Identification of hepatitis A virus non-structural protein 2B and its release by the major virus protease 3C

Unlike all other picornaviruses, the primary cleavage of the hepatitis A virus (HAV) polyprotein occurs at the 2A/2B junction and is carried out by the only proteinase encoded by the virus, 3Cpro . The resulting P1-2A capsid protein precursor is subsequently cleaved by 3C pro to generate VP0, VP3, and VP1-2A, which associate as pentamers. An unidentified cellular proteinase acting at the VP1/2A junction releases the mature capsid protein VP1 from VP1-2A later in the morphogenesis process. Although these aspects of polyprotein processing are well characterized, the function of 2A is unknown. To study its role in the viral life cycle, we assessed the infectivity of synthetic, genome-length RNAs containing 11 different in-frame deletions in the 2A region. Deletions in the N-terminal 40% of 2A abolished infectivity, whereas deletions in the C-terminal 60% resulted in viruses with a small-focus replication phenotype. C-terminal deletions in 2A had no effect on RNA replication kinetics under one-step growth conditions, nor did they have an effect on capsid protein synthesis and 3C pro -mediated processing. However, C-terminal deletions in 2A altered the VP1/2A cleavage, resulting in accumulation of uncleaved VP1-2A precursor in virions and possibly accounting for a delay in the appearance of infectious particles with these mutants, as well as a fourfold decrease in specific infectivity of the virus particles. When the capsid proteins were expressed from recombinant vaccinia viruses, the N-terminal part of 2A was required for efficient cleavage of the P1-2A precursor by 3C pro and assembly of structural precursors into pentamers. These data indicate that the N-terminal domain of 2A must be present as a C-terminal extension of P1 for folding of the capsid protein precursor to allow efficient 3C pro -mediated cleavages and to promote pentamer assembly, after which cleavage at the VP1/2A junction releases the mature VP1 protein, a process that appears to be necessary to produce highly infectious particles.

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

Analysis of Deletion Mutants Indicates that the 2A Polypeptide of Hepatitis A Virus Participates in Virion Morphogenesis

Cohen

Bénichou

Martin

2002

“…It was later demonstrated that the 2B nonstructural protein is considerably larger than that predicted by the original processing model, extending into the upstream sequences of the polyprotein. These studies demonstrated that the 2A polypeptide is smaller than originally predicted and that 3C pro is responsible for the cleavage at the newly identified 2A/2B junction (17,30). Although this cleavage results in an amino-terminal P1-2A segment that includes the three major capsid proteins, as well as a VP1-2A precursor that has been identified in capsid morphogenesis intermediates (9), it is not known whether this is the primary cleavage within the polyprotein or whether it follows other cleavages further downstream in the polyprotein.…”

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confidence: 99%

Characterization of Recombinant Hepatitis A Virus Genomes Containing Exogenous Sequences at the 2A/2B Junction

Beard

Cohen

Lemon

et al. 2001

The family Picornaviridae is comprised of a large number of single-stranded RNA viruses that are currently classified into five major genera of human and veterinary pathogens and several lesser genera (38). These include the genera Enterovirus (of which poliovirus is a member and the prototype picornavirus), Rhinovirus, Cardiovirus (including encephalomyocarditis virus [EMCV]), Aphthovirus (foot-and-mouth disease virus), and Hepatovirus. Hepatitis A virus (HAV) is the only virus species within the genus Hepatovirus. A human virus that is transmitted by the fecal-oral route, HAV is responsible for approximately 50% of all cases of acute viral hepatitis within the United States (23). The genomic RNA of HAV shares a general molecular organization and replication strategy with that of other picornaviruses. The positive-strand RNA genome is approximately 7.5 kb in length (12,26,34). It is linked covalently to a small polypeptide (VPg) at its 5Ј terminus (46), contains a 3Ј polyadenylated tail, and is capable of functioning directly as mRNA when introduced into the cytoplasm of host cells (13). A lengthy 5Ј terminal nontranslated RNA segment contains numerous highly ordered RNA structures and a functional internal ribosome entry site (IRES) (10). As with the IRES elements of other picornaviruses, the HAV IRES directs the cap-independent translation of a polyprotein encoded within a single large open reading frame (10, 47). Picornaviral IRES elements have been classified as either type 1 (the enteroviruses and rhinoviruses) or type 2 (cardioviruses, aphthoviruses, and hepatoviruses) on the basis of substantial differences in their higher ordered RNA structure, yet they all function to facilitate internal ribosome entry (25,48).Despite these similarities to other picornaviruses, the polyprotein of HAV presents some striking and unique features. In enteroviral and rhinoviral replication, the primary polyprotein cleavage takes place at the P1/P2 junction (VP1/ 2A) and is mediated by a cis-acting viral proteinase activity associated with 2A (2A pro ). In the case of the cardioviruses and aphthoviruses, the primary scission is also dependent upon 2A but occurs by a unique autocatalytic mechanism at the 2A/2B junction (36,40). Although the first model proposed for the polyprotein processing of HAV resembled the enterovirus cleavage cascade (12), subsequent studies demonstrated that the only virally encoded proteinase, 3Cpro , was able to cleave in the middle of what was originally considered to be the HAV 2A coding region (19,41). It was later demonstrated that the 2B nonstructural protein is considerably larger than that predicted by the original processing model, extending into the upstream sequences of the polyprotein. These studies demonstrated that the 2A polypeptide is smaller than originally predicted and that 3C pro is responsible for the cleavage at the newly identified 2A/2B junction (17,30). Although this cleavage results in an amino-terminal P1-2A segment that includes the three major capsid proteins, as well as ...

“…In contrast, primary polyprotein processing for Hepatovirus takes place between 2A and 2B and is carried out by the single viral proteinase 3C pro , which displays a different substrate specificity from that of the enteroviral 3C proteinases, marked by radically different amino acid preferences at the P4 and P2= positions of the substrate (8)(9)(10)(11). HAV 3C pro cleaves the 2A/2B junction between residues Gln836 and Ala837 (numbering according to the GenBank entry for HM175 [M14707.1; 12]), a region located 144 residues upstream of the enteroviral 2A/2B junction, resulting in a smaller 2A protein (71 to 73 amino acids long) and a considerably larger 2B protein (251 amino acids long) (11,(13)(14)(15). The functions of the 2A and 2B proteins are also different in distinct viruses.…”

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confidence: 99%

Structural Basis for Host Membrane Remodeling Induced by Protein 2B of Hepatitis A Virus

Vives-Adrián

Garriga

Buxaderas

et al. 2015

The complexity of viral RNA synthesis and the numerous participating factors require a mechanism to topologically coordinate and concentrate these multiple viral and cellular components, ensuring a concerted function. Similarly to all other positivestrand RNA viruses, picornaviruses induce rearrangements of host intracellular membranes to create structures that act as functional scaffolds for genome replication. The membrane-targeting proteins 2B and 2C, their precursor 2BC, and protein 3A appear to be primarily involved in membrane remodeling. Little is known about the structure of these proteins and the mechanisms by which they induce massive membrane remodeling. Here we report the crystal structure of the soluble region of hepatitis A virus (HAV) protein 2B, consisting of two domains: a C-terminal helical bundle preceded by an N-terminally curved five-stranded antiparallel ␤-sheet that displays striking structural similarity to the ␤-barrel domain of enteroviral 2A proteins. Moreover, the helicoidal arrangement of the protein molecules in the crystal provides a model for 2B-induced host membrane remodeling during HAV infection. IMPORTANCENo structural information is currently available for the 2B protein of any picornavirus despite it being involved in a critical process in viral factory formation: the rearrangement of host intracellular membranes. Here we present the structure of the soluble domain of the 2B protein of hepatitis A virus (HAV). Its arrangement, both in crystals and in solution under physiological conditions, can help to understand its function and sheds some light on the membrane rearrangement process, a putative target of future antiviral drugs. Moreover, this first structure of a picornaviral 2B protein also unveils a closer evolutionary relationship between the hepatovirus and enterovirus genera within the Picornaviridae family. Due to their limited genome size, viruses are obligate intracellular parasites that recruit cell host components for their multiplication. All currently known positive-strand RNA viruses, as well as some DNA viruses, rearrange host intracellular membranes to create the so-called viral factories, vesicular structures that concentrate the viral and host proteins required for replication as well as the viral genomes and, at the same time, shield them from host antiviral defenses (1).Despite the key role of viral factories in viral replication, the molecular processes leading to their formation are only partially understood. This is especially true for picornaviruses (PVs), one of the largest families of pathogens known, for which the lack of information on host membrane rearrangement processes contrasts with the highly detailed data available for other stages of the picornavirus viral cycle (e.g., receptor binding, viral entry, and RNA synthesis).The Picornaviridae family includes many important human and animal pathogens such as polioviruses, rhinoviruses, footand-mouth disease virus, and hepatitis A virus (HAV). HAV is the only species and only serotype in t...