Characteristics of Helicase-Primase Inhibitor Amenamevir-Resistant Herpes Simplex Virus
Characteristics of Helicase-Primase Inhibitor Amenamevir-Resistant Herpes Simplex Virus
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DOI：
10.1128/aac.00494-21
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发表时间：
2021-07
影响因子：
4.9
通讯作者：
Y. Sato;T. Suenaga;Makoto Kobayashi;Nozomu Miyazaki;Takato Suzuki;Ken Ishioka;T. Suzutani
中科院分区：
文献类型：
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作者：
Y. Sato;T. Suenaga;Makoto Kobayashi;Nozomu Miyazaki;Takato Suzuki;Ken Ishioka;T. Suzutani
抗疱疹药物阿米诺韦(Amenamevir，AMNV)直接抑制单纯疱疹病毒1型(HSV-1)、单纯疱疹病毒2型(HSV-2)和水痘-带状疱疹病毒的解旋酶-启动酶复合体，并抑制这些病毒的复制。虽然已有几种突变的HSV病毒对解旋酶-引物酶抑制剂产生抗药性的报道，但导致抗药性的突变尚不清楚，因为含有单一突变的重组病毒尚未被分析。摘要抗疱疹药物阿米诺韦(AMNV)直接抑制单纯疱疹病毒1型(HSV-1)、单纯疱疹病毒2型(HSV-2)和水痘-带状疱疹病毒的解旋酶-引物酶复合体，并抑制这些病毒的复制。虽然已有几种突变的HSV病毒对解旋酶-引物酶抑制剂产生抗药性的报道，但导致抗药性的突变尚不清楚，因为含有单一突变的重组病毒尚未被分析。在AMNV处理下，我们通过几代氨基酸取代获得了AMNV抗性病毒。分离到20株单纯疱疹病毒1型和19例单纯疱疹病毒2型突变株，其中UL5解旋酶和/或UL52Primase突变(S)，而辅因子UL8突变不明显。UL5突变位于基序IV的下游，其中UL5 K356N在HSV-1和K355N在HSV-2中的突变频率最高，分别为9/20和9/19。我们利用细菌人工染色体(BAC)诱变技术，用单一氨基酸取代获得了抗AMNV的重组HSV-1。结果发现UL5解旋酶的G352C和UL52Primase的F360C/V和N902T是新的突变。在UL5中含有K356N的病毒对AMNV的抗性是其他突变体的10倍，在体外和体内表现出与亲本HSV-1相同的病毒生长和毒力，而其他突变体的毒力则有所减弱。所有重组病毒对其他抗疱疹药物阿昔洛韦和磷甲酸钠敏感。总之，在BAC突变的基础上，本研究首次发现了导致AMNV抗性的UL5和UL52突变，并发现HSV-1中K356N突变频率最高的突变保持了病毒的生长和相当于亲本病毒的毒力。
The antiherpetic drug amenamevir (AMNV) inhibits the helicase-primase complex of herpes simplex virus 1 (HSV-1), HSV-2, and varicella-zoster virus directly as well as inhibiting the replication of these viruses. Although several mutated HSV viruses resistant to helicase-primase inhibitors have been reported, the mutations contributing to the resistance remain unclear, as recombinant viruses containing a single mutation have not been analyzed. ABSTRACT The antiherpetic drug amenamevir (AMNV) inhibits the helicase-primase complex of herpes simplex virus 1 (HSV-1), HSV-2, and varicella-zoster virus directly as well as inhibiting the replication of these viruses. Although several mutated HSV viruses resistant to helicase-primase inhibitors have been reported, the mutations contributing to the resistance remain unclear, as recombinant viruses containing a single mutation have not been analyzed. We obtained AMNV-resistant viruses with amino acid substitutions by several passages under AMNV treatment. Twenty HSV-1 and 19 HSV-2 mutants with mutation(s) in UL5 helicase and/or UL52 primase, but not in cofactor UL8, were isolated. The mutations in UL5 were located downstream of motif IV, with UL5 K356N in HSV-1 and K355N in HSV-2, in particular, identified as having the highest frequency, which was 9/20 and 9/19, respectively. We generated recombinant AMNV-resistant HSV-1 with a single amino acid substitution using bacterial artificial chromosome (BAC) mutagenesis. As a result, G352C in UL5 helicase and F360C/V and N902T in UL52 primase were identified as novel mutations. The virus with K356N in UL5 showed 10-fold higher AMNV resistance than did other mutants and showed equivalent viral growth in vitro and virulence in vivo as the parent HSV-1, although other mutants showed attenuated virulence. All recombinant viruses were susceptible to the other antiherpetic drugs, acyclovir and foscarnet. In conclusion, based on BAC mutagenesis, this study identified, for the first time, mutations in UL5 and UL52 that contributed to AMNV resistance and found that a mutant with the most frequent K356N mutation in HSV-1 maintained viral growth and virulence equivalent to the parent virus.