Limits...
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

In vitro synthesized pORF2 and mutant proteins were subjected to immunoprecipitation before (A) or after (B) cross-linking. The mixtures were analyzed on a nonreducing 7.5% polyacrylamide gel. Molecular size markers are shown inkilodaltons (MW).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC129057&req=5

Figure 3: In vitro synthesized pORF2 and mutant proteins were subjected to immunoprecipitation before (A) or after (B) cross-linking. The mixtures were analyzed on a nonreducing 7.5% polyacrylamide gel. Molecular size markers are shown inkilodaltons (MW).

Mentions: To determine if the in vitro expressed pORF2 can also oligomerize, the proteins were immunoprecipitated before or after cross-linking and analyzed on a nonreducing 3.5% gel (Figure 3). About 50% of the wild type ORF2 protein was found as dimers, with a small fraction as higher oligomers as well. The deletion of 111 N-terminal amino acids again showed no effect on this distribution. However, the deletion of amino acids 585–610 resulted in a drastic loss of pORF2 dimers. This effect was even more pronounced in the Δ2–111/Δ585–610 double mutant. The Δ2–34 and 137/310/562 mutants showed wild type patterns. To further evaluate the complexes formed, sucrose gradient sedimentation of in vitro expressed wild type and mutant ORF2 proteins was carried out (Figure 4). Following immunoprecipitation and nonreducing SDS-PAGE, two predominant forms, corresponding to monomers and dimers, were again observed. As expected, the proportion of dimers was higher in fractions towards the bottom of the gradient. When compared to the wild type protein, the Δ2–111 mutant being significantly smaller, sedimented to a lower density in the gradient. In agreement with earlier results, while the deletion of 111 N-terminal residues showed no effect on the monomer-to-dimer ratio, the deletion of amino acids 585–610 resulted in loss of the dimeric species. Taken together, these results suggested that residues 585 to 610 were required for pORF2 oligomerization.


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)

In vitro synthesized pORF2 and mutant proteins were subjected to immunoprecipitation before (A) or after (B) cross-linking. The mixtures were analyzed on a nonreducing 7.5% polyacrylamide gel. Molecular size markers are shown inkilodaltons (MW).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: In vitro synthesized pORF2 and mutant proteins were subjected to immunoprecipitation before (A) or after (B) cross-linking. The mixtures were analyzed on a nonreducing 7.5% polyacrylamide gel. Molecular size markers are shown inkilodaltons (MW).
Mentions: To determine if the in vitro expressed pORF2 can also oligomerize, the proteins were immunoprecipitated before or after cross-linking and analyzed on a nonreducing 3.5% gel (Figure 3). About 50% of the wild type ORF2 protein was found as dimers, with a small fraction as higher oligomers as well. The deletion of 111 N-terminal amino acids again showed no effect on this distribution. However, the deletion of amino acids 585–610 resulted in a drastic loss of pORF2 dimers. This effect was even more pronounced in the Δ2–111/Δ585–610 double mutant. The Δ2–34 and 137/310/562 mutants showed wild type patterns. To further evaluate the complexes formed, sucrose gradient sedimentation of in vitro expressed wild type and mutant ORF2 proteins was carried out (Figure 4). Following immunoprecipitation and nonreducing SDS-PAGE, two predominant forms, corresponding to monomers and dimers, were again observed. As expected, the proportion of dimers was higher in fractions towards the bottom of the gradient. When compared to the wild type protein, the Δ2–111 mutant being significantly smaller, sedimented to a lower density in the gradient. In agreement with earlier results, while the deletion of 111 N-terminal residues showed no effect on the monomer-to-dimer ratio, the deletion of amino acids 585–610 resulted in loss of the dimeric species. Taken together, these results suggested that residues 585 to 610 were required for pORF2 oligomerization.

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