title: 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.
authors.affiliation: Department of Molecular and Medical Pharmacology, University of California at Los Angeles, 90095, USA.
authors.forename: Vaithilingaraja
authors.initials: V
authors.lastname: Arumugaswami
authors.forename: Ronika
authors.initials: R
authors.lastname: Sitapara
authors.forename: Seungmin
authors.initials: S
authors.lastname: Hwang
authors.forename: Moon Jung
authors.initials: MJ
authors.lastname: Song
authors.forename: Tuyet Ngoc
authors.initials: TN
authors.lastname: Ho
authors.forename: Nancy Qi
authors.initials: NQ
authors.lastname: Su
authors.forename: Eric Y
authors.initials: EY
authors.lastname: Sue
authors.forename: Vidhya
authors.initials: V
authors.lastname: Kanagavel
authors.forename: Fangfang
authors.initials: F
authors.lastname: Xing
authors.forename: Xiaolin
authors.initials: X
authors.lastname: Zhang
authors.forename: Minglei
authors.initials: M
authors.lastname: Zhao
authors.forename: Hongyu
authors.initials: H
authors.lastname: Deng
authors.forename: Ting-Ting
authors.initials: TT
authors.lastname: Wu
authors.forename: Sudhakar
authors.initials: S
authors.lastname: Kanagavel
authors.forename: LuLu
authors.initials: L
authors.lastname: Zhang
authors.forename: Sugandha
authors.initials: S
authors.lastname: Dandekar
authors.forename: Jeanette
authors.initials: J
authors.lastname: Papp
authors.forename: Ren
authors.initials: R
authors.lastname: Sun
mesh_terms: D000818:Animals; D002460:Cell Line; D005347:Fibroblasts / Q000821:virology; D005816:Genetic Complementation Test; D051379:Mice; D016254:Mutagenesis, Insertional; D018144:Rhadinovirus / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D015534:Trans-Activators / Q000235:genetics / Q000502:physiology*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000502:physiology*; D014779:Virus Replication
chemical_list: D015534:Trans-Activators; D017361:Viral Nonstructural Proteins