Detection and identification of U69 gene mutations encoded by ganciclovir-resistant human herpesvirus 6 using denaturing high-performance liquid chromatography

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• 作者：Kazushi Nakano ; Kazuko Nishinaka ; Tatsuya Tanaka ; Atsushi Ohshima ; Nakaba Sugimoto ; Yuji Isegawa
• 关键词：U69 ; Protein kinase ; HHV-6 ; GCV resistant ; dHPLC
• 刊名：Journal of Virological Methods
• 出版年：2009
• 出版时间：November 2009
• 年：2009
• 卷：161
• 期：2
• 页码：223-230
• 全文大小：1220 K

文摘

A denaturing high-performance liquid chromatography (dHPLC) assay was developed to detect antiviral drug-resistance mutations of human herpesvirus 6 (HHV-6). Recombinant baculoviruses were created that contained wild-type and mutant forms of the HHV-6 U69 gene, which determines sensitivity to the antiviral drug ganciclovir (GCV). The mutations causing GCV resistance in HHV-6 U69 were single-base mutations adapted from known GCV-resistant DNA sequences of HCMV, and their ability to confer GCV resistance on recombinant baculoviruses was confirmed. Six characterized mutant sequences, including one reported previously that encodes a GCV-sensitive kinase-activity mutant, were used. DNA was extracted, and the levels of homoduplex and heteroduplex DNA in the PCR products from mixed wild-type and mutant viral DNAs were determined using dHPLC. The optimized assay could distinguish the different mutants, and could detect mutants representing only 10 % of the DNAs. The new assay with dHPLC readout permitted the rapid (4 h), objective, and reproducible detection of HHV-6 drug-resistance mutations.