We have applied sequence-based computer programs and our own databases to select 75 nt oligonucleotide sequences within the HSV-1 genome that are specific for the expression of individual viral transcripts. Better than half of the total viral transcripts can be uniquely specified with sets of 2--3 short sequences. We have arrayed these probes on chemically activated glass slides to generate a """"""""first generation"""""""" HSV-1 DNA microarray (HSV-chip). We have used nick-translated viral DNA and cloned DNA fragments to optimize hybridization conditions and demonstrate the high specificity of this first generation chip. We have also used oligo-dT primed cDNA labeled with Cy3 and Cy5-luorescent nucleoside derivatives generated from RNA isolated from cells infected with HSV under various conditions, which influence the class of genes expressed. Inhibition of de novo protein synthesis allows only expression of immediate-early genes, blockage of DNA replication inhibits expression of strict late genes, isolation of RNA at short times after infection results in high enrichment of early phase transcripts, etc. Our preliminary results confirm that all classes of viral transcripts can be detected with good efficiency and very high specificity. We will now: 1. Optimize an HSV-1 DNA microarray by designing additional viral and cellular probes and labeling regimens to increase specificity and especially the discrimination between overlapping viral genes. 2. Apply the chips to the in-depth analysis of the influence of specific viral genes, cell types, and state of cell differentiation on the global program of HSV gene expression. These experiments will be carried out using well- defined recombinant viruses.3. Assay global patterns of viral gene expression during productive and reactivating infection in the mouse. Here we will take advantage of the well documented roles of specific viral genes in aspects of viral pathogenesis and latency. 4. Apply these methods towards design of DNA microarrays specific for other members of the human alpha herpesvirus group.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA090287-02
Application #
6514937
Study Section
Experimental Virology Study Section (EVR)
Program Officer
Wong, May
Project Start
2001-06-01
Project End
2005-05-31
Budget Start
2002-06-01
Budget End
2003-05-31
Support Year
2
Fiscal Year
2002
Total Cost
$309,639
Indirect Cost
Name
University of California Irvine
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
161202122
City
Irvine
State
CA
Country
United States
Zip Code
92697
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