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A C-Terminal Hydrophobic Region is Required for Homo-Oligomerization of the Hepatitis E Virus Capsid (ORF2) Protein.

Xiaofang L, Zafrullah M, Ahmad F, Jameel S - J. Biomed. Biotechnol. (2001)

Bottom Line: When expressed in vitro or in transfected cells, the ORF2 protein assembled as dimers, trimers and higher order forms.While N-terminal deletions upto 111 amino acids had no effect, the deletion of amino acids 585-610 led to reduced homo-oligomerization.This deletion also resulted in aberrant folding of the protein, as determined by its sensitivity to trypsin.This study suggests that a C-terminal hydrophobic region encompassing amino acids 585-610 of the ORF2 protein might be critical for capsid biogenesis.

View Article: PubMed Central - HTML - PubMed

ABSTRACT
Hepatitis E virus (HEV) is the causative agent of hepatitis E, an acute form of viral hepatitis. The open reading frame 2 (ORF2) of HEV encodes the viral capsid protein, which can self-oligomerize into virus-like particles. To understand the domains within this protein important for capsid biogenesis, we have carried out in vitro analyses of association and folding patterns of wild type and mutant ORF2 proteins. When expressed in vitro or in transfected cells, the ORF2 protein assembled as dimers, trimers and higher order forms.While N-terminal deletions upto 111 amino acids had no effect, the deletion of amino acids 585-610 led to reduced homo-oligomerization. This deletion also resulted in aberrant folding of the protein, as determined by its sensitivity to trypsin. This study suggests that a C-terminal hydrophobic region encompassing amino acids 585-610 of the ORF2 protein might be critical for capsid biogenesis.

No MeSH data available.


Related in: MedlinePlus

COS-1 cells transfected with wild type or Δ2–111ORF2 expression plasmids were lysed 48 hours post-transfection, the lysates cross-linked with glutaraldehyde and separated on a sucrose gradient as described in materials and methods. The gradient fractions were immunoprecipitated with anti-pORF2 antibodies and analyzed on nonreducing 3.5% polyacrylamide gels.
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Figure 2: COS-1 cells transfected with wild type or Δ2–111ORF2 expression plasmids were lysed 48 hours post-transfection, the lysates cross-linked with glutaraldehyde and separated on a sucrose gradient as described in materials and methods. The gradient fractions were immunoprecipitated with anti-pORF2 antibodies and analyzed on nonreducing 3.5% polyacrylamide gels.

Mentions: To determine if pORF2 can oligomerize, COS-1 cells were transiently transfected with vectors expressing wild type ORF2 or the N-terminal deletion mutant ORF2[Δ2–111]. After cross-linking and sucrose gradient sedimentation, the distribution of ORF2 proteins across the gradient was determined by immunoprecipitation of each fraction. Multiple species ranging from monomers to trimers were evident (Figure 2), showing that pORF2 has the ability to homo-oligomerize. For wild type pORF2, very little monomeric and some dimeric forms were evident; most of the protein was seen as trimers or higher oligomers. On deletion of the N-terminal 111 amino acids, the dimeric forms were significantly reduced. However, the trimers and higher oligomers were still observed towards the bottom of the gradient. The ORF2 protein oligomers were also analyzed after a 3 h chase following the 30 min labeling period. Though the signals were weaker, there was no change in the oligomer patterns following the chase period. This suggested that pORF2 turnover in cells was rapid and that oligomer formation took place either cotranslationally or very soon following translation. These results also indicated that up to 111 amino acids at the N-terminus were dispensable for the homo-oligomerization of pORF2.


A C-Terminal Hydrophobic Region is Required for Homo-Oligomerization of the Hepatitis E Virus Capsid (ORF2) Protein.

Xiaofang L, Zafrullah M, Ahmad F, Jameel S - J. Biomed. Biotechnol. (2001)

COS-1 cells transfected with wild type or Δ2–111ORF2 expression plasmids were lysed 48 hours post-transfection, the lysates cross-linked with glutaraldehyde and separated on a sucrose gradient as described in materials and methods. The gradient fractions were immunoprecipitated with anti-pORF2 antibodies and analyzed on nonreducing 3.5% polyacrylamide gels.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC129057&req=5

Figure 2: COS-1 cells transfected with wild type or Δ2–111ORF2 expression plasmids were lysed 48 hours post-transfection, the lysates cross-linked with glutaraldehyde and separated on a sucrose gradient as described in materials and methods. The gradient fractions were immunoprecipitated with anti-pORF2 antibodies and analyzed on nonreducing 3.5% polyacrylamide gels.
Mentions: To determine if pORF2 can oligomerize, COS-1 cells were transiently transfected with vectors expressing wild type ORF2 or the N-terminal deletion mutant ORF2[Δ2–111]. After cross-linking and sucrose gradient sedimentation, the distribution of ORF2 proteins across the gradient was determined by immunoprecipitation of each fraction. Multiple species ranging from monomers to trimers were evident (Figure 2), showing that pORF2 has the ability to homo-oligomerize. For wild type pORF2, very little monomeric and some dimeric forms were evident; most of the protein was seen as trimers or higher oligomers. On deletion of the N-terminal 111 amino acids, the dimeric forms were significantly reduced. However, the trimers and higher oligomers were still observed towards the bottom of the gradient. The ORF2 protein oligomers were also analyzed after a 3 h chase following the 30 min labeling period. Though the signals were weaker, there was no change in the oligomer patterns following the chase period. This suggested that pORF2 turnover in cells was rapid and that oligomer formation took place either cotranslationally or very soon following translation. These results also indicated that up to 111 amino acids at the N-terminus were dispensable for the homo-oligomerization of pORF2.

Bottom Line: When expressed in vitro or in transfected cells, the ORF2 protein assembled as dimers, trimers and higher order forms.While N-terminal deletions upto 111 amino acids had no effect, the deletion of amino acids 585-610 led to reduced homo-oligomerization.This deletion also resulted in aberrant folding of the protein, as determined by its sensitivity to trypsin.This study suggests that a C-terminal hydrophobic region encompassing amino acids 585-610 of the ORF2 protein might be critical for capsid biogenesis.

View Article: PubMed Central - HTML - PubMed

ABSTRACT
Hepatitis E virus (HEV) is the causative agent of hepatitis E, an acute form of viral hepatitis. The open reading frame 2 (ORF2) of HEV encodes the viral capsid protein, which can self-oligomerize into virus-like particles. To understand the domains within this protein important for capsid biogenesis, we have carried out in vitro analyses of association and folding patterns of wild type and mutant ORF2 proteins. When expressed in vitro or in transfected cells, the ORF2 protein assembled as dimers, trimers and higher order forms.While N-terminal deletions upto 111 amino acids had no effect, the deletion of amino acids 585-610 led to reduced homo-oligomerization. This deletion also resulted in aberrant folding of the protein, as determined by its sensitivity to trypsin. This study suggests that a C-terminal hydrophobic region encompassing amino acids 585-610 of the ORF2 protein might be critical for capsid biogenesis.

No MeSH data available.


Related in: MedlinePlus