The Herpesviridae family of viruses includes several significant human pathogens, including herpes simplex virus, varicella-zoster virus, Epstein-Barr virus, Kaposi's sarcoma associated herpesvirus, and human cytomegalovirus (HCMV). DNA replication in these viruses produces large concatemeric intermediates, from which unit genomes are cleaved at specific sequences and packaged into viral particles. The halogenated benzimidazole ribonucleosides are a new class of antiviral drugs which block this process. Unfortunately, our understanding of herpesvirus cleavage and packing is scant and the mechanism of action of these drugs remains unknown. Using murine cytomegalovirus (MCMV), we have begun to define the cis elements that are recognized at the cleavage site. Using guinea pig cytomegalovirus (GPCMV), we have demonstrated that the mechanism of cleavage duplicates sequences at the viral termini. Recent findings in GPCMV and HCMV suggest that the benzimidazoles induce a premature cleavage to produce truncated genomes.
In aim 1 of this proposal, we will more accurately define the cis cleavage/packaging signals of MCMV and initiate efforts to analyze cis elements in HCMV.
In aim 2, experiments to elucidate the mechanisms of cleavage and packaging will define the phenotypes of cis mutations in MCMV with respect to formation of intracellular replicative DNAs and extracellular viral genomes. The benzimidazole-induced accumulation of truncated genomes will be investigated by further analysis of the novel ends formed, characterization of capsid composition and structure, and by electron microscopy. A model proposed for the mechanism of repeat duplication will be tested both by analysis of replicative intermediates and by construction of genetic mutations in cleavage/packaging cis elements.
In aim 3, viral cleavage/packaging proteins will be characterized both in vitro and in vivo using recombinant expression and their functions studied using either in vitro or permeabilized cell cleavage assays. As cleavage and packaging are highly conserved among herpesviruses, this information should be directly applicable to pathogenic human herpesviruses and may reveal the mechanism of action of the benzimidazoles or facilitate the discovery of novel compounds that target the cleavage and packaging processes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI046668-04
Application #
6726113
Study Section
Experimental Virology Study Section (EVR)
Program Officer
Beisel, Christopher E
Project Start
2001-05-15
Project End
2006-03-31
Budget Start
2004-04-01
Budget End
2005-03-31
Support Year
4
Fiscal Year
2004
Total Cost
$250,600
Indirect Cost
Name
Virginia Commonwealth University
Department
Pediatrics
Type
Schools of Medicine
DUNS #
105300446
City
Richmond
State
VA
Country
United States
Zip Code
23298
Ourahmane, Amine; Sauer, Anne; Nixon, Daniel E et al. (2018) A Guinea pig cytomegalovirus resistant to the DNA maturation inhibitor BDCRB. Antiviral Res 154:44-50
Wang, Jian Ben; Zhu, Yali; McVoy, Michael A et al. (2012) Changes in subcellular localization reveal interactions between human cytomegalovirus terminase subunits. Virol J 9:315
Wang, Jian Ben; McVoy, Michael A (2011) A 128-base-pair sequence containing the pac1 and a presumed cryptic pac2 sequence includes cis elements sufficient to mediate efficient genome maturation of human cytomegalovirus. J Virol 85:4432-9
Sauer, Anne; Wang, Jian Ben; Hahn, Gabriele et al. (2010) A human cytomegalovirus deleted of internal repeats replicates with near wild type efficiency but fails to undergo genome isomerization. Virology 401:90-5
Cui, Xiaohong; McGregor, Alistair; Schleiss, Mark R et al. (2008) Cloning the complete guinea pig cytomegalovirus genome as an infectious bacterial artificial chromosome with excisable origin of replication. J Virol Methods 149:231-9
Wang, Jian Ben; Nixon, Daniel E; McVoy, Michael A (2008) Definition of the minimal cis-acting sequences necessary for genome maturation of the herpesvirus murine cytomegalovirus. J Virol 82:2394-404
Schleiss, Mark R; McGregor, Alistair; Choi, K Yeon et al. (2008) Analysis of the nucleotide sequence of the guinea pig cytomegalovirus (GPCMV) genome. Virol J 5:139
McVoy, Michael A; Nixon, Daniel E (2005) Impact of 2-bromo-5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole riboside and inhibitors of DNA, RNA, and protein synthesis on human cytomegalovirus genome maturation. J Virol 79:11115-27
Nixon, Daniel E; McVoy, Michael A (2004) Dramatic effects of 2-bromo-5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole riboside on the genome structure, packaging, and egress of guinea pig cytomegalovirus. J Virol 78:1623-35
Hahn, Gabriele; Jarosch, Margit; Wang, Jian Ben et al. (2003) Tn7-mediated introduction of DNA sequences into bacmid-cloned cytomegalovirus genomes for rapid recombinant virus construction. J Virol Methods 107:185-94

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