Department of Zoology and Life Sciences Institute, University of British Columbiagrid.17091.3e, Vancouver, British Columbia, Canada.
J Virol. 2022 Sep 14;96(17):e0111822. doi: 10.1128/jvi.01118-22. Epub 2022 Aug 11.
Being nonpathogenic to humans, rodent parvoviruses (PVs) are naturally oncolytic viruses with great potential as anti-cancer agents. As these viruses replicate in the host cell nucleus, they must gain access to the nucleus during infection. The PV minute virus of mice (MVM) and several other PVs transiently disrupt the nuclear envelope (NE) and enter the nucleus through the resulting breaks. However, the molecular basis of this unique nuclear entry pathway remains uncharacterized. In this study, we used MVM as a model to investigate the molecular mechanism by which PVs induce NE disruption during viral nuclear entry. By combining bioinformatics analyses, metabolic labeling assays, mutagenesis, and pharmacological inhibition, we identified a functional myristoylation site at the sequence GGKVGH of the unique portion of the capsid protein VP1 (VP1u) of MVM. Performing proteolytic cleavage studies with a peptide containing this myristoylation site or with purified virions, we found tryptophan at position 77 of MVM VP1u is susceptible to chymotrypsin cleavage, implying this cleavage exposes G (glycine) 78 at the N-terminus of VP1u for myristoylation. Subsequent experiments using inhibitors of myristoylation and cellular proteases with MVM-infected cells, or an imaging-based quantitative NE permeabilization assay, further indicate protein myristoylation and a chymotrypsin-like activity are essential for MVM to locally disrupt the NE during viral nuclear entry. We thus propose a model for the nuclear entry of MVM in which NE disruption is mediated by VP1u myristoylation after the intact capsid undergoes proteolytic processing to expose the required N-terminal G for myristoylation. Rodent parvoviruses (PVs), including minute virus of mice (MVM), have the ability to infect and kill cancer cells and thereby possess great potential in anti-cancer therapy. In fact, some of these viruses are currently being investigated in both preclinical studies and clinical trials to treat a wide variety of cancers. However, the detailed mechanism of how PVs enter the cell nucleus remains unknown. In this study, we for the first time demonstrated a chemical modification called "myristoylation" of a MVM protein plays an essential role in the nuclear entry of the virus. We also showed, in addition to protein myristoylation, a chymotrypsin-like activity, which may come from cellular proteasomes, is required for MVM to get myristoylated and enter the nucleus. These findings deepen our understanding on how MVM and other related PVs infect host cells and provide new insights for the development of PV-based anti-cancer therapies.
作为对人类无致病性的啮齿动物微小病毒（PVs），是具有巨大潜力的天然溶瘤病毒，可作为抗癌药物。由于这些病毒在宿主细胞的细胞核内复制，因此在感染过程中必须进入细胞核。小鼠微小病毒（MVM）和其他几种微小病毒会短暂破坏核膜（NE），并通过由此产生的裂口进入细胞核。然而，这种独特的核进入途径的分子基础仍未被描述。在这项研究中，我们使用 MVM 作为模型，研究了 PV 在病毒核进入过程中诱导 NE 破坏的分子机制。通过结合生物信息学分析、代谢标记测定、诱变和药理学抑制，我们鉴定了 MVM 衣壳蛋白 VP1（VP1u）独特区序列 GGKVGH 处的功能性豆蔻酰化位点。用含有该豆蔻酰化位点的肽或纯化的病毒进行蛋白水解切割研究，我们发现 MVM VP1u 位置 77 的色氨酸容易被糜蛋白酶切割，这意味着该切割使 VP1u 的 N 端甘氨酸 78 暴露出来进行豆蔻酰化。随后使用 MVM 感染细胞的豆蔻酰化抑制剂和细胞蛋白酶、或基于成像的定量 NE 通透性测定实验，进一步表明蛋白豆蔻酰化和糜蛋白酶样活性对于 MVM 在病毒核进入过程中局部破坏 NE 是必需的。因此，我们提出了一种 MVM 核进入模型，其中完整衣壳经历蛋白水解处理以暴露所需的 N 端甘氨酸进行豆蔻酰化后，VP1u 的豆蔻酰化介导了 NE 的破坏。
啮齿动物微小病毒（PVs），包括小鼠微小病毒（MVM），具有感染和杀死癌细胞的能力，因此在抗癌治疗中有很大的潜力。事实上，其中一些病毒目前正在临床前研究和临床试验中进行研究，以治疗多种癌症。然而，PV 进入细胞的详细机制仍然未知。在这项研究中，我们首次证明了一种称为“豆蔻酰化”的 MVM 蛋白化学修饰在病毒核进入中起着至关重要的作用。我们还表明，除了蛋白豆蔻酰化外，还需要一种糜蛋白酶样活性，可能来自细胞蛋白酶体，以使 MVM 进行豆蔻酰化并进入细胞核。这些发现加深了我们对 MVM 和其他相关 PV 感染宿主细胞的理解，并为基于 PV 的抗癌疗法的发展提供了新的见解。
