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

Wild type or mutant ORF2 proteins were synthesized in vitro in the absence (lanes 1–3) or presence (lanes 5–7) of canine pancreatic membranes. Subsequently, each reaction mix was divided into three parts, treated withtrypsin and Triton-X100 as shown and analyzed on reducing SDS-7.5% polyacrylamide gels. Molecular size markers (lane 4) are, from top to bottom, 200 kDa, 97.4 kDa, 66 kDa, and 46 kDa.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC129057&req=5

Figure 5: Wild type or mutant ORF2 proteins were synthesized in vitro in the absence (lanes 1–3) or presence (lanes 5–7) of canine pancreatic membranes. Subsequently, each reaction mix was divided into three parts, treated withtrypsin and Triton-X100 as shown and analyzed on reducing SDS-7.5% polyacrylamide gels. Molecular size markers (lane 4) are, from top to bottom, 200 kDa, 97.4 kDa, 66 kDa, and 46 kDa.

Mentions: Wild type and mutant ORF2 proteins were synthesized in vitro, in the absence or presence of added microsomal membranes and then subjected to trypsin digestion (Figure 5). The wild type and Δ585–610 proteins, with an intact N-terminus, were found to be protected from trypsin digestion when synthesized in the presence of membranes (lanes 5–7). The Δ2–111 and Δ2–111/Δ585–610 mutant proteins, lacking the N-terminus, were not protected under identical conditions. Further, the trypsin protection was lost when the membrane vesicles were lysed with detergent. These results were in agreement with our earlier findings of a functional membrane-translocating signal sequence at the N-terminus of pORF2. In the absence of membranes, the wild type as well as mutant proteins were susceptible to trypsin, but the polypeptide patterns generated were vastly different (lanes 1–3). The wild type protein showed at least four prominent polypeptidesranging in size from about 40–60 kDa. The Δ2–111 mutant showed two major polypeptides in the 50–60 kDa size range. The Δ585–610 mutant protein, however, was completely sensitive to trypsin, an effect seen with the Δ2–111/Δ585–610 protein as well. These results suggested that the region encompassing amino acids 585–610 also influenced the folding 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)

Wild type or mutant ORF2 proteins were synthesized in vitro in the absence (lanes 1–3) or presence (lanes 5–7) of canine pancreatic membranes. Subsequently, each reaction mix was divided into three parts, treated withtrypsin and Triton-X100 as shown and analyzed on reducing SDS-7.5% polyacrylamide gels. Molecular size markers (lane 4) are, from top to bottom, 200 kDa, 97.4 kDa, 66 kDa, and 46 kDa.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Wild type or mutant ORF2 proteins were synthesized in vitro in the absence (lanes 1–3) or presence (lanes 5–7) of canine pancreatic membranes. Subsequently, each reaction mix was divided into three parts, treated withtrypsin and Triton-X100 as shown and analyzed on reducing SDS-7.5% polyacrylamide gels. Molecular size markers (lane 4) are, from top to bottom, 200 kDa, 97.4 kDa, 66 kDa, and 46 kDa.
Mentions: Wild type and mutant ORF2 proteins were synthesized in vitro, in the absence or presence of added microsomal membranes and then subjected to trypsin digestion (Figure 5). The wild type and Δ585–610 proteins, with an intact N-terminus, were found to be protected from trypsin digestion when synthesized in the presence of membranes (lanes 5–7). The Δ2–111 and Δ2–111/Δ585–610 mutant proteins, lacking the N-terminus, were not protected under identical conditions. Further, the trypsin protection was lost when the membrane vesicles were lysed with detergent. These results were in agreement with our earlier findings of a functional membrane-translocating signal sequence at the N-terminus of pORF2. In the absence of membranes, the wild type as well as mutant proteins were susceptible to trypsin, but the polypeptide patterns generated were vastly different (lanes 1–3). The wild type protein showed at least four prominent polypeptidesranging in size from about 40–60 kDa. The Δ2–111 mutant showed two major polypeptides in the 50–60 kDa size range. The Δ585–610 mutant protein, however, was completely sensitive to trypsin, an effect seen with the Δ2–111/Δ585–610 protein as well. These results suggested that the region encompassing amino acids 585–610 also influenced the folding 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