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

Abstract

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.

Characterization of GB Virus B Polyprotein Processing Reveals the Existence of a Novel 13-kDa Protein with Partial Homology to Hepatitis C Virus p7 Protein

“…Generation of Recombinant Vaccinia Viruses and Expression Assays in CV1 Cells-Recombinant vaccinia viruses vv-C-E2 701 -V5 and vv-C-E2 673 -V5 were generated by homologous recombination in CV1 cells infected with the temperature-sensitive ts7 strain of vaccinia virus and co-transfected with pTM/C-E2 701 -V5 or pTM/C-E2 673 -V5, respectively, as well as wild-type Copenhagen vaccinia virus DNA, essentially as described previously (34). Recombinant viruses were plaque-purified twice on 143B thymidine kinase-deficient cells under selective conditions.…”

The unique role of domain 2A of the hepatitis A virus precursor polypeptide P1-2A in viral morphogenesis

“…Additional experimental data showed that HAV assembly not only depends on the N-terminal part of domain 2A, but that also its C-terminus influences particle formation. In fact, deletions within the 2A C-terminal region alter the cleavability of the VP1/2A site, impairing proper assembly and capsid maturation (2). In spite of these unusual features, the existing evidence still does not provide clear indications for the various parts of domain 2A in HAV particle formation.…”

“…The present and previous immunological data and biochemical evidence imply that the C-terminus of VP1 and the N-terminus of domain 2A contain oligomerization and procapsid maturation signals (2,5). In particular, the arginine residue at position 278 of VP1-2A seems to be involved in both functions, as substitution of arginine 278 by methionine interferes with oligomerization of P1-2A and maturation of procapsids, probably by hindering the correct structure of the assembly and maturation signals.…”

“…While protein 2A of poliovirus (the prototypic picornavirus) is a proteinase, the respective domain in the HAV polyprotein represents the C-terminal extension of the structural protein VP1-2A that lacks any proteolytic activity. Domain 2A functions as the pri-http://bmbreports.org BMB reports mary signal for viral particle assembly, being involved in P1-2A oligomerization to pentamers and subsequent particle maturation (1,2). As part of VP1-2A, domain 2A is exposed to the surface of immature or empty HAV particles, while mature particles containing viral RNA are almost devoid of 2A.…”

Human hepatitis A virus is united with a host of relations

“…It has been suggested that the function of this codon bias is to reduce the rate of protein synthesis and allow for more accurate folding. Another distinguishing feature of HAV is that the structural protein virus protein 1 (VP1) is extended at its C terminus with an extra domain as VPX, which is important in the virus assembly process (7). The extra sequence of the PVX is then removed by cellular proteases, a process not seen in other picornaviruses, where the cleavage at this junction is carried out by viral protease.…”