phenotypes of v-bcl-2 mutants may be identified using different route of infection. Why are both v-bcl-2 and v-cyclin important for efficient ex vivo reactivation from latency? We speculate that la- tently infected cells are in a resting G0 state, and that the process of ex vivo reactivation requires the v-cyclin for in- duction of cell cycle progression. In this scenario, the v-bcl-2 would prevent apoptosis induced either by expression of vi- ral genes critical for ex vivo reactivation or by proapoptotic host genes that come into play during ex vivo reactivation. It is interesting that the KSHV v-cyclin can induce apopto- sis in cells when overexpressed, and that this is blocked by coexpression of the KSHV v-bcl-2 (43). Similarly, the HV68 v-cyclin induces cell cycle progression in transgenic thymocytes and is oncogenic, despite causing increased apoptosis (26). Thus, the v-bcl-2 may be needed to prevent an apoptotic response caused by v-cyclin expression. It is also possible that the v-bcl-2 prevents apoptosis in- duced by host proteins. For example, the HV68 and HVS v-bcl-2 can block both Fas and TNF- mediated apoptosis when over-expressed (6, 44), and the EBV v-bcl-2 can in- hibit granzyme-mediated apoptosis (45). This latter is par- ticularly significant since deficiency of perforin (which is key for induction of apoptosis by host granzymes) results in increased number of latently infected cells (unpublished data). It will be important to determine the precise host and viral pathways for apoptosis induction that are inhibited by v-bcl-2 expression from the viral genome, as opposed to overexpression in cultured cells. It is possible that the v-bcl-2 is critical for blocking apoptosis induced by both host and viral proteins. Identification of apoptotic pathways inhibited by v-bcl-2 may lead to therapies for -herpesvi- rus associated diseases, as enhancement of such pathways, for example by ablating v-bcl-2 function, may prevent viral reactivation or inhibit persistent replication. Different Genetic Requirements for Acute and Persistent Repli- cation In Vivo. The demonstration that both the v-bcl-2 and the v-cyclin are critical for persistent replication in an immunocompromised host, but not for acute infection, identifies persistent infection as a genetically distinct phase of herpesvirus infection in vivo. Thus, -herpesvirus infec- tion in vivo has the following experimentally distinguish- able components: acute replication, establishment of la- tency, maintenance of latency, persistent replication, and reactivation from latency. The presence of conserved genes with a specialized function during persistent replication rather than replication during acute infection suggests that persistent replication is a physiologically important compo- nent of herpesvirus infection. It is likely that persistent replication contributes to spread of the virus within the population. In addition, it has been suggested that persistent replication may contribute to tum- origenesis by -herpesviruses (46). It is therefore possible that genes of the virus that are necessary for persistent repli- cation will also contribute to tumorigenesis even if they are not independently oncogenic. This hypothesis predicts that induction of tumors by -herpesviruses could be prevented by targeting the function of genes essential for persistent replication, even when the genes have no role in replica- tion in tissue culture, replication during acute infection, or detectable transforming activity. It is important to note that there is no direct evidence to date that persistent replication occurs in normal hosts. However, intermittent reactivation does occur in normal hosts, and considerable evidence shows that T cells recognizing lytic HV68 antigens are continuously stimulated during latency, suggesting that lytic viral gene expression, and perhaps persistent replica- tion at a level undetectable by current assays, does occur in normal hosts (47–53). It is interesting that two genes that are important for effi- cient reactivation ex vivo (v-cyclin and v-bcl-2) are also im- portant for persistent replication in an immunocompro- mised host. This strongly supports the hypothesis that persistent virus is derived from reactivation events rather than continued passage of infectious virus from one lyti- cally infected cell to the next. In this model, persistent virus would derive from reactivation events with expansion of the reactivation-derived virus limited by components of the immune system. One such host factor is IFN- , which controls latency, persistence, and chronic disease due to in- fection with both the -herpesvirus HV68 (54; unpub- lished data) and the -herpesvirus murine cytomegalovirus (32, 38, 55). It is possible that effective control of chronic -herpesvirus infection would best be accomplished by si- multaneous blockade of the function of genes critical for reactivation and persistent replication and enhancement of immune functions such as IFN- that are critical for con- trolling reactivation from latency and persistence. We also thank members of the laboratories of H.W. Virgin, S.H. Speck, David Leib, and Lynda Morrison for constructive comments on this research. Helpful comments on the manuscript were made by Beth Levine, Scott A. Tibbetts, and Joy T. Loh. H.W. Virgin was supported by grant RPG-97-134-01-MBC from the American Cancer Society and National Institutes of Health grants AI39616, CA74730, and HL60090. S.H. Speck was supported by National Institutes of Health grants CA43143, CA52004, CA58524, CA74730. L.F. van Dyk was supported by grant 5T32 AI 07163 from the National Institutes of Health. Submitted: 31 October 2001 Revised: 2 January 2002 Accepted: 4 February 2002 References 1. Pipas, J.M., and A.J. Levine. 2001. Role of T antigen interac- tions with p53 in tumorigenesis. Semin. Cancer Biol. 11:23– 30. 2. Jung, J.U., J.K. Choi, A. Ensser, and B. Biesinger. 1999. Her- pesvirus saimiri as a model for gammaherpesvirus oncogene- sis. Semin. Cancer Biol. 9:231–239. 3. Wolf, J.K., and P.T. Ramirez. 2001. The molecular biology of cervical cancer. Cancer Invest. 19:621–629. 4. Kao, W.H., S.L. Beaudenon, A.L. Talis, J.M. Huibregtse, and P.M. Howley. 2000. Human papillomavirus type 16 E6 induces self-ubiquitination of the E6AP ubiquitin-protein li- gase. J. Virol. 74:6408–6417. 5. Virgin, H.W., P. Latreille, P. Wamsley, K. Hallsworth, K.E. 938 v-bcl-2 and v-cyclin Are Chronic Disease Herpesvirus Genes