DNA Microarrays of the Complex Human Cytomegalovirus Genome: Profiling Kinetic Class with Drug Sensitivity of Viral Gene Expression

Abstract

We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the largest member of the herpesvirus family, human cytomegalovirus (HCMV). In this study, an HCMV chip was fabricated and used to characterize the temporal class of viral gene expression. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of oligonucleotides on glass for ORFs in the HCMV genome. Viral gene expression was monitored by hybridization to the oligonucleotide microarrays with fluorescently labelled cDNAs prepared from mock-infected or infected human foreskin fibroblast cells. By using cycloheximide and ganciclovir to block de novo viral protein synthesis and viral DNA replication, respectively, the kinetic classes of array elements were classified. The expression profiles of known ORFs and many previously uncharacterized ORFs provided a temporal map of immediate-early (α), early (β), early-late (γ1), and late (γ2) genes in the entire genome of HCMV. Sequence compositional analysis of the 5′ noncoding DNA sequences of the temporal classes, performed by using algorithms that automatically search for defined and recurring motifs in unaligned sequences, indicated the presence of potential regulatory motifs for β, γ1, and γ2 genes. In summary, these fabricated microarrays of viral DNA allow rapid and parallel analysis of gene expression at the whole viral genome level. The viral chip approach coupled with global biochemical and genetic strategies should greatly speed the functional analysis of established as well as newly discovered large viral genomes.

Molecular analysis of cerebrospinal fluid in viral diseases of the central nervous system

“…Despite high costs and current limited availability of technology and instrumentation, the DNA chip technology is in rapid development in virology, especially in the field of research, e.g. for measuring viral gene expression (Chambers et al, 1999;Jenner et al, 2001). Sequences from plasma or other clinical samples have initially been tested for epidemiological or diagnostic purposes, such as screening for multiple HIV-1 drug resistance mutations (Wilson et al, 2000).…”

A role for arrays in clinical virology: fact or fiction?

“…Microarrays have been used in two other ways in virology, firstly, to study the RNA expression profile of the virus following infection, including drug effects in vitro (Chambers et al, 1999;Jenner et al, 2001) and secondly, to study the RNA expression profile of the host following infection, including drug effects in vitro (Corbeil et al, 2001;Domachowske et al, 2002;Geiss et al, 2000;Iizuka et al, 2002;Zhu et al, 1998). The applications of these two complementary approaches in microbiology have been reviewed by (Cummings and Relman, 2000;Kato-Maeda et al, 2001;Manger and Relman, 2000).…”

“…The applications of these two complementary approaches in microbiology have been reviewed by (Cummings and Relman, 2000;Kato-Maeda et al, 2001;Manger and Relman, 2000). The RNA expression of HCMV in cell culture in the presence or absence of cycloheximide or ganciclovir was analysed with an array of oligonucleotides representing HCMV ORFs made on glass slides (Chambers et al, 1999). Similarly, an array of 288 amplicons representing 88 HHV-8/KSHV ORFs was made on nylon membranes, and used to compare the expression of viral mRNAs during latency and TPA-induced lytic replication (Jenner et al, 2001).…”

“…The interaction of the virus and the host cell (6) Study of the RNA expression profile of virus following infection, including drug effects in vitro (e.g. HCMV (Chambers et al, 1999), KSHV (Jenner et al, 2001)) (7) Study RNA expression profile of host following infection, including drug effects in vitro (e.g. HCMV (Zhu et al, 1998), PVM (Domachowske et al, 2002), HIV-1 (Corbeil et al, 2001;Geiss et al, 2000)) a More useful for bacterial pathogens.…”

Functional genomics in virology and antiviral drug discovery

“…Viral genome microarrays are useful for determining the complex transcriptional program of larger viruses, especially members of the herpesvirus family. For instance, the transcriptional program of the 250 kilobase human cytomegalovirus (HCMV) genome [10] was mapped by Chambers and colleagues using oligonucleotide arrays representing all 226 HCMV open reading frames (ORFs) [11]. Because the function and expression pattern of many of the HCMV genes are still unknown, such classification might help in characterizing unknown ORFs (e.g.…”