Single-molecule packaging initiation in real time by a viral DNA packaging machine from bacteriophage T4

Proc Natl Acad Sci U S A. 2014 Oct 21;111(42):15096-101. doi: 10.1073/pnas.1407235111. Epub 2014 Oct 6.


Viral DNA packaging motors are among the most powerful molecular motors known. A variety of structural, biochemical, and single-molecule biophysical approaches have been used to understand their mechanochemistry. However, packaging initiation has been difficult to analyze because of its transient and highly dynamic nature. Here, we developed a single-molecule fluorescence assay that allowed visualization of packaging initiation and reinitiation in real time and quantification of motor assembly and initiation kinetics. We observed that a single bacteriophage T4 packaging machine can package multiple DNA molecules in bursts of activity separated by long pauses, suggesting that it switches between active and quiescent states. Multiple initiation pathways were discovered including, unexpectedly, direct DNA binding to the capsid portal followed by recruitment of motor subunits. Rapid succession of ATP hydrolysis was essential for efficient initiation. These observations have implications for the evolution of icosahedral viruses and regulation of virus assembly.

Keywords: molecular motors; single-molecule fluorescence imaging; virus packaging.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adenosine Triphosphatases / chemistry
  • Adenosine Triphosphate / chemistry
  • Bacteriophage T4 / physiology*
  • Capsid / chemistry
  • Capsid Proteins / chemistry
  • DNA Packaging
  • DNA, Viral / chemistry*
  • Genome, Viral
  • Microscopy, Fluorescence
  • Molecular Motor Proteins / chemistry
  • Photobleaching
  • Protein Conformation
  • Virus Assembly*


  • Capsid Proteins
  • DNA, Viral
  • Molecular Motor Proteins
  • Adenosine Triphosphate
  • Adenosine Triphosphatases