High-resolution functional profiling of a gammaherpesvirus RTA locus in the context of the viral genome.
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.
Arumugaswami
Vaithilingaraja
V
Department of Molecular and Medical Pharmacology, University of California at Los Angeles, 90095, USA.
Sitapara
Ronika
R
Hwang
Seungmin
S
Song
Moon Jung
MJ
Ho
Tuyet Ngoc
TN
Su
Nancy Qi
NQ
Sue
Eric Y
EY
Kanagavel
Vidhya
V
Xing
Fangfang
F
Zhang
Xiaolin
X
Zhao
Minglei
M
Deng
Hongyu
H
Wu
Ting-Ting
TT
Kanagavel
Sudhakar
S
Zhang
LuLu
L
Dandekar
Sugandha
S
Papp
Jeanette
J
Sun
Ren
R
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
D015534:Trans-Activators; D017361:Viral Nonstructural Proteins