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

Abstract

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-penetrating 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 and molecular 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 demonstrate 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 VP4 might be essential for the penetration of host endosomal membranes and release of the viral genome during HAV entry. IMPORTANCEHepatitis A virus causes acute hepatitis in humans through the fecal-oral route and is particularly prevalent in underdeveloped regions with poor hygienic conditions. Although a vaccine for HAV exists, its high cost makes it unsuitable for universal application in developing countries. Studies on host-virus interaction for HAV have been hampered due to a lack of starting material, since the virus is extremely slow growing in culture. Among the unknown aspects of the HAV life cycle is its manner of host membrane penetration, which is one of the most important initial steps in viral infection. Here, we present data to suggest that a small peptide, VP4, a component of the HAV structural polyprotein, might be essential in helping the viral genome cross cell membranes during entry. It is hoped that this work might help in elucidating the manner of initial host cell interaction by HAV.

Cryo-EM study of slow bee paralysis virus at low pH reveals iflavirus genome release mechanism

“…During cell entry, nonenveloped viruses have to breach a biological membrane to deliver their genomes to the cell cytoplasm. Previously, it has been shown that both myristoylated and unmodified VP4 minor capsid proteins (which are 60-80 residues long) of several picornaviruses and dicistroviruses can induce the lysis of liposomes (26,40,41). In contrast, the VP4 subunits of SBPV have been predicted to be only 20 residues long, and lack the myristoylation signal sequence (42).…”

“…The N-terminal regions of VP1 subunits of enteroviruses contain sequences, which were proposed to form amphipathic α-helices that disrupt endosome membranes, and together with VP4 subunits enable translocation of the virus genome to the cytoplasm (26,27,41). Twelve residues from the N terminus of SBPV VP1 become disordered upon genome release (Fig.…”

The C-terminal region of the non-structural protein 2B from Hepatitis A Virus demonstrates lipid-specific viroporin-like activity

“…While the 2B peptide existed primarily as a random coil in phosphate buffer, it adapted to a significantly alpha-helical (45.4%) conformation in the presence of 50% tetrafluoroethylene (TFE), which is known to mimic a hydrophobic environment ( Fig. 1B ) 17 18 . This indicated the possibility of a helical transition in the C-terminal region of 2B in the hydrophobic environment of cellular membranes.…”

“…2B ). The release and dequenching of SulfoB fluorescence was measured as described 17 . In order to detect whether membrane disruption by the 2B peptide is lipid composition-specific, we produced liposomes mimicking the mammalian plasma membrane, and membranes of cellular organelles like endoplasmic reticulum (ER), golgi bodies, mitochondria as well as the outer nuclear membrane.…”

Spot Immunomagnetic Enrichment Device for Rapid Detection of Pathogens in Peripheral Blood

“…Cell–MNP binding was confirmed using a transmission electron microscope (TEM) (Tecnai TF20, FEI) operated at an acceleration voltage of 200 kV. The TEM grid was prepared by applying a 10 μL aliquot of the sample to a 300 square mesh carbon‐coated copper grid (Electron Microscopy Sciences) followed by washing and addition of uranyl acetate as a negative staining agent …”