High-Resolution Functional Profiling of a Gammaherpesvirus RTA Locus in the Context of the Viral Genome

Abstract

Gammaherpesviruses Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus are associated with multiple human cancers. Our goal was to develop a quantitative, high-throughput functional profiling system to identify viral cis-elements and protein subdomains critical for virus replication in the context of the herpesvirus genome. In gamma-2 herpesviruses, the transactivating factor RTA is essential for initiation of lytic gene expression and viral reactivation. We used the RTA locus as a model to develop the functional profiling approach. The mutant murine gammaherpesvirus 68 viral library, containing 15-bp random insertions in the RTA locus, was passaged in murine fibroblast cells for multiple rounds of selection. The effect of each 15-bp insertion was characterized using fluorescent-PCR profiling. We identified 1,229 insertions in the 3,845-bp RTA locus, of which 393, 282, and 554 were critically impaired, attenuated, and tolerated, respectively, for viral growth. The functional profiling phenotypes were verified by examining several individual RTA mutant clones for transactivating function of the RTA promoter and transcomplementing function of the RTA-null virus. Thus, the profiling approach enabled us to identify several novel functional domains in the RTA locus in the context of the herpesvirus genome. Importantly, our study has demonstrated a novel system to conduct high-density functional genetic mapping. The genome-scale expansion of the genetic profiling approach will expedite the functional genomics research on herpesvirus. Human gammaherpesviruses Epstein-Barr virus (EBV) andKaposi's sarcoma-associated herpesvirus (KSHV) are involved in the development of epithelial, mucosal, hematopoietic, and endothelial cell cancers (7, 28). Murine gammaherpesvirus 68 (MHV-68), a pathogen affecting wild rodents, has been used as an animal model to study the fundamental principles underlying replication of gammaherpesviruses (32,35,37). For gamma-2 herpesviruses, including KSHV and MHV-68, the balance between latent and lytic phases of the viral life cycle is controlled by the immediate-early viral protein, RTA (replication and transcription activator) (11,25,26,34,41); hence, RTA plays a pivotal role in the viral life cycle. RTA is a b-zipper protein consisting of N-terminal DNA binding (DBD) and dimerization domains and a C-terminal transactivation (TA) domain (9,10,13,15,36). Understanding both the regulation of RTA transcription and the mechanism of RTA function is important. The promoter of RTA contains many regulatory cis-elements that are bound by viral and cellular factors, resulting in transcriptional activation or repression of RTA. Protein kinase C, cyclic AMP/protein kinase A, AP-1, Ras/Raf/ MEK/ERK/Ets-1, and Notch signaling pathways have been demonstrated to induce KSHV reactivation through direct or indirect activation of RTA expression (8,23,38,43,45). NF-B and LANA negatively regulate the RTA expression (4,19,20).The promoter elements of RTA have been studied extensively using transient transfe...

Construction and Characterization of an Infectious Murine Gammaherpesivrus‐68 Bacterial Artificial Chromosome

“…In order to efficiently and systematically study the functions of individual viral genes encoded by MHV-68, we decided to clone the sequenced WUMS isolate [ 1 ] as a BAC plasmid. After obtaining the MHV-68 BAC plasmid, we conducted transposon-mediated random insertional mutagenesis to systematically identify the viral ORFs essential for viral replication [ 19 ] as well as to functionally profile a genetic locus in great details [ 20 ]. Due to the extensive use of our MHV-68 BAC plasmid in the field, we describe here the construction and characterization of this MHV-68 BAC.…”

Murine Gammaherpesvirus 68 ORF48 Is an RTA-Responsive Gene Product and Functions in both Viral Lytic Replication and Latency during In Vivo Infection

“…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 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.…”

High-Resolution Functional Profiling of the Norovirus Genome

“…Transposon (Tn)-mediated insertional mutagenesis has been widely used to identify the essential functional domains of many viral proteins (23,35,37,54,69) and cis-acting elements (3,4) and more recently for comprehensive functional analysis of entire viral genomes, such as the hepatitis C virus genome (3,9,44). In comparison to targeted mutagenesis, Tn mutagenesis allows the structure and function of viral proteins to be randomly probed.…”

“…In comparison to targeted mutagenesis, Tn mutagenesis allows the structure and function of viral proteins to be randomly probed. When combined with insertion profiling techniques, this approach can be used to simultaneously determine the effect of a large number of insertions on viral replication (3,4,9,44).…”