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Journal of Virology, January 2004, p. 539-543, Vol. 78, No. 1
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.1.539-543.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Rapid Genetic Engineering of Human Cytomegalovirus by Using a Lambda Phage Linear Recombination System: Demonstration that pp28 (UL99) Is Essential for Production of Infectious Virus

William J. Britt,1,2,3* Michael Jarvis,4 Jun-Young Seo,2 Derek Drummond,4 and Jay Nelson4,5

Departments of Pediatrics,1 Microbiology,2 Neurobiology, School of Medicine, University of Alabama in Birmingham, Birmingham, Alabama,3 Department of Molecular Microbiology and Immunology,5 Vaccine and Gene Therapy Institute, Oregon Health Sciences Center, Portland, Oregon4

Received 8 July 2003/ Accepted 16 September 2003

A highly efficient lambda phage recombination system previously utilized for studies of bacterial artificial chromosome (BAC)-maintained mouse chromosomal DNA was adapted for the study of the role of human cytomegalovirus (HCMV)-encoded pp28 (UL99) in virus replication. Incorporating a two-step mutagenesis strategy with blue/white selection in Escherichia coli containing a HCMV AD169 BAC, we have shown that we can rapidly introduce point mutations into the HCMV BAC using linear PCR fragments. All manipulations were carried out in bacteria, which greatly accelerated the introduction and analysis of mutations in the viral genome. Our results indicated that HCMV pp28 was essential for the production of infectious virus and that introduction of a single base change that resulted in loss of the myristylation site on pp28 was also associated with the lack of production of infectious virus. Although the block in the viral morphogenesis cannot be determined from these studies, the latter finding suggested that authentic intracellular localization of pp28, not only the expression of the protein, is required for virus assembly.

* Corresponding author. Mailing address: 1600 7th Ave. South, Children's Harbor Building 107, Birmingham, AL 35233-1711. Phone: (205) 996-7762. Fax: (205) 975-6549. E-mail: wbritt{at}peds.uab.edu.

Journal of Virology, January 2004, p. 539-543, Vol. 78, No. 1
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.1.539-543.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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