Studies of the mechanism of origin activation in Herpes Viruses

HSV-1. Herpes Simplex type-1 virus (HSV-1) provides an excellent system for study of the early replication steps in a mammalian system. HSV-1 genome encodes seven proteins required for origin-dependent DNA replication consisting of a DNA polymerase and its accessory protein, a heterotrimeric helicase-primase, a single-strand (ss) DNA-binding protein (ICP8) and an origin-binding protein, UL9 protein. HSV-1 contains three functional origins of DNA replication. One, oriL, is present in the long unique segment of the genome while the other highly homologous origin, oriS, is present twice in the repeat region flanking the short unique segment. The minimal functional oriS sequence (79 bp) consists of a 45 bp palindrome containing a central AT-rich element flanked on each side by two high affinity UL9 protein-binding sites designated box I and box II.  We have now isolated all of the HSV-1 proteins which catalyze Herpes replication and these proteins are in hand in sufficient amounts to carry out studies similar to what we have been able to do in the T4 and T7 systems. These studies will for the first time show whether or not there is a trombone looping mechanism in eukaryotes. Further it will help define the role of each of these proteins in replication, information essential to the design of new anti-viral drugs.

When cells are infected by HSV-1, the viral DNA is replicated in condensed bodies in the cell nuclei (replication bodies or ND10 bodies). These bodies contain not only all of the replication factors, but also many of the host, human DNA repair proteins.  We will purify these bodies from HSV-1 infected human cells (Vero), on sucrose gradients. Using EM it will be possible to investigate their 3D structure and composition (using gold tagged antibodies). This will provide a critically needed view into the architecture and mechanism of DNA replication factories in a human cell.

We have spend considerable effort over the years to solve the structure of the filaments formed by the HSV-1 recombinase/SSB protein called ICP8. The study with the Egelman laboratory (see recent papers) provides the most detailed structure yet, and work to be published shortly will refine these structures.  In the classic thinking, “proteins come to the DNA, act and leave”.  More and more we suspect that in the dense replication bodies in HSV-1 infected cells, pre-formed filaments of ICP8 protein exist as protein scaffolds, waiting for “DNA to come to the protein and then leave”. In this model the DNA would meld into these protein scaffolds, undergo annealing and recombination events and then be released. This new view will be tested by a series of both in vitro and in vivo studies. If true, it would greatly reshape our thinking of replication.

KSHV. Kaposi’s sarcoma herpes virus (HHV8) is a newly discovered herpes virus linked to several human cancers. There is a very active effort among several groups at the Lineberger Comprehensive Cancer Center to investigate the replication and life cycle of this virus. Our group has recently isolated the KSHV SSB/recombinase (similar to ICP8) and has shown that it can form protein filaments in solution (Ozgur, Damania, and Griffith, 2011). We are currently further characterizing this protein by biochemical approaches and will be carrying out mutant studies as well as studies of how the origin binding protein of KSHV binds and remodels the KSHV origin. Future work will entail the cloning and characterization of each of the KSHV replication proteins.

Selected References:

  • Alexander Makhov, Paul Boehmer, I. Robert Lehman and Jack Griffith. The Herpes Simplex Virus type I UL9 protein carries out origin specific DNA unwinding and forms stem/loop structures. EMBO J. 15: 1742-1750, l996.
  • Alexander Makhov, Paul Boehmer, I. Robert Lehman and Jack Griffith. Visualization of the Unwinding of Long DNA Chains by the Herpes Simplex Virus type-I UL9 and ICP8. J. Mol. Biol. 258: 789- 799, 1996.
  • Nina B. Reuven, Smaranda Willcox, Jack D. Griffith, and Sandra K. Weller. Catalysis of strand exchange by the HSV-1 UL12 and ICP8 proteins: potent ICP8 recombinase activity is revealed upon resection of dsDNA substrate by nucleases. J Mol Biol. 342: 57-71. 2004
  • Alexander Makhov and Jack Griffith. Visualization of the Annealing of Complementary Single-stranded DNA Catalyzed by the Herpes Simplex Virus Type 1 ICP8 SSB/Recombinase. J Mol Biol. 355:911-22. 2006
  • Makhov AM, Sen A, Yu X, Simon MN, Griffith JD, Egelman EH. The bipolar filaments formed by herpes simplex virus type 1 SSB/recombination protein (ICP8) suggest a mechanism for DNA annealing. J Mol Biol. 2009  386(2):273-9 PMID: 9138689
  • Manolaridis I, Mumtsidu E, Konarev P, Makhov AM, Fullerton SW, Sinz A, Kalkhof S, McGeehan JE, Cary PD, Griffith JD, Svergun D, Kneale GG, Tucker PA. Structural and biophysical characterization of the proteins interacting with the herpes simplex virus 1 origin of replication. J Biol Chem. 2009, 284(24):16343-53.  PMID: 19329432
  • Ozgur S, Damania B, Griffith J. The Kaposi’s sarcoma-associated herpesvirus ORF6 DNA binding protein forms long DNA-free helical protein filaments.  J Struct Biol. 2011 174(1):37-43.  PMID:  21047556