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

Abstract

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.

Control of the negative IREStrans-acting factor KHSRP by ubiquitination

“…In this study, we found that KHSRP colocalized in the cytoplasm with KLHL12 in EV71-infected cells (Figure 2D). We also noted that both KLHL12 and viral protein 2B accumulated in specific cellular compartments around the nucleus: KLHL12 is involved in ER-Golgi transport and localizes to the endoplasmic reticulum (ER) (31), while the picornavirus 2B protein also localizes in the ER and Golgi complex (37,38). These findings suggest that KLHL12 and viral protein 2B may colocalize in the ER, and this could be related to formation of the viral replication complex.…”

Inhibitor-Based Therapeutics for Treatment of Viral Hepatitis

“…Oligomerization levels can range from dimeric association ( e.g. in HAV 2B) 97 to large heptameric assemblies ( e.g . in HCV p7).…”

“…In fact, recent work from our laboratory has characterized the membrane activity of capsid component VP4 and non-structural protein 2B from HAV. 97 , 101 Utilizing a combination of biophysical studies, MD simulation and electron microscopy, it was shown that VP4 forms discreet pores of 5∼9 nm in artificial membranes. Our data thus indicates that VP4 is a membrane penetrating peptide which may allow HAV to escape from endosomes and gain access to the cellular cytosol.…”

“…We further investigated the non-structural 2B protein of HAV, which was found to contain a stretch of 60 residues in its C-terminus, and which has viroporin-like activity. 97 A combination of crosslinking studies, biophysical assays and simulation was employed to determine that 2B probably forms dimers and generates ∼3 nm pores in membranes. The resulting alteration of membrane permeability is thought to be essential for HAV replication.…”

A direct role for hepatitis B virus X protein in inducing mitochondrial membrane permeabilization

“…Hepatitis A virus (HAV) 2B and hepatitis C virus (HCV) p7 directly induce membrane permeabilization, collectively termed viroporins. 13,14 These are small, nonstructural proteins forming nonselective channels to transport cations and large molecules and have been proposed to modulate the intracellular electrochemical balance of host cells by inducing membrane permeabilization. 13,15 In a related study, HBV was also shown to induce mitochondrial membrane permeabilization.…”