Viral genome packaging is a complex, non-spontaneous, multi-step enzymatic reaction that in tailed bacteriophages and herpesviruses proceeds via formation of an empty precursor capsid (or procapsid) that is filled with genetic material by the action of two proteins, known as large and small terminase. Though commonly studied as individual subunits, viral terminases bind, pump and cleave viral DNA assembled into large macromolecular complexes of poorly characterized structure, function and composition. The recent discovery of a potent antiviral agent specific to Human Cytomegalovirus small terminase subunit (pUL56) has grown further interest in viral packaging motors. In this grant, combining hybrid methods in structural biology (i.e. X-crystallography, cryo-electron microscopy, hydrogen/deuterium exchange mass spectrometry) with modern biochemical approaches (i.e. conformation- specific synthetic Fabs, site directed mutagenesis, yeast 1-hybrid), we seek to understand the principles governing viral genome packaging through the comparative analysis of motors from different DNA viruses. We are particularly interested in deciphering the atomic structure of macromolecular assemblies formed by terminase subunits during the packaging reaction and the role of S-terminase that is functionally conserved from bacterial viruses to herpesviruses. This research tries to fill a significant and growing knowledge gap between the enzymology of genome packaging, which is increasing well-understood thanks to single molecule biophysical studies, and the molecular machines catalyzing packaging. Building upon the work initiated in the previous funding cycle, we seek to: 1.) Elucidate the architecture of terminase assemblies formed during viral genome packaging; 2.) Determine the conserved architecture of Human Cytomegalovirus small terminase (pUL56) and its interaction with viral DNA and the antiviral drug letermovir.

Public Health Relevance

Viral genome packaging motors are complex molecular machines essential for viral replication and propagation. In this grant, we study the genome packaging motors of Salmonella-phage P22 and human cytomegalovirus (HCMV). Our research will contribute to delineate fundamental mechanisms of viral genome packaging and decipher how chemical inhibition of small terminase in pathogenic herpesviruses can be exploited to develop potent and specific antivirals.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM100888-05A1
Application #
9382346
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Flicker, Paula F
Project Start
2012-08-01
Project End
2021-07-31
Budget Start
2017-08-01
Budget End
2018-07-31
Support Year
5
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Thomas Jefferson University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
053284659
City
Philadelphia
State
PA
Country
United States
Zip Code
19107
Parent, Kristin N; Schrad, Jason R; Cingolani, Gino (2018) Breaking Symmetry in Viral Icosahedral Capsids as Seen through the Lenses of X-ray Crystallography and Cryo-Electron Microscopy. Viruses 10:
Lokareddy, Ravi K; Sankhala, Rajeshwer S; Roy, Ankoor et al. (2017) Portal protein functions akin to a DNA-sensor that couples genome-packaging to icosahedral capsid maturation. Nat Commun 8:14310
Cingolani, Gino; Panella, Andrea; Perrone, Maria Grazia et al. (2017) Structural basis for selective inhibition of Cyclooxygenase-1 (COX-1) by diarylisoxazoles mofezolac and 3-(5-chlorofuran-2-yl)-5-methyl-4-phenylisoxazole (P6). Eur J Med Chem 138:661-668
Motwani, Tina; Lokareddy, Ravi K; Dunbar, Carmen A et al. (2017) A viral scaffolding protein triggers portal ring oligomerization and incorporation during procapsid assembly. Sci Adv 3:e1700423
Wu, Wei; Sankhala, Rajeshwer S; Florio, Tyler J et al. (2017) Synergy of two low-affinity NLSs determines the high avidity of influenza A virus nucleoprotein NP for human importin ? isoforms. Sci Rep 7:11381
Sankhala, Rajeshwer S; Lokareddy, Ravi K; Begum, Salma et al. (2017) Three-dimensional context rather than NLS amino acid sequence determines importin ? subtype specificity for RCC1. Nat Commun 8:979
Bhardwaj, Anshul; Sankhala, Rajeshwer S; Olia, Adam S et al. (2016) Structural Plasticity of the Protein Plug That Traps Newly Packaged Genomes in Podoviridae Virions. J Biol Chem 291:215-26
Sankhala, Rajeshwer S; Lokareddy, Ravi K; Cingolani, Gino (2016) Divergent Evolution of Nuclear Localization Signal Sequences in Herpesvirus Terminase Subunits. J Biol Chem 291:11420-33
Sankhala, Rajeshwer S; Lokareddy, Ravi K; Cingolani, Gino (2015) A Greasy Aid to Capsid Assembly: Lessons from a Salty Virus. Structure 23:1777-1779
Pumroy, Ruth A; Ke, Song; Hart, Darren J et al. (2015) Molecular determinants for nuclear import of influenza A PB2 by importin ? isoforms 3 and 7. Structure 23:374-84

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