Unbiased Mutagenesis of MHV68 LANA Reveals a DNA-Binding Domain Required for LANA Function In Vitro and In Vivo

PLoS Pathogens

Clinton R. Paden¹  Scott A. Tibbetts²  Samuel H. Speck³  J. Craig Forrest⁴ 

[1] Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, United States of America;Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America;Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, United States of America;Immunology and Molecular Pathogenesis Graduate Program, Emory University, Atlanta, Georgia, United States of America

关键词: Luciferase;    DNA-binding proteins;    Polymerase chain reaction;    Viral replication;    DNA transcription;    Transcriptional control;    Viral persistence;    latency;    DNA sequence analysis;   

DOI  :  10.1371/journal.ppat.1002906

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

The Latency-Associated Nuclear Antigen (LANA), encoded by ORF73, is a conserved gene among the γ2-herpesviruses (rhadinoviruses). The Kaposi's Sarcoma-Associated Herpesvirus (KSHV) LANA is consistently expressed in KSHV-associated malignancies. In the case of the rodent γ2-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 phenotypes, we generated an unbiased library of mLANA point mutants using error-prone PCR, and screened a large panel of mutants for repression of the mLANA-responsive promoter to identify loss of function mutants. Notably, among the mutant mLANA proteins recovered, many of the mutations are in a predicted EBNA-1-like DNA-binding domain. Consistent with this prediction, those tested displayed loss of DNA binding activity. We engineered six of these mLANA mutants into the MHV68 genome and tested the resulting mutant viruses for: (i) replication fitness; (ii) efficiency of latency establishment; and (iii) reactivation from latency. Interestingly, each of these mLANA-mutant viruses exhibited phenotypes similar to the mLANA-null mutant virus, indicating that DNA-binding is critical for mLANA function.

【 授权许可】

CC BY