Identification of an immunodominant antigenic site involving the capsid protein VP3 of hepatitis A virus.

ABSTRACT Most details of the processing of the hepatitis A virus (HAV) polyprotein are known. Unique among members of the familyPicornaviridae, the primary cleavage of the HAV polyprotein is mediated by 3Cpro, the only proteinase known to be encoded by the virus, at the 2A/2B junction. All other cleavages of the polyprotein have been considered to be due to 3Cpro, although the precise location and mechanism responsible for the VP1/2A cleavage have been controversial. Here we present data that argue strongly against the involvement of the HAV 3Cproproteinase in the maturation of VP1 from its VP1-2A precursor. Using a heterologous expression system based on recombinant vaccinia viruses directing the expression of full-length or truncated capsid protein precursors, we show that the C terminus of the mature VP1 capsid protein is located near residue 764 of the polyprotein. However, a proteolytically active HAV 3Cpro that was capable of directing both VP0/VP3 and VP3/VP1 cleavages in vaccinia virus-infected cells failed to process the VP1-2A precursor. Using site-directed mutagenesis of an infectious molecular clone of HAV, we modified potential VP1/2A cleavage sites that fit known 3Cprorecognition criteria and found that a substitution that ablates the presumed 3Cpro dipeptide recognition sequence at Glu764-Ser765 abolished neither infectivity nor normal VP1 maturation. Altered electrophoretic mobility of VP1 from a viable mutant virus with an Arg764 substitution indicated that this residue is present in VP1 and that the VP1/2A cleavage occurs downstream of this residue. These data indicate that maturation of the HAV VP1 capsid protein is not dependent on 3Cpro processing and may thus be uniquely dependent on a cellular proteinase.

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Redundant Late Domain Functions of Tandem VP2 YPX3L Motifs in Nonlytic Cellular Egress of Quasi-enveloped Hepatitis A Virus

Journal of Virology ◽

10.1128/jvi.01308-18 ◽

2018 ◽

Vol 92 (23) ◽

Cited By ~ 10

Author(s):

Olga González-López ◽

Efraín E. Rivera-Serrano ◽

Fengyu Hu ◽

Lucinda Hensley ◽

Kevin L. McKnight ◽

...

Keyword(s):

Life Cycle ◽

Capsid Protein ◽

Hepatitis A ◽

Hepatitis A Virus ◽

Host Protein ◽

Endosomal Sorting ◽

Late Domain ◽

Membrane Budding ◽

Late Domains ◽

Endosomal Vesicles

ABSTRACTThe quasi-envelopment of hepatitis A virus (HAV) capsids in exosome-like virions (eHAV) is an important but incompletely understood aspect of the hepatovirus life cycle. This process is driven by recruitment of newly assembled capsids to endosomal vesicles into which they bud to form multivesicular bodies with intraluminal vesicles that are later released at the plasma membrane as eHAV. The endosomal sorting complexes required for transport (ESCRT) are key to this process, as is the ESCRT-III-associated protein, ALIX, which also contributes to membrane budding of conventional enveloped viruses. YPX1or3L late domains in the structural proteins of these viruses mediate interactions with ALIX, and two such domains exist in the HAV VP2 capsid protein. Mutational studies of these domains are confounded by the fact that the Tyr residues (important for interactions of YPX1or3L peptides with ALIX) are required for efficient capsid assembly. However, single Leu-to-Ala substitutions within either VP2 YPX3L motif (L1-A and L2-A mutants) were well tolerated, albeit associated with significantly reduced eHAV release. In contrast, simultaneous substitutions in both motifs (L1,2-A) eliminated virus release but did not inhibit assembly of infectious intracellular particles. Immunoprecipitation experiments suggested that the loss of eHAV release was associated with a loss of ALIX recruitment. Collectively, these data indicate that HAV YPX3L motifs function as redundant late domains during quasi-envelopment and viral release. Since these motifs present little solvent-accessible area in the crystal structure of the naked extracellular capsid, the capsid structure may be substantially different during quasi-envelopment.IMPORTANCENonlytic release of hepatitis A virus (HAV) as exosome-like quasi-enveloped virions is a unique but incompletely understood aspect of the hepatovirus life cycle. Several lines of evidence indicate that the host protein ALIX is essential for this process. Tandem YPX3L “late domains” in the VP2 capsid protein could be sites of interaction with ALIX, but they are not accessible on the surface of an X-ray model of the extracellular capsid, raising doubts about this putative late domain function. Here, we describe YPX3L domain mutants that assemble capsids normally but fail to bind ALIX and be secreted as quasi-enveloped eHAV. Our data support late domain function for the VP2 YPX3L motifs and raise questions about the structure of the HAV capsid prior to and following quasi-envelopment.

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5430135 DNA sequence encoding a cynomolgus monkey hepatitis a virus capsid protein

Biotechnology Advances ◽

10.1016/0734-9750(96)83031-x ◽

1996 ◽

Vol 14 (3) ◽

pp. 379

Keyword(s):

Capsid Protein ◽

Dna Sequence ◽

Cynomolgus Monkey ◽

Hepatitis A ◽

Hepatitis A Virus ◽

Virus Capsid ◽

Sequence Encoding ◽

Virus Capsid Protein

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5430135 DNA sequence encoding a cynomolgus monkey hepatitis a virus capsid protein

Biotechnology Advances ◽

10.1016/0734-9750(96)82800-x ◽

1996 ◽

Vol 14 (3) ◽

pp. 320

Keyword(s):

Capsid Protein ◽

Dna Sequence ◽

Cynomolgus Monkey ◽

Hepatitis A ◽

Hepatitis A Virus ◽

Virus Capsid ◽

Sequence Encoding ◽

Virus Capsid Protein

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New protein comprising at least one hepatitis A virus epitope; capsid protein expression in vaccinia virus vector for use as recombinant vaccine

Vaccine ◽

10.1016/0264-410x(92)90034-h ◽

1992 ◽

Vol 10 (2) ◽

pp. 133

Keyword(s):

Protein Expression ◽

Vaccinia Virus ◽

Capsid Protein ◽

Hepatitis A ◽

Hepatitis A Virus ◽

Recombinant Vaccine ◽

Virus Vector ◽

Vaccinia Virus Vector ◽

Virus Epitope ◽

New Protein

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Fusion of vesicles induced by a sequence from the VP3 capsid protein of Hepatitis A virus

Biomedical Chromatography ◽

10.1002/(sici)1099-0801(199904)13:2<161::aid-bmc849>3.0.co;2-v ◽

1999 ◽

Vol 13 (2) ◽

pp. 161-162

Author(s):

A. Chávez ◽

M. A. Busquets ◽

M. Pujol ◽

M. A. Alsina ◽

Y. Cajal

Keyword(s):

Capsid Protein ◽

Hepatitis A ◽

Hepatitis A Virus

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

Journal of Virology ◽

10.1128/jvi.76.15.7495-7505.2002 ◽

2002 ◽

Vol 76 (15) ◽

pp. 7495-7505 ◽

Cited By ~ 43

Author(s):

Lisette Cohen ◽

Danièle Bénichou ◽

Annette Martin

Keyword(s):

Capsid Protein ◽

Hepatitis A ◽

Hepatitis A Virus ◽

Protein A ◽

Growth Conditions ◽

Protein Precursor ◽

Synthetic Genome ◽

Vaccinia Viruses ◽

One Step ◽

Capsid Protein Vp1

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 3Cpro 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 3Cpro-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 3Cpro 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 3Cpro-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.

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