Non-enveloped viruses require membrane-penetrating peptides for gaining entry inside the cytoplasm of host cells during the early stages of infection. Although several such peptides have been identified as essential components for non-enveloped virus entry, the molecular mechanism of membrane destabilization by these peptides is not well established. Here, we investigate the putative membrane penetrating peptide VP4 of Hepatitis A Virus (HAV) using a combination of molecular dynamics simulation and mutational studies. Using all-atom molecular dynamics simulation, we show that effective membrane disruption requires specific oligomeric forms (pentameric or hexameric) of VP4, while the monomeric form cannot cause similar disruption in target membranes. Reduction in hydrophobicity of VP4 significantly affects membrane penetration properties in silico, with even the oligomeric associations showing decreased membrane penetration efficiency. A synthetic peptide with a concurrent reduction in hydrophobicity is unable to disrupt liposomes in vitro, while the introduction of these mutations in the context of the viral genome adversely affects the propagation of HAV in cell culture. Taken together, our studies highlight hydrophobicity and oligomerization as some of the crucial mechanistic aspects of membrane penetration by capsid components of non-enveloped viruses.

非包膜病毒需要膜穿透肽才能在感染的早期阶段进入宿主细胞的细胞质内。尽管已经确定了几种这样的肽作为非包膜病毒进入的必要组分，但是这些肽引起的膜失稳的分子机理尚不十分清楚。在这里，我们结合分子动力学模拟和突变研究调查甲型肝炎病毒（HAV）的假定的膜穿透肽VP4。使用全原子分子动力学模拟，我们显示有效的膜破坏需要VP4的特定寡聚形式（五聚体或六聚体），而单体形式不能在靶膜中引起类似的破坏。VP4疏水性的降低会显着影响膜的渗透性能在计算机上，甚至低聚物缔合也显示出降低的膜穿透效率。同时疏水性降低的合成肽不能在体外破坏脂质体，而在病毒基因组的背景下引入这些突变会对HAV在细胞培养中的繁殖产生不利影响。综上所述，我们的研究突出了疏水性和低聚性，这是非包膜病毒衣壳成分对膜渗透的重要机制。