Di Antonio Veronica, Palù Giorgio, Alvisi Gualtiero
Department of Molecular Medicine, University of Padua, 35122 Padova, Italy.
Microorganisms. 2021 Apr 26;9(5):928. doi: 10.3390/microorganisms9050928.
Human cytomegalovirus (HCMV) genome replication is a complex and still not completely understood process mediated by the highly coordinated interaction of host and viral products. Among the latter, six different proteins form the viral replication complex: a single-stranded DNA binding protein, a trimeric primase/helicase complex and a two subunit DNA polymerase holoenzyme, which in turn contains a catalytic subunit, pUL54, and a dimeric processivity factor ppUL44. Being absolutely required for viral replication and representing potential therapeutic targets, both the ppUL44-pUL54 interaction and ppUL44 homodimerization have been largely characterized from structural, functional and biochemical points of view. We applied fluorescence and bioluminescence resonance energy transfer (FRET and BRET) assays to investigate such processes in living cells. Both interactions occur with similar affinities and can take place both in the nucleus and in the cytoplasm. Importantly, single amino acid substitutions in different ppUL44 domains selectively affect its dimerization or ability to interact with pUL54. Intriguingly, substitutions preventing DNA binding of ppUL44 influence the BRET of protein-protein interactions, implying that binding to dsDNA induces conformational changes both in the ppUL44 homodimer and in the DNA polymerase holoenzyme. We also compared transiently and stably ppUL44-expressing cells in BRET inhibition assays. Transient expression of the BRET donor allowed inhibition of both ppUL44 dimerization and formation of the DNA polymerase holoenzyme, upon overexpression of FLAG-tagged ppUL44 as a competitor. Our approach could be useful both to monitor the dynamics of assembly of the HCMV DNA polymerase holoenzyme and for antiviral drug discovery.
人巨细胞病毒（HCMV）基因组复制是一个复杂且尚未完全理解的过程，由宿主和病毒产物的高度协调相互作用介导。在后者中，六种不同的蛋白质形成病毒复制复合体：一种单链DNA结合蛋白、一种三聚体引发酶/解旋酶复合体和一种双亚基DNA聚合酶全酶，后者又包含一个催化亚基pUL54和一个二聚体持续合成因子ppUL44。由于对病毒复制绝对必需且代表潜在治疗靶点，ppUL44-pUL54相互作用和ppUL44同二聚化在很大程度上已从结构、功能和生化角度进行了表征。我们应用荧光和生物发光共振能量转移（FRET和BRET）测定法来研究活细胞中的此类过程。两种相互作用以相似的亲和力发生，并且可以在细胞核和细胞质中发生。重要的是，不同ppUL44结构域中的单个氨基酸取代选择性地影响其二聚化或与pUL54相互作用的能力。有趣的是，阻止ppUL44与DNA结合的取代会影响蛋白质-蛋白质相互作用的BRET，这意味着与双链DNA的结合会诱导ppUL44同二聚体和DNA聚合酶全酶的构象变化。我们还在BRET抑制测定中比较了瞬时和稳定表达ppUL44的细胞。当作为竞争者过量表达FLAG标签的ppUL44时，BRET供体的瞬时表达可抑制ppUL44二聚化和DNA聚合酶全酶的形成。我们的方法对于监测HCMV DNA聚合酶全酶组装的动力学以及抗病毒药物发现可能都有用。
