Krosky P M, Underwood M R, Turk S R, Feng K W, Jain R K, Ptak R G, Westerman A C, Biron K K, Townsend L B, Drach J C
Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
J Virol. 1998 Jun;72(6):4721-8. doi: 10.1128/JVI.72.6.4721-4728.1998.
2,5,6-Trichloro-1-beta-D-ribofuranosyl benzimidazole (TCRB) is a potent and selective inhibitor of human cytomegalovirus (HCMV) replication. TCRB acts via a novel mechanism involving inhibition of viral DNA processing and packaging. Resistance to the 2-bromo analog (BDCRB) has been mapped to the UL89 open reading frame (ORF), and this gene product was proposed as the viral target of the benzimidazole nucleosides. In this study, we report the independent isolation of virus that is 20- to 30-fold resistant to TCRB (isolate C4) and the characterization of the virus. The six ORFs known to be essential for viral DNA cleavage and packaging (UL51, UL52, UL56, UL77, UL89, and UL104) were sequenced from wild-type HCMV, strain Towne, and from isolate C4. Mutations were identified in UL89 (D344E) and in UL56 (Q204R). The mutation in UL89 was identical to that previously reported for virus resistant to BDCRB, but the mutation in UL56 is novel. Marker transfer analysis demonstrated that each of these mutations individually caused approximately 10-fold resistance to the benzimidazoles and that the combination of both mutations caused approximately 30-fold resistance. The rate and extent of replication of the mutants was the same as for wild-type virus, but the viruses were less sensitive to inhibition of DNA cleavage by TCRB. Mapping of resistance to UL56 supports and extends recent work showing that UL56 codes for a packaging motif binding protein which also has specific nuclease activity (E. Bogner et al., J. Virol. 72:2259-2264, 1998). Resistance which maps to two different genes suggests that their putative proteins interact and/or that either or both have a benzimidazole ribonucleoside binding site. The results also suggest that the gene products of UL89 and UL56 may be antiviral drug targets.
2,5,6-三氯-1-β-D-呋喃核糖基苯并咪唑（TCRB）是一种强效且具有选择性的人类巨细胞病毒（HCMV）复制抑制剂。TCRB通过一种涉及抑制病毒DNA加工和包装的新机制发挥作用。对2-溴类似物（BDCRB）的抗性已定位到UL89开放阅读框（ORF），并且该基因产物被认为是苯并咪唑核苷的病毒靶点。在本研究中，我们报告了对TCRB具有20至30倍抗性病毒（分离株C4）的独立分离及该病毒的特性。从野生型HCMV毒株Towne和分离株C4中对已知对病毒DNA切割和包装至关重要的6个ORF（UL51、UL52、UL56、UL77、UL89和UL104）进行了测序。在UL89（D344E）和UL56（Q204R）中鉴定到了突变。UL89中的突变与先前报道的对BDCRB耐药的病毒中的突变相同，但UL56中的突变是新的。标记转移分析表明，这些突变中的每一个单独都会导致对苯并咪唑产生约10倍的抗性，并且两个突变的组合会导致约30倍的抗性。突变体的复制速率和程度与野生型病毒相同，但这些病毒对TCRB抑制DNA切割的敏感性较低。对UL56的抗性定位支持并扩展了最近的研究工作，该研究表明UL56编码一种包装基序结合蛋白，该蛋白也具有特定的核酸酶活性（E. Bogner等人，《病毒学杂志》72:2259 - 2264，1998）。定位于两个不同基因的抗性表明它们的推定蛋白相互作用和/或它们中的一个或两个都具有苯并咪唑核糖核苷结合位点。结果还表明，UL89和UL56的基因产物可能是抗病毒药物靶点。
