF31 blocked late lytic protein expression. Our results demonstrate that MHV-68 ORF31 is required for viral lytic replication, and its function is conserved in its KSHV homologue.

* Corresponding author. Mailing address: Department of Molecular and Medical Pharmacology, University of California at Los Angeles, Los Angeles, CA 90095. Phone: (310) 794-5557. Fax: (310) 794-5123. E-mail: rsun{at}mednet.ucla.edu.

Journal of Virology, June 2004, p. 6610-6620, Vol. 78, No. 12
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.12.6610-6620.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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