Resistance of herpes simplex virus type 1 to peptidomimetic ribonucleotide reductase inhibitors: Selection and characterization of mutant isolates
Resistance of herpes simplex virus type 1 to peptidomimetic ribonucleotide reductase inhibitors: Selection and characterization of mutant isolates
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DOI：
10.1128/jvi.70.2.787-793.1996
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发表时间：
1996-02-01
影响因子：
5.4
通讯作者：
Cordingley, MG
中科院分区：
文献类型：
--
作者：
Bonneau, AM;Kibler, P;Cordingley, MG
单纯疱疹病毒(HSV)编码自己的核糖核苷酸还原酶(RR)，提供病毒DNA复制所需的高水平脱氧核苷三磷酸。HSV RR由两个不同的亚基组成，即R1和R2，它们的结合是酶活性所必需的。模拟R2 C端氨基酸的模拟肽抑制剂通过阻止R1和R2的结合来抑制HSV RR，这些化合物是候选的抗病毒治疗药物。在这里，我们描述了体外筛选对RR抑制剂Bild733敏感性降低三到九倍的1型HSV KOS变异体。耐药株的体外生长特性与野生型Kos相似，但对阿昔洛韦更敏感，可能是由于其RR功能受损所致。R1区的单一氨基酸替换(Ala-1091到Ser)与对Bild 733的三重抗性相关，而更高水平的抗性需要额外的替换(Pro-1090到Leu)。这些突变被重新引入到HSV 1型KOS中，并被证明足以赋予耐药表型，体外与从这些突变病毒感染的细胞中分离的RR的研究表明，这些RR与Bild 733的结合比野生型酶更弱，并且功能受损，表现出R1-R2亚单位结合的解离常数(K-d)增加和/或活性(k(CAT))降低。这项工作提供了证据表明，单纯疱疹病毒R1的C末端(残基1090和1091)参与了R2结合作用，并证明了对亚单位结合抑制剂的抗性可能与靶酶的活性受损有关。
Herpes simplex virus (HSV) encodes its own ribonucleotide reductase (RR), which provides the high levels of deoxynucleoside triphosphates required for viral DNA replication in infected cells. HSV RR is composed of two distinct subunits, R1 and R2, whose association is required for enzymatic activity. Peptidomimetic inhibitors that mimic the C-terminal amino acids of R2 inhibit HSV RR by preventing the association of R1 and R2, These compounds are candidate antiviral therapeutic agents. Here we describe the in vitro selection of HSV type 1 KOS variants with three- to ninefold-decreased sensitivity to the RR inhibitor BILD 733. The resistant isolates have growth properties in vitro similar to those of wild-type KOS but are more sensitive to acyclovir, possibly as a consequence of functional impairment of their RRs. A single amino acid substitution in R1 (Ala-1091 to Ser) was associated with threefold resistance to BILD 733, whereas an additional substitution (Pro-1090 to Leu) was required for higher levels of resistance. These mutations were reintroduced into HSV type 1 KOS and shown to be sufficient to confer the resistance phenotype, Studies in vitro with RRs isolated from cells infected with these mutant viruses demonstrated that these RRs bind BILD 733 more weakly than the wild-type enzyme and are also functionally impaired, exhibiting an elevated dissociation constant (K-d) for R1-R2 subunit association and/or reduced activity (k(cat)). This work provides evidence that the C-terminal end of HSV R1 (residues 1090 and 1091) is involved in R2 binding interactions and demonstrates that resistance to subunit association inhibitors may be associated with compromised activity of the target enzyme.