Herpes simplex virus Research
Microarray Analysis of Cellular Transcript
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
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"
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.
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.
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
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.
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.