Identiﬁcation of the In Vivo Role of a Viral bcl-2 Shivaprakash Gangappa,1 Linda F. van Dyk,1 Travis J. Jewett,2 Samuel H. Speck,3 and Herbert W.Virgin IV1, 2 1Department of Pathology and Immunology, and 2Department of Molecular Microbiology,Washington University School of Medicine, St. Louis, MO 63110 3Division of Microbiology and Immunology,Yerkes Regional Primate Research Center, Emory University, Atlanta, GA 30329 Abstract Many -herpesviruses encode candidate oncogenes including homologues of host bcl-2 and cyclin proteins (v-bcl-2, v-cyclin), but the physiologic roles of these genes during infection are not known. We show for the first time in any virus system the physiologic role of v-bcl-2. A -herpesvirus v-bcl-2 was essential for efficient ex vivo reactivation from latent infection, and for both persistent replication and virulence during chronic infection of immunocompromised (interferon [IFN]- / ) mice. The v-cyclin was also critical for the same stages in pathogenesis. Strikingly, while the v-bcl-2 and v-cyclin were important for chronic infection, these genes were not essential for viral replication in cell culture, viral replication during acute infection in vivo, establishment of latent infection, or virulence during acute infection. We conclude that v-bcl-2 and v-cyclin have important roles during latent and persistent -herpesvirus infection and that herpesviruses encode genes with specific roles during chronic infection and disease, but not acute infection and disease. As -herpesviruses primarily cause human disease during chronic infection, these chronic disease genes may be important targets for therapeutic intervention. Key words: viral genes • viral latency • reactivation • persistent replication • chronic infection Introduction Regulation of both cell cycle progression and apoptosis is critical for many aspects of virus infection, particularly for oncogenic DNA viruses such as adenoviruses, polyomavi- ruses, papillomaviruses, and -herpesviruses. Adenovi- ruses, polyomaviruses, and papillomaviruses target the functions of pRb and p53 to regulate cellular functions such as cell cycle and apoptosis with significant conse- quences including oncogenesis (1–4). Oncogenic -herpes- viruses manipulate the same aspects of cell function via expression of multiple genes including v-bcl-2 and v-cyclin. While these genes clearly regulate important aspects of cellular physiology, the physiologic role of these genes during infection is unknown. v-bcl-2 genes are encoded by oncogenic -herpesviruses including the human -herpesviruses EBV and Kaposi’s sarcoma herpesvirus (KSHV),* the primate -herpesvirus Address correspondence to H.W. Virgin, Department of Pathology and Immunology, Washington University School of Medicine, Box 8118, 660 South Euclid Ave., Phone: 314-362-9223; Fax: 314-362-4096; E-mail: virgin@immunology.wustl.edu or S.H. Speck, Division of Mi- crobiology and Immunology, Yerkes Regional Primate Research Center, Emory University, Atlanta, GA 30329. Phone: 404-727-7665; Fax: 404- 727-7768; E-mail: sspeck@rmy.emory.edu *Abbreviations used in this paper: CPE, cytopathic effect; HV68, -her- herpesvirus saimiri (HVS), and the closely related murine virus -herpesvirus 68 ( HV68) (5–10). Adenovirus and African swine fever virus also encode anti-apoptotic v-bcl-2 proteins (11–16). The in vivo role of v-bcl-2 genes has never been defined. However, the capacity of these pro- teins to inhibit apoptosis is clear since transient expression of -herpesvirus v-bcl-2 proteins is anti-apoptotic for cells in culture (6–10). An EBV v-bcl-2 mutant has been con- structed, but had no detectable phenotype in immortaliza- tion of primary human B cells (17). This lack of phenotype suggests that the v-bcl-2 gene may play a role in infection that can only be detected by in vivo experimentation. Consistent with a role for regulation of cell cycle pro- gression in -herpesvirus infection and disease, KSHV, HVS, and HV68 encode a v-cyclin, and EBV regulates the expression of host cyclin molecules (5, 18–25). To deter- mine the in vivo role for v-cyclin we characterized HV68 v-cyclin mutants. We found that the HV68 v-cyclin is onco- pesvirus 68; HVS, herpesvirus saimiri; KSHV, Kaposi’s sarcoma herpesvi- rus; MEF, mouse embryonic fibroblast; RAG, recombination activating gene; wt, wild-type. 931 J. Exp. Med.  The Rockefeller University Press • 0022-1007/2002/04/931/10 $5.00 Volume 195, Number 7, April 1, 2002 931–940 http://www.jem.org/cgi/content/full/195/7/931