Maturation of the Hepatitis A Virus Capsid Protein VP1 Is Not Dependent on Processing by the 3CproProteinase

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.

Download Full-text

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.

Download Full-text

A single mutation in capsid protein VP1 (Q145E) of a genogroup C4 strain of human enterovirus 71 generates a mouse-virulent phenotype

Journal of General Virology ◽

10.1099/vir.0.043893-0 ◽

2012 ◽

Vol 93 (9) ◽

pp. 1935-1940 ◽

Cited By ~ 36

Author(s):

Zainun Zaini ◽

Peter McMinn

Keyword(s):

Capsid Protein ◽

Virulent Strain ◽

Infectious Clone ◽

Enterovirus 71 ◽

Site Directed Mutagenesis ◽

Human Enterovirus ◽

Single Mutation ◽

Human Enterovirus 71 ◽

Capsid Protein Vp1 ◽

Vp1 Capsid Protein

We modified the capsid protein of a human enterovirus 71 (HEV71) belonging to subgenogroup C4 (HEV71-C4) to generate a mouse virulent strain, based on the genetic information derived from our previous subgenogroup B3 mouse-adapted virus. Infectious clone-derived mutant virus populations containing the capsid protein mutations VP1-Q145E and VP1-Q145G were generated by site-directed mutagenesis of an infectious clone of a subgenogroup C4 strain. Viruses expressing the VP1-Q145E were virulent in 5-day-old BALB/c mice with 100 % mortality rate observed. Skeletal muscle appears to be the primary site of replication of this virus with limb muscle showing severe myositis. Virus was also isolated from spleen, liver, heart and brain of infected mice. This study demonstrates that introducing a key mutation into the HEV71 VP1 capsid protein is able to generate a mouse virulent HEV71 strain from a different genogroup as well as providing an alternative strategy for the generation of mouse virulent HEV71.

Download Full-text

Expression and immunogenic analysis of recombinant polypeptides derived from capsid protein VP1 for developing subunit vaccine material against hepatitis A virus

Protein Expression and Purification ◽

10.1016/j.pep.2014.04.011 ◽

2014 ◽

Vol 100 ◽

pp. 1-9 ◽

Cited By ~ 3

Author(s):

Kyoung Ok Jang ◽

Jong-Hwa Park ◽

Hyun Ho Lee ◽

Dae Kyun Chung ◽

Wonyong Kim ◽

...

Keyword(s):

Capsid Protein ◽

Subunit Vaccine ◽

Hepatitis A ◽

Hepatitis A Virus ◽

Capsid Protein Vp1 ◽

Recombinant Polypeptides

Download Full-text

Hepatitis A Virus Capsid Protein VP1 Has a Heterogeneous C Terminus

Journal of Virology ◽

10.1128/jvi.73.7.6015-6023.1999 ◽

1999 ◽

Vol 73 (7) ◽

pp. 6015-6023 ◽

Cited By ~ 43

Author(s):

Judith Graff ◽

Oliver C. Richards ◽

Kristine M. Swiderek ◽

Michael T. Davis ◽

Felicia Rusnak ◽

...

Keyword(s):

Amino Acid ◽

Capsid Protein ◽

Hepatitis A ◽

Hepatitis A Virus ◽

Peptide Sequence ◽

Efficient Production ◽

Terminal Amino Acid ◽

Viral Protease ◽

C Terminus ◽

Terminal Amino

ABSTRACT Hepatitis A virus (HAV) encodes a single polyprotein which is posttranslationally processed into the functional structural and nonstructural proteins. Only one protease, viral protease 3C, has been implicated in the nine protein scissions. Processing of the capsid protein precursor region generates a unique intermediate, PX (VP1-2A), which accumulates in infected cells and is assumed to serve as precursor to VP1 found in virions, although the details of this reaction have not been determined. Coexpression in transfected cells of a variety of P1 precursor proteins with viral protease 3C demonstrated efficient production of PX, as well as VP0 and VP3; however, no mature VP1 protein was detected. To identify the C-terminal amino acid residue of HAV VP1, we performed peptide sequence analysis by protease-catalyzed [18O]H2O incorporation followed by liquid chromatography ion-trap microspray tandem mass spectrometry of HAV VP1 isolated from purified virions. Two different cell culture-adapted isolates of HAV, strains HM175pE and HM175p35, were used for these analyses. VP1 preparations from both virus isolates contained heterogeneous C termini. The predominant C-terminal amino acid in both virus preparations was VP1-Ser274, which is located N terminal to a methionine residue in VP1-2A. In addition, the analysis of HM175pE recovered smaller amounts of amino acids VP1-Glu273 and VP1-Thr272. In the case of HM175p35, which contains valine at amino acid position VP1-273, VP1-Thr272 was found in addition to VP1-Ser274. The data suggest that HAV 3C is not the protease responsible for generation of the VP1 C terminus. We propose the involvement of host cell protease(s) in the production of HAV VP1.

Download Full-text

Expression of goose parvovirus VP1 capsid protein by a baculovirus expression system and establishment of fluorescent antibody test to diagnose goose parvovirus infection

Archives of Virology ◽

10.1007/s007050050617 ◽

1999 ◽

Vol 144 (8) ◽

pp. 1639-1645 ◽

Cited By ~ 16

Author(s):

K. Takehara ◽

T. Nakata ◽

K. Takizawa ◽

C. K. Limn ◽

K. Mutoh ◽

...

Keyword(s):

Capsid Protein ◽

Fluorescent Antibody ◽

Expression System ◽

Antibody Test ◽

Baculovirus Expression System ◽

Baculovirus Expression ◽

Goose Parvovirus ◽

Vp1 Capsid Protein

Download Full-text

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.85.21.8281 ◽

1988 ◽

Vol 85 (21) ◽

pp. 8281-8285 ◽

Cited By ~ 52

Author(s):

L. H. Ping ◽

R. W. Jansen ◽

J. T. Stapleton ◽

J. I. Cohen ◽

S. M. Lemon

Keyword(s):

Capsid Protein ◽

Hepatitis A ◽

Hepatitis A Virus ◽

Antigenic Site

Download Full-text

Genetic Analysis of the Major Capsid Protein of the Archaeal Fusellovirus SSV1: Mutational Flexibility and Conformational Change

10.20944/preprints201711.0086.v2 ◽

2017 ◽

Author(s):

Eric A. Iverson ◽

David A. Goodman ◽

Madeline E. Gorchels ◽

Kenneth M. Stedman

Keyword(s):

Capsid Protein ◽

Major Capsid Protein ◽

Point Mutations ◽

Site Directed Mutagenesis ◽

Vp1 Gene ◽

Capsid Protein Gene ◽

Vp1 Protein ◽

Transposon Insertion ◽

N Terminus ◽

Capsid Protein Vp1

Viruses with spindle or lemon-shaped virions are rare in the world of viruses, but are common in viruses of archaeal extremophiles, possibly due to the extreme conditions in which they thrive. However, the structural and genetic basis for the unique spindle shape is unknown. The best-studied spindle-shaped virus, SSV1, is composed mostly of the major capsid protein VP1. Similar to many other viruses, proteolytic cleavage of VP1 is thought to be critical for virion formation. Unlike half of the genes in SSV1, including the minor capsid protein gene vp3, the vp1 gene does not tolerate deletion or transposon insertion. In order determine the role of the vp1 gene and its proteolysis for virus function, we developed techniques for site-directed mutagenesis of the SSV1 genome and complemented deletion mutants with vp1 genes from other SSVs. By analyzing these mutants we demonstrate that the N-terminus of the VP1 protein is required, but the N-terminus, or entire SSV1 VP1 protein, can be exchanged with VP1s from other SSVs. However, the conserved glutamate at the cleavage site is not essential for infectivity. Interestingly, viruses containing point mutations at this position generate mostly abnormal virions.

Download Full-text

Genetic Analysis of the Major Capsid Protein of the Archaeal Fusellovirus SSV1: Mutational Flexibility and Conformational Change

10.20944/preprints201711.0086.v1 ◽

2017 ◽

Author(s):

Eric A. Iverson ◽

David A. Goodman ◽

Madeline E. Gorchels ◽

Kenneth M. STEDMAN

Keyword(s):

Capsid Protein ◽

Major Capsid Protein ◽

Point Mutations ◽

Site Directed Mutagenesis ◽

Vp1 Gene ◽

Vp1 Protein ◽

Transposon Insertion ◽

N Terminus ◽

Capsid Protein Vp1 ◽

Virion Formation

Viruses with spindle or lemon-shaped virions are rare in the world of viruses, but are common in viruses of archaeal extremophiles, possibly due to the extreme conditions in which they thrive. However, the structural and genetic basis for the unique spindle shape is unknown. The best-studied spindle-shaped virus, SSV1, is composed mostly of the major capsid protein VP1. Similar to many other viruses, proteolytic cleavage of VP1 is thought to be critical for virion formation. Unlike half of the genes in SSV1, including the minor capsid protein VP3, the vp1 gene does not tolerate deletion or transposon insertion. In order determine the role of the vp1 gene and its proteolysis for virus function, we developed techniques for site-directed mutagenesis of the SSV1 genome and complemented deletion mutants with vp1 genes from other SSVs. By analyzing these mutants we demonstrate that the N-terminus of the VP1 protein is required, but the N-terminus, or entire SSV1 VP1 protein, can be exchanged with VP1s from other SSVs. However, the conserved glutamate at the cleavage site is not essential. Interestingly, viruses containing point mutations at this position generate mostly abnormal virions.

Download Full-text

Expression and processing of the canine calicivirus capsid precursor

Microbiology ◽

10.1099/0022-1317-81-1-195 ◽

2000 ◽

Vol 81 (1) ◽

pp. 195-199 ◽

Cited By ~ 10

Author(s):

Yuichi Matsuura ◽

Yukinobu Tohya ◽

Mihoko Onuma ◽

Frank Roerink ◽

Masami Mochizuki ◽

...

Keyword(s):

Capsid Protein ◽

Mammalian Cells ◽

Expression System ◽

Immunoblot Analysis ◽

Site Directed Mutagenesis ◽

Feline Calicivirus ◽

Putative Cleavage Site ◽

Mammalian Expression ◽

Capsid Precursor ◽

Infected Cells

The ORF2 product of canine calicivirus (CaCV) was identified and its processing in mammalian cells was analysed. Immunoblot analysis revealed the presence of the 75 kDa capsid precursor in addition to a 57 kDa capsid protein and a 22 kDa N-terminal polypeptide in CaCV-infected cells treated at an elevated temperature. When the CaCV ORF2 was expressed in a transient mammalian expression system, only the 75 kDa precursor was detected in immunoblot analysis, suggesting that no post-translational processing occurred in this system. However, the precursor was processed to a 57 kDa protein and a 22 kDa polypeptide by the proteinase of feline calicivirus (FCV) when this was co-expressed with ORF2. Processing was blocked by site-directed mutagenesis of the putative cleavage site in the capsid precursor. The results indicate that the proteinase of FCV can cleave the capsid precursor of CaCV to produce the mature capsid protein and that CaCV may have a similar proteinase.

Download Full-text