Array analysis of viral gene transcription during lytic infection of cells in tissue culture with Varicella-Zoster virus.
Varicella-zoster virus (VZV), a neurotropic alphaherpesvirus, causes childhood chickenpox (varicella), becomes latent in dorsal root and autonomic ganglia, and reactivates decades later to cause shingles (zoster) and other neurologic complications. Although the sequence and configuration of VZV DNA have been determined, relatively little is known about viral gene expression in productively infected cells. This is in part because VZV is highly cell associated, and sufficient titers of cell-free virus for use in synchronizing infection do not develop. PCR-based transcriptional arrays were constructed to simultaneously determine the relative abundance of the approximately 70 predicted VZV open reading frames (ORFs). Fragments (250 to 600 bp) from the 5' and 3' end of each ORF were PCR amplified and inserted into plasmid vectors. The virus DNA inserts were amplified, quantitated, and spotted onto nylon membranes. Probing the arrays with radiolabeled cDNA synthesized from VZV-infected cells revealed an increase in the magnitude of the expressed VZV genes from days 1 to 3 after low-multiplicity virus infection but little change in their relative abundance. The most abundant VZV transcripts mapped to ORFs 9/9A, 64, 33/33A, and 49, of which only ORF 9 corresponded to a previously identified structural gene. Array analysis also mapped transcripts to three large intergenic regions previously thought to be transcriptionally silent, results subsequently confirmed by Northern blot and reverse transcription-PCR analysis. Array analysis provides a formidable tool to analyze transcription of an important ubiquitous human pathogen.
Cohrs
Randall J
RJ
Department of Neurology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA. randall.cohrs@uchsc.edu
Hurley
Michael P
MP
Gilden
Donald H
DH
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D021901:DNA, Intergenic / Q000032:analysis; D015967:Gene Expression Regulation, Viral; D014645:Herpesvirus 3, Human / Q000254:growth & development / Q000472:pathogenicity*; D006801:Humans; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014775:Virus Activation; D017735:Virus Latency
D021901:DNA, Intergenic; D014764:Viral Proteins