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Development of novel antivirals, which requires knowledge of the viral life cycle in molecular detail, is a daunting task, involving extensive investments, and frequently resulting in failure. As there exist significant commonalities among virus families in the manner of host interaction, identifying and targeting common rather than specific features may lead to the development of broadly useful antivirals. Here, we have targeted the 3C protease of Hepatitis A Virus (HAV), a feco-orally transmitted virus of the family Picornaviridae, for identification of potential antivirals. The 3C protease is a viable drug target as it is required by HAV, as well as by other picornaviruses, for post-translational proteolysis of viral polyproteins and for inhibiting host innate immune pathways. Computational screening, followed by chemical synthesis and experimental validation resulted in identification of a few compounds which, at low micromolar concentrations, could inhibit HAV 3C activity. These compounds were further tested experimentally against the 3C protease of Human Rhinovirus, another member of the Picornaviridae family, with comparable results. Computational studies on 3C proteases from other members of the picornavirus family have indicated that the compounds identified could potentially be generic inhibitors for picornavirus 3C proteases., (© 2018 Federation of European Biochemical Societies.)

Viroporins are virally encoded, membrane-active proteins, which enhance viral replication and assist in egress of viruses from host cells. The 2B proteins in the picornaviridae family are known to have viroporin-like properties, and play critical roles during virus replication. The 2B protein of Hepatitis A Virus (2B), an unusual picornavirus, is somewhat dissimilar from its analogues in several respects. HAV 2B is approximately 2.5 times the length of other 2B proteins, and does not disrupt calcium homeostasis or glycoprotein trafficking. Additionally, its membrane penetrating properties are not yet clearly established. Here we show that the membrane interacting activity of HAV 2B is localized in its C-terminal region, which contains an alpha-helical hairpin motif. We show that this region is capable of forming small pores in membranes and demonstrates lipid specific activity, which partially rationalizes the intracellular localization of full-length 2B. Using a combination of biochemical assays and molecular dynamics simulation studies, we also show that HAV 2B demonstrates a marked propensity to dimerize in a crowded environment, and probably interacts with membranes in a multimeric form, a hallmark of other picornavirus viroporins. In sum, our study clearly establishes HAV 2B as a bona fide viroporin in the picornaviridae family.