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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

Schematic presentation of protease TEV cleavage site, V8 cleavage site at the N-terminus of connector in His-procapsid, primary sequences and secondary structure of wild-type phenotype pRNA I-i′. (A) Primary sequences and predicted secondary structure of wild-type phenotype pRNA I-i′. Right and left loops involved in hand-in-hand interaction are boxed and in boldface. Two functional domains are outlined. (B) TEV cleavage site, V8 cleavage site and amino acid sequence at the N-terminal of His-gp10 as shown in a His-procapsid model. (C) Illustration for the formation of a pRNA dimer (A-b′) (B-a′) via interlocking right and left loop. (D) Sequence in the right loop and left loop of individual specific pRNA.
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fig1: Schematic presentation of protease TEV cleavage site, V8 cleavage site at the N-terminus of connector in His-procapsid, primary sequences and secondary structure of wild-type phenotype pRNA I-i′. (A) Primary sequences and predicted secondary structure of wild-type phenotype pRNA I-i′. Right and left loops involved in hand-in-hand interaction are boxed and in boldface. Two functional domains are outlined. (B) TEV cleavage site, V8 cleavage site and amino acid sequence at the N-terminal of His-gp10 as shown in a His-procapsid model. (C) Illustration for the formation of a pRNA dimer (A-b′) (B-a′) via interlocking right and left loop. (D) Sequence in the right loop and left loop of individual specific pRNA.

Mentions: The viral DNA-packaging motor of dsDNA bacteriophage (e.g. phi29, lambda, T4 and T7) can translocate and compress genomic DNA with tremendous velocity into a preformed protein shell, called the procapsid, fueled by hydrolysis of ATP during assembly (1–6). The procapsid of Bacillus subtilis bacteriophage phi29 consists of the major capsid protein gp8 (235 copies), connector protein gp10 (12 copies), head fiber protein gp8.5 (55 copies) and the scaffolding protein gp7 (180 copies) (7–9). Investigation of the bacterial virus phi29 DNA-packaging motor revealed a molecule known as pRNA (packaging RNA) that plays an essential role in packaging DNA into procapsid (10). The pRNA contains two functional domains (Figure 1A) (11,12). One domain, composed of the central region of the pRNA, binds to the procapsid. The other domain, which functions as a DNA translocation domain, is located at the 5′/3′ paired ends. Six copies of pRNA have been hypothesized to form a hexameric ring via monomer to dimer and then hexamer (13–15) to drive the DNA-packaging motor.


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)

Schematic presentation of protease TEV cleavage site, V8 cleavage site at the N-terminus of connector in His-procapsid, primary sequences and secondary structure of wild-type phenotype pRNA I-i′. (A) Primary sequences and predicted secondary structure of wild-type phenotype pRNA I-i′. Right and left loops involved in hand-in-hand interaction are boxed and in boldface. Two functional domains are outlined. (B) TEV cleavage site, V8 cleavage site and amino acid sequence at the N-terminal of His-gp10 as shown in a His-procapsid model. (C) Illustration for the formation of a pRNA dimer (A-b′) (B-a′) via interlocking right and left loop. (D) Sequence in the right loop and left loop of individual specific pRNA.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1636484&req=5

fig1: Schematic presentation of protease TEV cleavage site, V8 cleavage site at the N-terminus of connector in His-procapsid, primary sequences and secondary structure of wild-type phenotype pRNA I-i′. (A) Primary sequences and predicted secondary structure of wild-type phenotype pRNA I-i′. Right and left loops involved in hand-in-hand interaction are boxed and in boldface. Two functional domains are outlined. (B) TEV cleavage site, V8 cleavage site and amino acid sequence at the N-terminal of His-gp10 as shown in a His-procapsid model. (C) Illustration for the formation of a pRNA dimer (A-b′) (B-a′) via interlocking right and left loop. (D) Sequence in the right loop and left loop of individual specific pRNA.
Mentions: The viral DNA-packaging motor of dsDNA bacteriophage (e.g. phi29, lambda, T4 and T7) can translocate and compress genomic DNA with tremendous velocity into a preformed protein shell, called the procapsid, fueled by hydrolysis of ATP during assembly (1–6). The procapsid of Bacillus subtilis bacteriophage phi29 consists of the major capsid protein gp8 (235 copies), connector protein gp10 (12 copies), head fiber protein gp8.5 (55 copies) and the scaffolding protein gp7 (180 copies) (7–9). Investigation of the bacterial virus phi29 DNA-packaging motor revealed a molecule known as pRNA (packaging RNA) that plays an essential role in packaging DNA into procapsid (10). The pRNA contains two functional domains (Figure 1A) (11,12). One domain, composed of the central region of the pRNA, binds to the procapsid. The other domain, which functions as a DNA translocation domain, is located at the 5′/3′ paired ends. Six copies of pRNA have been hypothesized to form a hexameric ring via monomer to dimer and then hexamer (13–15) to drive the DNA-packaging motor.

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