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Controlling bacteriophage phi29 DNA-packaging motor by addition or discharge of a peptide at N-terminus of connector protein that interacts with pRNA.

Sun J, Cai Y, Moll WD, Guo P - Nucleic Acids Res. (2006)

Bottom Line: However, the pRNA binding and virion assembly activity were greatly reduced.The DNA-packaging efficiency with dimeric pRNA was more seriously affected by the extension than with monomeric pRNA.These results reveal a potential to turn off and turn on the motor by attaching or removing, respectively, a component to outer part of the motor, and offers an approach for the inhibition of viral replication by using a drug or a small peptide targeted to motor components.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathobiology, Purdue Cancer Center and Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907, USA.

ABSTRACT
Bacteriophage phi29 utilizes a motor to translocate genomic DNA into a preformed procapsid. The motor contains six pRNAs, an enzyme and one 12-subunit connector with a central channel for DNA transportation. A 20-residue peptide containing a His-tag was fused to the N-terminus of the connector protein gp10. This fusion neither interfered with procapsid assembly nor affected the morphology of the prolate-shaped procapsid. However, the pRNA binding and virion assembly activity were greatly reduced. Such decreased functions can be switched back on by the removal of the tag via protease cleavage, supporting the previous finding that the N-terminus of gp10 is essential for the pRNA binding. The DNA-packaging efficiency with dimeric pRNA was more seriously affected by the extension than with monomeric pRNA. It is speculated that the fusion of the tag generated physical hindrance to pRNA binding, with greater influence for the dimers than the monomers due to their size. These results reveal a potential to turn off and turn on the motor by attaching or removing, respectively, a component to outer part of the motor, and offers an approach for the inhibition of viral replication by using a drug or a small peptide targeted to motor components.

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Related in: MedlinePlus

SDS–PAGE (10%) profile of the His-procapsid before and after TEV cleavage.
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fig2: SDS–PAGE (10%) profile of the His-procapsid before and after TEV cleavage.

Mentions: Procapsid proteins gp7, gp8, gp8.5 and His-gp10 were co-expressed in E.coli cells, harboring the plasmid pHis-gp7-8-8.5-10. These proteins self-assembled into His-procapsid. After purification, the His-tagged procapsid was analyzed with SDS–PAGE, revealing that all the procapsid structural components gp7, gp8, gp8.5 and gp10 of the His-procapsid were present with a similar ratio as compared to wild-type phi29 procapsid. The molecular weight of gp7, gp8 and gp8.5 were identical to the corresponding proteins in wild-type procapsid, while His-gp10 showed higher molecular weight than wild-type gp10 due to the 20 additional amino acids (Figure 2). His-procapsid, His-procapsid cleaved by TEV and wild-type procapsid were examined by negative stain electron microscopy. Results showed that there were no significant changes in the structure among them; all showed the typical prolate-shape of phi29 procapsid (Figure 3), suggesting that the His-procapsid possesses the same morphology as procapsid assembled from untagged gp10 (Figure 3A).


Controlling bacteriophage phi29 DNA-packaging motor by addition or discharge of a peptide at N-terminus of connector protein that interacts with pRNA.

Sun J, Cai Y, Moll WD, Guo P - Nucleic Acids Res. (2006)

SDS–PAGE (10%) profile of the His-procapsid before and after TEV cleavage.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636484&req=5

fig2: SDS–PAGE (10%) profile of the His-procapsid before and after TEV cleavage.
Mentions: Procapsid proteins gp7, gp8, gp8.5 and His-gp10 were co-expressed in E.coli cells, harboring the plasmid pHis-gp7-8-8.5-10. These proteins self-assembled into His-procapsid. After purification, the His-tagged procapsid was analyzed with SDS–PAGE, revealing that all the procapsid structural components gp7, gp8, gp8.5 and gp10 of the His-procapsid were present with a similar ratio as compared to wild-type phi29 procapsid. The molecular weight of gp7, gp8 and gp8.5 were identical to the corresponding proteins in wild-type procapsid, while His-gp10 showed higher molecular weight than wild-type gp10 due to the 20 additional amino acids (Figure 2). His-procapsid, His-procapsid cleaved by TEV and wild-type procapsid were examined by negative stain electron microscopy. Results showed that there were no significant changes in the structure among them; all showed the typical prolate-shape of phi29 procapsid (Figure 3), suggesting that the His-procapsid possesses the same morphology as procapsid assembled from untagged gp10 (Figure 3A).

Bottom Line: However, the pRNA binding and virion assembly activity were greatly reduced.The DNA-packaging efficiency with dimeric pRNA was more seriously affected by the extension than with monomeric pRNA.These results reveal a potential to turn off and turn on the motor by attaching or removing, respectively, a component to outer part of the motor, and offers an approach for the inhibition of viral replication by using a drug or a small peptide targeted to motor components.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathobiology, Purdue Cancer Center and Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907, USA.

ABSTRACT
Bacteriophage phi29 utilizes a motor to translocate genomic DNA into a preformed procapsid. The motor contains six pRNAs, an enzyme and one 12-subunit connector with a central channel for DNA transportation. A 20-residue peptide containing a His-tag was fused to the N-terminus of the connector protein gp10. This fusion neither interfered with procapsid assembly nor affected the morphology of the prolate-shaped procapsid. However, the pRNA binding and virion assembly activity were greatly reduced. Such decreased functions can be switched back on by the removal of the tag via protease cleavage, supporting the previous finding that the N-terminus of gp10 is essential for the pRNA binding. The DNA-packaging efficiency with dimeric pRNA was more seriously affected by the extension than with monomeric pRNA. It is speculated that the fusion of the tag generated physical hindrance to pRNA binding, with greater influence for the dimers than the monomers due to their size. These results reveal a potential to turn off and turn on the motor by attaching or removing, respectively, a component to outer part of the motor, and offers an approach for the inhibition of viral replication by using a drug or a small peptide targeted to motor components.

Show MeSH
Related in: MedlinePlus