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Herpes simplex virus Research

Microarray Analysis of Cellular Transcript Abundance
as a Function of HSV Infection

HSV infection leads to profound alterations of cellular metabolism including a general inhibition of cellular RNA transcription. Further, the virus encodes specific functions such as the virion-associated shut-off protein (vhs) that degrades mRNA. Despite this, the level of a number of cellular transcripts are, at least transitorily, increased following infection--indicating specific activation by viral infection. Many of these are cellular "defense" systems.

The oligonucleotide-based microarray used for HSV genes contains a few diagnostic cellular genes as well, a number of these are representative of defense pathways, such as infereron and MHC-mediated antigen presentation.  An example of the induction of a sub-set of such defense genes can be seen below where the normalized levels of several cellular transcripts seen in HeLa cells at 6 hr after infection with different multiplicities of HSV is shown.

levels of cellular transcripts

Of course, a full catalogue of the cellular transcripts both up- and down-regulated by HSV infection can only be obtained by analysis of transcript abundance using more extensive cellular chips. As a start towards such studies, we have utilized chips printed by the GTI with 5000 cDNAs made by PCR amplification of a library constructed by workers at the NIH using mouse embryonic tissue as a source of mRNAs.

As one test of technique, we have compared the hybridization of colloidal gold and silver-tagged cDNA generated by random-priming of poly(A)-containing RNA isolated from mouse neural bulb cells infected with either 1 PFU or 5 PFU of HSV-1 or HSV-2.  Examples of RLS detection of cDNA generated from 200 ng samples of poly(A)-containing RNA from cells infected with 1 PFU/cell of either HSV-1 or HSV-2 and hybridized to the 5,000 gene chips as compared to mock-infected samples are shown in false color below.  In these views, the gold channel is shown in red and the silver-scattering channel in green.

microarray photo
click image for a larger view

Scatter analysis of transcript abundances which differ significantly (p<0.05) in HSV-1 and HSV-2 infected cells at both multiplicities is shown below:

scatter analysis of transcipt abundances

These measures of transcript abundance demonstrate the more efficient shut-off of all infected cell RNA induced by HSV-2 as compared to HSV-1, and the induction of a small number of cellular transcripts at the lower multiplicity of infection. The identity of these cellular transcripts is generally different following infections with the two closely related viruses, but at least two identical ones of unknown function are activated under both conditions. Clearly, much further analysis is required, but the sensitivity of the method and its potential value in understanding differences in the patterns of HSV-1 and HSV-2 pathogenesis are clear.

Future Studies

The application of DNA-microarray analysis of HSV gene expression to the study of the regulation of viral gene expression in productive and latent infection, and its role in pathogenesis is an exciting approach towards the study of the interaction between viral and cellular genes that defines viral pathogenesis.  In conjunction with the construction of carefully defined viral mutants, and the application of increasingly sophisticated differentiated cell cultures and animal models, we will generate an increasingly realistic view of the parameters of viral infection and the response of the host to it.