The Homepage of Dr. Edward K. Wagner 
border corner

Herpes simplex virus Research

The Use of DNA Microarrays
to Analyze Gene Expression in HSV infected Cells

The analysis of cellular gene expression in response to various stimuli has been given tremendous impetus with the recent development of DNA microarrays.  Applied to the study of large viruses, this technology empowers us with the ability to carry out rapid, global studies of viral gene expression and cellular responses to infection under varying conditions of infection.

We have established a long-term collaborative program with Professor Peter Ghazal, Director of the Scottish Genome Centre at the University of Edinburgh to design and construct oligonucleotide-based DNA microarrays in order to study global changes in viral and cellular transcript abundance following infections with neurotropic herpesviruses, especially HSV-1. Our current approach is outlined below.

HSV Probe Map
Click image to view enlargement

The chip is made up of triplicate spots 75-mer oligonucleotides specific probes for each HSV-1 transcription unit. Because of the fact that a number of HSV transcript partially overlap each other, sharing 3'-polyadenylation sites, not all transcripts can be uniquely detected, but siting probes in the unique 5'-regions of the distal member of overlapping transcript groups, and the fact that a number of overlapping sets share a common kinetic class has allowed the resolution of 50 individual transcripts out of the 70-odd transcripts expressed with appreciable abundance during productive infection.

Application of DNA microarray analysis to the study of HSV transcript abundance.

The replication of HSV in infected cells proceeds through several phases or "waves" of expression of transcripts encoding various viral functions. This is readily seen by analyzing patterns of viral transcript abundance in RNA isolated at various times following infection of cultured cells, such as human fibroblasts (HFF cells). For such an experiment, poly(A)-containing RNA is isolated, and used to prime cDNA synthesis using dye-substituted nucleotides as outlined in the animated illustration of the microarray technique.

Each probe is spotted in triplicate on the chips, and the median value of the three separate hybridization values, less the background seen from the median values of a number of "blank" (i.e., SSC only) spots is determined, along with the standard error (SD) of the values.

rna time graph

The hybridization values for each probe group can best be shown as a table (see some recent publications), or the values for the resolved transcripts can be plotted with a bar graph (as shown at left) where the viral transcripts are grouped according to kinetic class.

Patterns of expression 30, 90, 150, 210, 330, and 510 min after adding virus show the increasing complexity of transcript abundance, and the fact that first immediate-early, then early, and finally late transcripts increase markedly in abundance.

View a time lapse animation of the time-dependant changes in HSV-1 transcript abundance during productive infection.
Click image for a larger view

The immediate-early and early stages of infection can be approximated with appropriate drug treatments. Thus, inhibition of de novo protein synthesis with cycloheximide leads to the continued expression of immediate-early transcripts as illustrated below. It is important to note, however, that the lack of viral protein synthesis under these conditions leads to expression of much higher levels of immediate-early transcripts than normally seen at the earliest times of a productive infection because the down-regulation of immediate-early transcription by ICP4 protein does not occur.

transcript abundance
click image for a larger view

Similarly, blockage of viral DNA replication with a drug such as Acyclovir (ACV) or phosphonoacetic acid (PAA) will lead to a situation where only immediate-early and early transcripts are expressed with normal abundance. These drugs do not fully inhibit the expression of the leaky-late transcripts. This is particularly evident when compared with the transcript abundance patterns seen during infection with a virus mutant that does not express viral DNA polymerase, and, thus, is completely blocked in DNA replication.

acyclovir blockage
click for a larger view

One very convenient way to compare changes in transcript abundance under varying conditions of infection, infection with metabolic inhibitors or between normal infection and that with a defined viral mutant is to carry out "scatter analysis". Here, chips are hybridized with cDNA made to RNA isolated under the conditions to be compared, and the normalized median values are plotted against each other in an X-Y scatter plot using excel or other graphical program. Those transcripts expressed at the same level under both sets of conditions will lie on or around a straight line, while those whose levels of abundance are altered under one condition or the other will fall off that line.

scatter analysis

An example of such analysis is shown above for the transcript abundances seen in cells infected with the DNA polymerase mutant as compared to treatment of cells infected with wt virus in the presence of ACV--note that the data are color coded as in the bar graphs, and that several values demonstrate significant deviation from a linear correlation.

scatter diagramSuch analysis is useful in studying the effects of different metabolic inhibitors. For example 4% DMSO and PAA both lead to profound inhibition of virus replication, and DMSO inhibits viral DNA replication, it is clear that transcript abundance is markedly different under the two conditions of infection. This leads naturally to the conclusion that the DMSO-based inhibition of viral replication is not solely the result of inhibition of viral DNA replication.  In fact, at least three specific viral processes necessary for normal levels of viral replication are inhibited with this compound.

Application of Resonance Light Scattering (RLS)
for the Detection of Hybrids

While the approaches outlined above are useful for the study of viral gene expression in cultured cells, it is clear that significant increases in sensitivity will enhance the methods applicability for the study of viral gene expression in animal models. Towards this end, higher sensitivity detection of hybridized cDNA can be accomplished by using colloidal gold and silver labeling of cDNA and resonance light scattering (RLS) measurements. The Invitrogen Corporation is marketing through its Genicon subsidiary both labeled nucleotides and a light scattering scanner. In our laboratory the colloidal gold and silver labeling protocol along with the use of a mixing hybridization chamber (Biomicrosystems) allows hybridization and ready analysis of samples of 100-200 ng of poly(A) RNA from infected cells; a 10-fold increase in sensitivity. Such methods coupled with other amplification methods should allow hybridization of RNA from a few 10s of cells or less from infected tissue.

Data Analysis
Statistical Validation of Microarray Data

All HSV probes are spotted in triplicate, and numerical values for hybridization are obtained by laser scanning of the chip to measure fluorescence (expressed in arbitrary units) derived from the spotted oligonucleotide.  This value is first adjusted by subtraction of the background fluorescence of an equivalent area within a concentric ring just outside the spotted sample, then this value is reduced to a net value by subtraction of the median of a large number (ca 100) regions spotted with SSC buffer alone during fabrication. The median of the three replicate samples and the standard deviation for each probe is expressed using Microsoft Excel.

With Laser scanners, there are two adjustable variables that must be set for each individual scan, the laser power and the photo multiplier gain.  While each can be adjusted to any value between 0 and 100%, useful values for the laser power lie between 40 and 90 with the photo multiplier set at some constant value or a set number of units more or less than laser power. Thus, in practice only one variable need be set.

The ratio of fluorescent signal to actual sample value is linear only to net (-SSC) values of 40,000 or so. While theoretically this allows a greater than 40-fold discrimination in hybridization values, the fact that weak fluorescent signals are inherently less reliable than strong ones makes the actual range of reciprocity considerably smaller.  Properly controlled, fluorescent signals differing by as much as 3 logs can be reliably differentiated--provided multiple scans are utilized.

In order to compare data from repeat and time-varying experiments, the chip hybridization data is normalized as follows.  Analysis begins by taking the median signal of probe values for each transcript set, and the 75th percentile rank for the total viral hybridization is calculated.  One experimental group in a set of replicates is chosen as representative, and the values of other groups are adjusted to an equivalent 75th percentile value.  This is simply a means of emphasizing the higher significance of high hybridization values as compared to lower ones.  An example of replicate data before and after normalization are shown below.

rna percentile

For publishing and statistical confidence in any findings, the minimum number of statistically independent samples is three.  This helps to indicate data variation representative of a population and allows rudimentary hypothesis testing by non-parametric statistical methods. Obviously, the reliability is greater with more independent samples.

Hybridization data can be expressed as a ratio between a control and an experimental determination (as graphically determined by scatter analysis as described above), as a numerical value related to fluorescence signal intensity (other examples shown above), or as a measure of relative abundance based upon the ratio of a given probe signal to the total viral signal under any given conditions.  In the latter case, it is important to include numerical signal strengths as a measure of hybridization no mater what the final form of presentation.  Further, knowledge of the background signal seen with non-specific probes or SSC-spotted blanks provides an index of general reliability of the hybridization in questions.

Resolution of overlapping transcripts.

Fluorescent-labeled nucleosides are quite bulky, and synthesis of cDNA using either oligo-dT or random-oligomer priming yields products averaging much less than 500 bp in length.  Thus, 75-mers probes representing sequences within 300 bp of polyadenylation site of each of the 50-odd transcription units can be readily detected using oligo-dT primers; however, oligomers representing sequences upstream of this region will not hybridize to oligo-dT primed cDNA made with dye-substituted nucleotides.  If polyadenylated RNA is isolated and cDNA generated by random oligomer primers, however, upstream oligonucleotide probes can provide data concerning transcripts represented by them.  This is illustrated below for several sets of overlapping transcripts.

overlapping transcripts

In the experiment shown, RNA was isolated at 4 hr following infection of HeLa cells--a time at which mainly early transcripts are abundant.  cDNA was synthesized either using an oligo-dT primer with 20 mg total RNA with Cy5-substituted dUTP, or with a random hexamer primer with 1 mg of poly(A)-selected RNA and Cy3-substituted dUTP.  The cDNAs were then hybridized to an HSV DNA microarray, and selected data for the UL27/28, UL39/40, UL44/45, US5/6/7, and US8/9 overlapping transcript sets.  In each case, the oligo-dT primed cDNA (green) only hybridized to the 3' probe.  The random-primed cDNA (red) provided signals for both probe except with the US5/6/7 transcript group where the distal US5 transcript is abundant at late times while the US7 is abundant early.