Replication and transcription activator (RTA) of gammaherpesvirus is an immediate early

Replication and transcription activator (RTA) of gammaherpesvirus is an immediate early gene product and regulates the expression of many downstream viral lytic genes. in ORF48 expression and through infection of laboratory mice, CP-868596 manufacturer we showed that CP-868596 manufacturer ORF48 plays important roles in different stages of viral infection and is etiologically associated with several human malignances, such as Kaposi’s sarcoma, multicentric Castleman’s disease, and primary effusion lymphoma (1). Like all other herpesviruses, KSHV exhibits two distinct life cycles: latency and lytic replication. During the latent stage, the viral genome resides in the nucleus as a circular episome and expresses only a subset of viral genes, with no production of infectious viral particles (2,C4). In contrast, during lytic replication, most, if not all viral genes, classified as immediate early, early, and late, are expressed in a highly regulated cascade manner, resulting in the release of progeny viruses for subsequent infection of naive cells and transmission to new hosts. Except in some types of multicentric Castleman’s disease, viral latency is the predominant form of infection in most KSHV-associated diseases and, hence, is considered to directly contribute to KSHV-related tumorigenesis. Even though viral lytic replication is found only in a small fraction of infected cells, it is a critical pathogenic step in the development of KSHV-related diseases. Lytic replication plays important roles in enhancing the latently infected cells in a paracrine fashion and also in replenishing the pool of latently infected cells by continuously infecting CP-868596 manufacturer naive cells (5, 6). KSHV, with a genome of approximately 165 kb, encodes more than 80 proteins (7, 8) although the function of many viral proteins remains mysterious. Until recently, studies on the function of KSHV lytic proteins had been severely hampered due to the lack of an effective lytic infection system and, more importantly, the lack of suitable small-animal models. Murine gammaherpesvirus 68 (MHV-68) is a member of the gammaherpesvirus subfamily, and it is genetically and biologically closely related to KSHV (9). MHV-68 replicates in permissive cell lines in a robust manner, and its bacterial artificial chromosome (BAC) system facilitates genetic manipulation and subsequent functional Mouse monoclonal to IgG2a Isotype Control.This can be used as a mouse IgG2a isotype control in flow cytometry and other applications assays. Moreover, as a natural pathogen to small rodents, MHV-68 can establish transient lytic CP-868596 manufacturer infection in the lung, followed by latent infection in B lymphocytes and macrophages in the spleen of laboratory mice (10, 11) after infection via the respiratory route. Consequently, MHV-68 has served as a model for studying its two human counterparts, KSHV and Epstein-Barr virus (EBV), in recent years. The replication and transcription activator (RTA), mainly encoded by ORF50, is conserved among gammaherpesviruses (12,C15). As an immediate early gene product, RTA serves as a molecular switch in the life cycle of KSHV and MHV-68 by initiating lytic replication and activating downstream gene transcription (15,C19). In order to better understand the cascade control of gammaherpesvirus lytic gene expression, it is critical to identify viral genes activated by RTA and to investigate the mechanisms by which RTA regulates the expression of these viral genes. In KSHV, a number of genes have been reported to be activated by RTA (19,C29), while in MHV-68, only three RTA-responsive genes have been identified, which are v-cyclin, ORF18, and ORF57 (30,C32). According to sequence alignment, ORF48 is conserved in all gammaherpesviruses. In KSHV, ORF48 was reported to be an immediate early gene with unknown function (33); in MHV-68, it was reported to be a tegument-associated protein (34) but was not essential for viral lytic replication or latency establishment, and its function was unclear (35, 36). However, rather than being based on site-specific mutagenesis, these results were mainly based on studies using deletion of large fragments (36) or insertions (35). Because this genomic region CP-868596 manufacturer is very compact, such large-scale genetic manipulations might affect the expression of several neighboring genes and thus alter the viral phenotype, masking the true function of ORF48. In this study, we investigated the transcriptional regulation and function of ORF48. We report here that ORF48 is a viral early gene product and can be activated by RTA. We further identified an RTA-responsive element (RRE) in the promoter (designated 48pRRE).