unbiased mutagenesis of mhv68 lana reveals a dna-binding domain required for lana function in vitro and in vivo无偏的诱变mhv68拉娜揭示了dna结合域所需拉娜函数体外和体内.pdfVIP

Unbiased Mutagenesis of MHV68 LANA Reveals a DNA- Binding Domain Required for LANA Function In Vitro and In Vivo 1,2 3 4 1 Clinton R. Paden , J. Craig Forrest , Scott A. Tibbetts , Samuel H. Speck * 1 Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, United States of America, 2 Immunology and Molecular Pathogenesis Graduate Program, Emory University, Atlanta, Georgia, United States of America, 3 Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America, 4 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, United States of America Abstract The Latency-Associated Nuclear Antigen (LANA), encoded by ORF73, is a conserved gene among the c2-herpesviruses (rhadinoviruses). The Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) LANA is consistently expressed in KSHV-associated malignancies. In the case of the rodent c2-herpesvirus, murine gammaherpesvirus 68 (MHV68), the LANA homolog (mLANA) is required for efficient virus replication, reactivation from latency and immortalization of murine fetal liver-derived B cells. To gain insights into mLANA function(s), knowing that KSHV LANA binds DNA and can modulate transcription of a variety of promoters, we sought out and identified a mLANA-responsive promoter which maps to the terminal repeat (TR) of MHV68. Notably, mLANA strongly repressed activity from this promoter. We extended these analyses to demonstrate direct, sequence-specific binding of recombinant mLANA to TR DNA by DNase I footprinting. To assess whether the DNA-binding and/or transcription modulating function is important in the known mLANA phenot