The VP4 Peptide of Hepatitis A Virus Ruptures Membranes through Formation of Discrete Pores

Membrane-active peptides, components of capsid structural proteins, assist viruses in overcoming the host membrane barrier in the initial stages of infection. Several such peptides have been identified, and their roles in membrane fusion or disruption have been characterized through biophysical studies. In several members of the Picornaviridae family, the role of the VP4 structural peptide in cellular-membrane penetration is well established. However, there is not much information on the membrane-pene-trating capsid components of hepatitis A virus (HAV), an unusual member of this family. The VP4 peptide of HAV differs from its analogues in other picornaviruses in being significantly shorter in length and in lacking a signal for myristoylation, thought to be a critical requisite for VP4-mediated membrane penetration. Here we report, for the first time, that the atypical VP4 in HAV contains significant membrane-penetrating activity. Using a combination of biophysical assays andmolecular dynamics simulation studies, we show that VP4 integrates into membrane vesicles through its N-terminal region to finally form discrete pores of 5- to 9-nm diameter, which induces leakage in the vesicles without altering their overall size or shape. We further dem-onstrate that the membrane activity of VP4 is specific toward vesicles mimicking the lipid content of late endosomes at acidic pH. Taken together, our data indicate that VP4might be essential for the penetration of host endosomal membranes and release of the viral genome during HAV entry. IMPORTANC