Coelho João, Martins Carlos, Ferreira Fernando, Leitão Alexandre
CIISA, Faculdade de Medicina Veterinária, ULisboa; Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal.
Instituto de Investigação Científica Tropical, CVZ, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal; CIISA, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal.
Virology. 2015 Jan 1;474:82-93. doi: 10.1016/j.virol.2014.10.034. Epub 2014 Nov 14.
Topoisomerases modulate the topological state of DNA during processes, such as replication and transcription, that cause overwinding and/or underwinding of the DNA. African swine fever virus (ASFV) is a nucleo-cytoplasmic double-stranded DNA virus shown to contain an OFR (P1192R) with homology to type II topoisomerases. Here we observed that pP1192R is highly conserved among ASFV isolates but dissimilar from other viral, prokaryotic or eukaryotic type II topoisomerases. In both ASFV/Ba71V-infected Vero cells and ASFV/L60-infected pig macrophages we detected pP1192R at intermediate and late phases of infection, cytoplasmically localized and accumulating in the viral factories. Finally, we used a Saccharomyces cerevisiae temperature-sensitive strain in order to demonstrate, through complementation and in vitro decatenation assays, the functionality of P1192R, which we further confirmed by mutating its predicted catalytic residue. Overall, this work strengthens the idea that P1192R constitutes a target for studying, and possibly controlling, ASFV transcription and replication.
拓扑异构酶在诸如复制和转录等导致DNA过度缠绕和/或解缠绕的过程中调节DNA的拓扑状态。非洲猪瘟病毒(ASFV)是一种核质双链DNA病毒,已证明其含有一个与II型拓扑异构酶具有同源性的开放阅读框(P1192R)。在这里,我们观察到pP1192R在ASFV分离株中高度保守,但与其他病毒、原核或真核II型拓扑异构酶不同。在ASFV/Ba71V感染的Vero细胞和ASFV/L60感染的猪巨噬细胞中,我们在感染的中晚期检测到pP1192R,其定位于细胞质并在病毒工厂中积累。最后,我们使用酿酒酵母温度敏感菌株,通过互补和体外解连环分析来证明P1192R的功能,我们通过突变其预测的催化残基进一步证实了这一点。总体而言,这项工作强化了P1192R构成研究并可能控制ASFV转录和复制的靶点这一观点。