Department of Genetics and Microbiology, Faculty of Science, Charles University, BIOCEV, 25250 Vestec, Czech Republic.
Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, 25250 Vestec, Czech Republic.
Viruses. 2020 Feb 18;12(2):227. doi: 10.3390/v12020227.
Viruses have evolved mechanisms to manipulate microtubules (MTs) for the efficient realization of their replication programs. Studying the mechanisms of replication of mouse polyomavirus (MPyV), we observed previously that in the late phase of infection, a considerable amount of the main structural protein, VP1, remains in the cytoplasm associated with hyperacetylated microtubules. VP1-microtubule interactions resulted in blocking the cell cycle in the G2/M phase. We are interested in the mechanism leading to microtubule hyperacetylation and stabilization and the roles of tubulin acetyltransferase 1 (αTAT1) and deacetylase histone deacetylase 6 (HDAC6) and VP1 in this mechanism. Therefore, HDAC6 inhibition assays, αTAT1 knock out cell infections, in situ cell fractionation, and confocal and TIRF microscopy were used. The experiments revealed that the direct interaction of isolated microtubules and VP1 results in MT stabilization and a restriction of their dynamics. VP1 leads to an increase in polymerized tubulin in cells, thus favoring αTAT1 activity. The acetylation status of MTs did not affect MPyV infection. However, the stabilization of MTs by VP1 in the late phase of infection may compensate for the previously described cytoskeleton destabilization by MPyV early gene products and is important for the observed inhibition of the G2→M transition of infected cells to prolong the S phase.
病毒已经进化出操纵微管(MTs)的机制,以有效地实现其复制程序。在研究小鼠多瘤病毒(MPyV)的复制机制时,我们之前观察到,在感染后期,大量主要结构蛋白 VP1 与高度乙酰化的微管一起留在细胞质中。VP1-微管相互作用导致细胞周期在 G2/M 期被阻断。我们对导致微管过度乙酰化和稳定的机制以及微管乙酰转移酶 1(αTAT1)和去乙酰化酶组蛋白去乙酰化酶 6(HDAC6)和 VP1 在该机制中的作用感兴趣。因此,使用了 HDAC6 抑制测定、αTAT1 敲除细胞感染、原位细胞分级分离以及共聚焦和 TIRF 显微镜。实验表明,分离的微管和 VP1 的直接相互作用导致 MT 稳定化和限制其动力学。VP1 导致细胞中聚合的微管蛋白增加,从而有利于αTAT1 活性。MTs 的乙酰化状态不会影响 MPyV 感染。然而,感染后期 VP1 对 MT 的稳定作用可能补偿了 MPyV 早期基因产物先前描述的细胞骨架不稳定,并且对观察到的感染细胞 G2→M 过渡的抑制很重要,以延长 S 期。