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A comprehensive library of mutations of Epstein–Barr virus

¹ Molecular Genetics Laboratory, Department of Microbiology and Immunology, Chang Gung University, Taoyuan 333, Taiwan
² Department of Applied Microbiology, National Chiayi University, Chiayi City 600, Taiwan
³ Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung 402, Taiwan
⁴ Division of Viral Infection, Department of Infectious Disease Control, International Research Center for Infections Diseases, The Institute of Medical Science, The University of Tokyo, 4–6–1 Shirokanedai, Minato-Ku, Tokyo 108–8639, Japan

Correspondence
Shih-Tung Liu
cgliu{at}mail.cgu.edu.tw

A mutant library of 249 mutants with mutations that span the entire Epstein–Barr virus (EBV) genome was generated by transposition with EZ : : TN <KAN-2> and insertion with an apramycin resistance gene by a PCR-targeting method. This study also demonstrates the feasibility of generating deletions and site-specific mutations in the BRLF1 promoter on the EBV genome to determine the regions in the promoter that are crucial to transcription. Analysing BZLF1 and BRLF1 mutants by microarray analysis revealed that these two genes regulate the transcription of EBV lytic genes differently. A BZLF1 mutation affects global expression of EBV lytic genes; almost no lytic gene is expressed by the mutant after lytic induction. However, although a BRLF1 mutant still transcribes most lytic genes, the expression of these lytic genes is inefficient. Furthermore, this study shows that the proximal Zta-response element in the BRLF1 promoter is crucial to BRLF1 transcription from the EBV genome, despite the fact that another work demonstrated that this site was unimportant in transient transfection analysis. Furthermore, mutants with a mutation in BDLF1 and BORF1 cannot assemble viral capsids. Results of this study demonstrate the usefulness of a comprehensive mutant library in genetic analyses of EBV.

Supplementary material and sequences of the primers used are available with the online version of this paper.

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