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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

Comparison of DNA-packaging activity with pRNA dimer and monomer using His-procapsid. (A) Native gel assay (8%) for the efficiency in dimer formation and (B) agarose gel assay for DNA-packaging efficiency.
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fig6: Comparison of DNA-packaging activity with pRNA dimer and monomer using His-procapsid. (A) Native gel assay (8%) for the efficiency in dimer formation and (B) agarose gel assay for DNA-packaging efficiency.

Mentions: An in vitro DNA-packaging system was used to test whether the His-procapsid is functional in DNA translocation. The results showed that the efficiency of DNA-packaging into His-procapsid decreased only 2- to 5-fold when using pRNA A-a′, Sph I A-a′, Sph I E-e′ or Sph I A-b′+Sph I B-a′ in the packaging assay (Figures 5 and 6B). Viral assembly activity of His-procapsid was assayed by plaque formation analyzed with the highly sensitive phi29 assembly system. It was found that phi29 assembly activity was reduced more than 10-fold in the presence of an additional 20 amino acids at the N-terminus of the connector (Figure 7).


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)

Comparison of DNA-packaging activity with pRNA dimer and monomer using His-procapsid. (A) Native gel assay (8%) for the efficiency in dimer formation and (B) agarose gel assay for DNA-packaging efficiency.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Comparison of DNA-packaging activity with pRNA dimer and monomer using His-procapsid. (A) Native gel assay (8%) for the efficiency in dimer formation and (B) agarose gel assay for DNA-packaging efficiency.
Mentions: An in vitro DNA-packaging system was used to test whether the His-procapsid is functional in DNA translocation. The results showed that the efficiency of DNA-packaging into His-procapsid decreased only 2- to 5-fold when using pRNA A-a′, Sph I A-a′, Sph I E-e′ or Sph I A-b′+Sph I B-a′ in the packaging assay (Figures 5 and 6B). Viral assembly activity of His-procapsid was assayed by plaque formation analyzed with the highly sensitive phi29 assembly system. It was found that phi29 assembly activity was reduced more than 10-fold in the presence of an additional 20 amino acids at the N-terminus of the connector (Figure 7).

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