Structure and Function of Essential Nucleoprotein Complexes Along a Viral Genome Packaging Pathway

PROJECT SUMMARY
A key step in the assembly of the large double-stranded DNA (dsDNA) viruses is packaging of a
genome into a pre-assembled procapsid by an ATP-driven motor complex. In the herpesviruses
and many bacteriophages, packaging is catalyzed by a terminase enzyme that utilizes a
concatemeric genome substrate. To accomplish this, terminase enzymes assemble into distinct
initiation, motor and termination complexes to processively excise an individual genome from the
concatemer, and concomitantly package it into the capsid. This requires that the enzymes cycle
between stable nuclease and dynamic motor intermediates. While our understanding of
packaging initiation and motor translocation is extensive, termination of genome packaging
remains ill-studied and poorly characterized in all viruses, primarily because defined experimental
systems have not been developed. Phage  is an exception wherein rigorous biochemical assays
allow molecular dissection of the entire assembly pathway. This multi-PI application proposes
to use phage  to interrogate termination, the final and most poorly characterized step in the
packaging pathway. Two fundamental questions central to genome packaging are addressed; (i)
how does the translocating motor recognize the genome end while also sensing that a sufficient
length of DNA has been packaged, transition to a site-specifically bound nuclease complex, and
(ii) how do “finishing proteins” promote end maturation and terminase ejection from the
nucleocapsid without loss of the tightly packaged DNA. We describe highly integrated and
synergistic biochemical, biophysical, single-molecule and structural approaches to characterize
this conserved and essential, yet largely unstudied step in virus assembly. Given that this process
is strongly conserved in all of the dsDNA viruses, both prokaryotic and eukaryotic, and the
commonality of initiation-translocation-termination pathways in biology, the results will have broad
implications in virology and cell biology.

Grant Number: 3R01GM127365-07S1
NIH Institute/Center: NIH
Principal Investigator: Carlos Catalano