BACKGROUND
the pseudorabies virus  is a member of the alpha-herpesviruses and an important pathogen of pigs, causing aujeszky's disease  <cit> .
the prv is a valuable model organism in herpesvirus research  <cit>  and also a powerful tool in neuroscience, employed to map neural circuits  and gene delivery  <cit> .
the prv life cycle is primarily controlled at the level of transcription.
the genes of α-herpesviruses are divided into three major temporal classes , which are regulated in a coordinated, cascade-like fashion .
first, the ie genes are expressed, independently of de novo protein synthesis from the virus.
the products of these genes are transcription factors and other regulatory proteins.
prv ie <dig> protein  is the major regulator of the transcription of the e genes, which in turn are mainly involved in replication of the viral dna.
blockers of dna synthesis inhibit the e gene expression to a much lesser extent than the l gene expression.
on a finer scale, the e genes can be subdivided into β <dig>  and β <dig>  genes.
finally, the l genes are transcribed, encoding mainly structural proteins involved in virion assembly.
the expression of the l genes is partially  or completely  dependent on the viral dna replication.
hsv encodes  <dig> ie genes: icp <dig>  icp <dig>  icp <dig>  icp <dig> and icp <dig>  by contrast, prv has been reported to express ep <dig>  proteins in the e kinetics, and it lacks the icp <dig> gene.
the ie <dig> gene of prv has been shown to be a true ie gene in various experimental systems ; however, there is no consensus as to whether the us <dig> protein  is expressed in the ie  <cit>  or the e kinetics  <cit> .
herpesviruses are capable of two types of infections: lytic  and latent  <cit> .
in the lytic pathway, the entire transcription machinery of the herpesvirus is initiated, and the progress of infection eventually leads to the production of new virions and the lysis of infected cells.
in contrast, in latency, only a limited segment  of the herpesvirus genome is transcriptionally active, no new virus particles are produced and the cells survive the infection.
a characteristic feature of the organization of the herpesvirus genome is the presence of nested genes producing 3'-coterminal transcripts.
the read-through of overlapping genes is regulated by the icp <dig> protein, which helps in the recognition of the internal polya signals, resulting in differential transcript lengths  <cit> .
the gene expression of α-herpesviruses, such as hsv types  <dig> and  <dig>  varicella-zoster virus, bovine herpesvirus type  <dig> and prv has been investigated earlier by traditional methods including northern blot analysis, ribonuclease protection assay, and end-point rt-pcr analysis.
however, each of these techniques is associated with a number of disadvantages.
for example, northern blot analysis is labor-intensive, allows only semiquantitative determination of the mrna level and is unsuitable for multiple mrna analysis.
moreover, hybridization-based membrane arrays profile changes in a nonlinear fashion, tending to overemphasize large alterations, and they are insensitive to smaller variations.
ribonuclease protection assay-based methods require the use of polyacrylamide gel electrophoresis and typically utilize radioactively labeled probes.
the limitations of endpoint pcr technique are the time-consuming procedure, the poor precision and the variable endpoints between samples.
over the past few years, microarray techniques have revolutionized practically all disciplines of molecular biology, including herpesvirus research.
as compared with traditional methods, microarray analysis is superior in that it is applicable for the simultaneous analysis of a large number of genes, and even whole genomes.
the disadvantage of dna chip technology is associated with the uncertain quality control: it is impossible to assess the identity of dna immobilized on any microarray.
further, there are many artifacts associated with image and data analysis.
real-time rt-pcr is an alternative to microarray techniques for the analysis of transcription from multiple genes.
the main advantage of real-time pcr is that it is more sensitive to low-fold changes than other high-throughput assays.
in addition, the real-time pcr technique provides a reproducible quantitation of dna copies, and has a large dynamic range, and various controls can be included to ensure accuracy, such as a loading control to verify equal cdna loading, a no-primer control to prove a measure of non-amplification-related background, a no-template control to screen for contamination of reagents or false amplification, and a no-rt control with confirm the absence of dna contamination.
in contrast to microarray techniques, in real-time pcr the parameters for each gene can be optimized individually.
moreover, the identity of pcr products can be confirmed through melting curve analysis, restriction endonuclease analysis, or dna sequencing.
in fact, real-time pcr is often used to verify gene expression data obtained by microarrays.
nevertheless, considerable pitfalls may be associated with this technique.
the major limitations of real-time rt-pcr relative to microarray techniques are the higher cost and labor intensiveness for a large number of samples.
another disadvantage of real-time pcr as compared with blotting techniques is that only the accumulation, but not the size of the transcripts can be monitored.
microarray techniques have recently been applied to investigate herpesvirus gene expression ; to analyze the effects of the deletion of particular viral genes or of the specific experimental conditions on whole-genome viral gene expression ; and to analyze the impact of virus infection on the expression of cellular genes .
the expression of prv genes has been studied by traditional methods, but many its genes have not yet been characterized at all.
flori and co-workers  <cit>  investigated the dialog between prv and epithelial cells, but obtained poor resolution for viral transcripts that did not provide conclusive data on the temporal expression of the prv genes.
to date, real-time rt-pcr has not been frequently utilized in herpesvirus research for global gene expression analysis.
with this technique oster and höllsberg  <cit>  carried out a kinetic analysis of  <dig> genes of human herpesvirus 6b, a β-herpesvirus, and dittmer and colleagues  <cit>  performed a whole-genome profiling of the rhesus monkey rhadinovirus, a γ-herpesvirus.
as far as we are aware, no genome-wide expression data obtained by qrt2-pcr have been published so far on α-herpesviruses.
in the present study, we describe the development and utilization of a real-time rt-pcr assay for the global analysis of prv gene expression.
we applied a novel method that allows the kinetic characterization of individual prv genes, and also the comparison of the expression dynamics of different viral genes.
expression profiles were constructed on the basis of relative expression ratios  calculated as described in the materials section.
this method is applicable for the analysis of gene expression in any genetic system that progressively changes in time.
RESULTS
experimental design
for each gene, a minimum of  <dig> independent replicates were carried out for statistical confidence, and the median of these values along with the standard error was calculated.
in these experiments, porcine kidney - <dig> epithelial cells were infected with prv with a low multiplicity of infection .
the reason for this was that our preliminary experiments indicated that infecting cells with a low dose of the virus produced a much better resolution of the cycle threshold  values for prv genes at different time points than infecting with a higher moi .
however, the use of a low moi led to a larger proportion of the cells remaining uninfected; accordingly, in order to avoid the initiation of a new infection cycle we chose a relatively short maximal incubation period.
initially, we attempted an 8-h investigation period, but observed a second infection wave of the virus , and therefore we restricted our analysis to  <dig> h post-infection .
the transcription of prv genes was monitored at  <dig> time points:  <dig>   <dig>   <dig>   <dig> and  <dig> h. prior to prv infection, cells were either untreated or treated with cycloheximide , a protein synthesis blocker, or phosphonoacetic acid , an inhibitor of dna synthesis.
we used strand-specific primers for the reverse transcription, one of the reasons for this being that we intended to exclude the distorting effects of potential overlapping antisense rnas transcribed from the antiparallel dna strands, which cannot be distinguished by oligo-dt- or random priming-based rt .
another reason was that it produced a much higher amount of specific cdnas than oligo-dt priming , which was especially important under our experimental conditions with a low moi.
we utilized the mathematical model for relative quantification described by soong et al.
<cit> ; however, we calculated the r values in a different way: we used the average of the maximal ect-sample values for each gene as a control, which was normalized with the average of the corresponding 28s values .
thus, in our calculation, a particular r value indicates the ratio of the mrna level of a gene at a given time point to the maximal rna level of this gene .
the relative amounts of the transcripts of different genes cannot be compared because the primer efficiencies may vary both in rt and in pcr.
even so, use of the maximal expression as the control value allows a comparison of the expression dynamics of viral genes both at individual time points and across the entire infection period.
the specificity of the pcr products was confirmed by quality control experiments, including tm analysis and page ; if some doubts remained, the amplification products were subjected to dna sequencing or restriction endonuclease analysis using amplicons containing recognition sites for a particular restriction endonuclease .
the accuracy of sampling and qrt2-pcr analysis were ensured by using loading controls, as well as no-rt, no-primer and no-template controls .
reproducibility of the internal control
a major concern regarding the choice of internal reference gene control in viral gene expression studies is the constancy of the reference gene products throughout the entire examination period.
we tested whether the 28s ribosomal rna of the pig fulfilled this requirement, and found that this gene was suitable as reference gene for the study of prv infection, at least in our 6-h examination period.
the 28s rrna values were highly reproducible: the mean value ± sd of all measurements the experiments was  <dig>  ±  <dig>  cycles throughout .
classification of prv genes in terms of dependence on de novo protein synthesis and dna replication
traditionally, lytic herpesvirus genes are classified into distinct kinetic groups on the basis of the effects of protein and dna synthesis inhibition on the gene expression.
in general, inhibitory drugs are applied to infected cells for a prolonged incubation period  and compared the inhibitory effects of these drugs by calculating the r value ratios for the treated and untreated samples.
chx analysis
to test the requirement of de novo protein synthesis for prv mrna production, pk- <dig> cells were untreated or treated with  <dig> μg/ml chx prior to the infection of cells with the virus.
at the indicated time points, rna was isolated and converted to cdna, which was subsequently analyzed by qrt2-pcr.
the degree of inhibition  was found to range between  <dig>  and 100% for all but one protein encoding prv gene  and two antisense transcripts , and therefore it cannot be regarded as an ie gene beyond doubt.
paa analysis
for examination of the dependence of the prv genes on dna replication, pk- <dig> cells were infected with the virus in the absence or presence of  <dig> μg/ml paa, an inhibitor of dna polymerase.
it was expected that paa would exert a more drastic effect on the l genes because the expressions of these genes are highly dependent on dna replication.
however, paa must affect the e gene expression, too.
the reason for this is that the gene expression is dependent not only on the promoter activity, but also on the copy numbers of the genes, which are higher after dna replication than in the initial phase of infection when the prv dna is represented in a single copy in a cell .
the inhibitory effect of paa on gene expression was calculated by using the ratio of the r values for the paa-treated/untreated samples at  <dig> h pi : a low ratio indicates a strong inhibitory effect and vice versa.
the prv genes were ranked from the least inhibited  to the most inhibited .
we expected that such a ranking of the prv genes on the basis of the inhibitory effect would provide natural clusters of e and l genes.
indeed, the results obtained conformed fairly well to the data published earlier on hsv and prv genes, though there is a certain degree of disagreement between the data published by the different authors on the hsv genes, and only incomplete expression data are available for the prv genes.
moreover, in many cases a prv gene is characterized without comparative analysis, e.g.
"it appears  <dig> h pi".
for comparison of the hsv and prv genes, we used five main sources for the hsv data: microarray data published by wagner  <cit> ; various expression data compiled by roizman  <cit> ; and the data collected by mettenleiter  <cit> .
for the prv genes, we used the data compiled by mettenleiter  <cit> .
we classified the analyzed but uncategorized prv genes via the following principles: if the mrna was detected between  <dig> and  <dig> h pi,, between  <dig> and  <dig> h pi, or  <dig> h pi, the particular gene was classified as e, e/l or l, respectively.
in the upper part of the ranking list , , we mostly find typical e genes, with typical l genes in the bottom part of the list.
however, we could not draw a clear-cut line between the e and l genes; instead, we put the  <dig> genes with intermediate values in the e/l group.
the precise locations of borderlines between kinetic classes were drawn arbitrarily, because we found smooth transitions between the ri-paa values.
we chose a typical e gene  of the hsv as the last member of the e group, and a typical l gene  of the hsv as the first member of the l group.
the  <dig> protein-encoding genes with the highest ri-paa values were ul <dig> , ul <dig>  and us <dig> .
these high ri-paa values indicate that paa does not exert a significant cytotoxic effect on the gene expression apart from blocking dna replication.
the  <dig> llt transcripts display a surprising response to paa treatment: the level of llt <dig> increases to  <dig> -fold at  <dig> h, and drops to  <dig> -fold at  <dig> h pi relative to the untreated sample; while the level of llt <dig> increases markedly close to 40-fold at  <dig> h, and 3-fold at  <dig> h pi .
the genes with the lowest ri-paa values are all but one  l genes in the hsv: ul <dig> , ul <dig>  and ul <dig> .
the strong inhibitory effect of paa on the us <dig> expression is surprising because it is an ie gene in the hsv.
the ie <dig> gene expression is also significantly inhibited by paa ; however, we found that ie <dig> and to a certain extent us <dig> were expressed in an "irregular" manner in other analyses, too .
ul <dig> and ul <dig>  which are l genes in hsv, do not appear to belong in the e kinetic class.
both genes were characterized as l genes in the prv by de wind et al.
<cit>  using northern blot analysis.
furthermore, ul <dig> is an l gene in the hsv, but its transcripts were detected at  <dig> h pi in the prv.
the ul <dig> and ul <dig> genes are e genes in the hsv, but were characterized as e/l and l genes, respectively, in the prv by dean and cheung  <cit> , respectively, which is in agreement with our results.
we classified the kinetically still uncharacterized prv genes as follows; e genes: us <dig>  ul <dig> and ul <dig>  l genes: ul <dig>  ul <dig>  ul <dig>  ul <dig>  ul <dig>  ul <dig>  ul <dig> and ul <dig>  peaking of the us <dig> gene expression at  <dig> h can be explained by the important role of us <dig> protein kinase in blocking the apoptotic pathway of the compromised cell in the e phase of infection  <cit> .
our kinetic classifications accorded more strongly with those published on the hsv than with those on the prv.
the reason may be that the expression profiling of prv genes was performed by others with low resolution techniques.
our data allow the following conclusions:  there is no sharp boundary between the e and l genes;  classification of the prv genes on the basis of the ri-paa values yielded similar results those for the homologous hsv genes.
a noteworthy exception is the us <dig> gene, which according to our paa analysis is expressed in l kinetics;  the ie <dig> and llt genes cannot be classified on the basis of the ri-paa data alone, because they display unique expression kinetics;  llt <dig> and llt <dig> respond differently to chx and paa treatment, indicating that these antisense transcripts are, at least partially, under different regulation.
we assume that the putative antisense promoter  controls the expression of llt <dig>
this table demonstrates the function and kinetic classification of prv genes in comparison with available data on hsv and prv genes.
a prv genes were classified on the bases of the ratio of the paa-treated and untreated samples, at  <dig> h pi.
b gene functions in italics rely on studies of the hsv- <dig> homologs.
kinetics of prv gene expression in untreated cells
real-time rt-pcr is a high-resolution technique, which allows a detailed analysis of gene expression dynamics and a reliable temporal classification of viral mrnas without drug treatment.
the data from these experiments are presented in detail in the text .
the us <dig> prv gene peaks at  <dig> h, llt <dig> at  <dig> h, while the other genes reach their maximum at  <dig> h within the 0- <dig> h examination period.
0- <dig> h incubation period
the accurate and reproducible data generated by real-time pcr allow the analysis of gene expression throughout the entire time course of infection.
in order to classify genes on the basis of their expression kinetics, we performed pearson's correlation analysis for each gene pair of the prv, using the net increase  in each time interval for the comparison .
genes were clustered into the same group if their pairwise correlation coefficients were high, ranging from r ≥  <dig>  to  <dig>  depending on the group.
surprisingly, genes belonging in a particular group very rarely give similarly high values as any of the genes belonging in other groups.
albeit pearson's coefficient expresses the kinetic properties of a gene in single numbers, the expression curves of genes belonging in the same group proved to be very similar .
we distinguished  <dig> gene sets.
group l <dig> of prv genes  contains  <dig> genes, of which  <dig> were identified as l and two as e/l genes by our paa analysis.
the only exception was ul <dig> which although an l gene in the hsv, behaved as an e gene in all of our analyses.
the profile curves of the genes clustered on the basis of pearson's correlation of pairwise rΔ values, gave very similar patterns of expression within the groups.
in each period of time, the expression curves can also be characterized by the formula: rΔ - rΔt .
for example, group l <dig> was characterized by hardly any change in the first  <dig> h, a moderate rise in mrna level between  <dig> and  <dig> h, and a sudden increase in mrna copy number from  <dig> to  <dig> h .
group l <dig>  contains one e/l and  <dig> l genes.
the expression profiles of these genes are characterized by a continuous intensive increase from  <dig> to  <dig> h .
our paa analysis revealed that all of the  <dig> members of group l <dig>  are l genes, except for us <dig>  which is an ie gene in the hsv.
in our system, therefore, most of the data , suggest that it is an l gene.
this group is characterized by the expression profile 0+/0-/+++.
group e <dig>  and group e <dig>  comprise e genes, except for ul <dig>  which is an l gene in the hsv.
however, this gene is characterized as an e gene in the prv  <cit> , which was confirmed by our paa analysis.
these genes are characterized by the expression profile: 0+/+/-.
group e <dig> , expression profile: +/+/0+; group e <dig> , profile: 0/+/ <dig>  group m <dig>  is composed of  <dig> members, shown by our paa analysis to exhibit either e or l kinetics.
the expression profiles of these genes are very similar , which draws attention to the fact that paa analysis alone is not sufficient for the grouping of genes with a similar expression profile.
the  <dig> members of group m <dig> exhibit a very high correlation  and similar expression profiles .
group llt  contains  <dig> llt genes that have an unusual expression profile .
the following  <dig> genes cannot be clustered on the basis of pearson's coefficients: ie <dig> , ul <dig> , ul <dig>  and us <dig> , which are likewise unusual indeed, these genes have peculiar expression profiles .
the expression profile can also be visualized by heatmap presentation, using the expression data generated with the rΔtvalues .
gene expression profiles of prv gene clusters were characterized by plus and minus signs at various time intervals .
the numbers of plus or minus signs indicate the relative rate of increase or decrease of transcripts.
co-regulated gene clusters
we utilized our data to analyze whether the expression properties of prv genes localized in adjacent genomic loci display similarities.
paa analysis
pearson's correlation
analysis of the gene clusters generated by using pearson's correlation coefficients revealed that genes within the same clusters are often located in neighboring genomic positions.
group l <dig> is composed of  <dig> members.
the adjacent ul <dig>  ul <dig>  ul <dig>  ul <dig> and ul <dig> genes are members of this group.
the high pairwise correlation coefficients and the similar profile curves of the rΔ values of these genes cannot be explained by the nested co-localization alone, because ul <dig> and ul <dig> are divergent genes and do not overlap with ul <dig>  ul <dig> and ul <dig>  the ul <dig> gene is convergent with ul <dig> and ul26; and ul <dig> is divergent with ul <dig>  moreover, ul <dig> is convergent with ul <dig> and ul <dig>  it should e borne in mind that ul <dig> and ul <dig>  an adjacent gene pair in the hsv, are relocalized into a remote position in the prv, due to a large inversion, but retain their expression properties.
this is also true for ul <dig>  ul <dig>  and ul <dig> .
several genes  in the us region of the prv genome belong in this group, too.
the ul <dig> and ul <dig> genes, which are oriented parallelly, but non-overlapping, likewise belong in group l <dig>  the ul <dig> and ul <dig> genes are in a divergent position with ul <dig>  group l <dig> is composed of  <dig> members.
the ul <dig> and ul <dig> genes are divergently oriented, while ul <dig> and ul <dig>  are convergent genes.
group l <dig> is a small group with  <dig> members.
the ul <dig> and ul <dig> genes are arranged in a divergent orientation.
group e2: two of the  <dig> members of this group, ul <dig> and ul <dig>  are overlapping genes.
group e3: two  of the  <dig> members of this group are divergently oriented with the third member, ul <dig>  group m1: ul <dig> and ul <dig> are a convergent gene pair.
the ul <dig>  and ul <dig> are l genes oriented in parallel and separated by the divergent early ul <dig> gene.
furthermore, ul <dig> and ul <dig> are a convergent adjacent gene pair.
genes with similar expression profiles appear to be under a common regulation, which cannot be explained solely by the nested localization  of genes.
group llt: the llt <dig> and llt <dig> expression patterns show a high degree of similarity.
however, there is noteworthy inverse relationship in the expression profiles of llt <dig> and llt <dig> with those of their complementary partners, ep <dig> and ie <dig> mrnas, respectively .
discussion
gene expressions of α-herpesviruses have already been analyzed by various techniques including northern-blot and microarray analysis.
different authors have often assigned the same genes to different kinetic groups, which may be explained by the poor resolution of the applied techniques.
only partial data are available on the expression of prv genes.
flori et al.
<cit>  carried out genome-wide gene expression analysis via a microarray technique, but this approach did not produce evaluable data on the kinetic properties of the prv genes.
we employed a novel qrt2-pcr technique using  strand-specific primers for the rt to obtain a higher yield and to eliminate the effects of the potential antisense transcripts; and  the average maximal ect values as the controls for the calculation of relative expression ratios .
other approaches, such as the models of pfaffl's  <cit>  and souazé et al.,  <cit> , and the Δct and ΔΔct models  <cit>  are simpler than the model we used; however, they all neglect the variation in the amplification efficiencies between cdna samples, which was present in our system.
traditionally, herpesvirus genes are grouped into kinetic classes on the basis of gene expression inhibitory effects of protein and dna synthesis-blocking reagents.
the ie genes should not be significantly inhibited by any of the above substances, the e genes are inhibited only by dna synthesis blockers, and the expression of the l genes is substantially inhibited by both protein and dna synthesis blockade.
we grouped the prv genes into ie, e, e/l and l categories on the basis of the results of chx and paa treatment , .
our findings conformed well with those published for the hsv genes.
the expression of the ie <dig> gene is enhanced by chx treatment.
the explanation of this phenomenon lies in the fact that the ie <dig> protein normally inhibits its own expression a short time after the onset of virus infection, a feature which is absent in chx-treated cells due to novel protein synthesis being blocked by the drug.
the other prv genes were inhibited to a significant extent by chx at every examined time point.
consequently, ie <dig> is the only ie gene of the prv.
the ep <dig> and ul <dig> genes  were shown to be e genes by others , and this was confirmed by our analyses.
interestingly, us <dig>  which is an ie gene in the hsv, appeared to be an l gene in most of our analyses: its expression was significantly inhibited by paa; it afforded a low amount of mrnas at  <dig> h pi, and a high rate of increase of transcript level at 4- <dig> h pi, all of these being l characteristics.
furthermore, together with a set of l genes, us <dig> belongs in group l <dig> according to the clustering of the genes on the basis of pairwise pearson correlation coefficients.
the moderately high relative amount of us <dig> transcripts at  <dig> h pi is the only e-like characteristic of this gene.
however, us <dig> displays a high ct value, suggesting a low absolute amount of transcripts at  <dig> h pi.
we may mention here that the ct value of a transcript is dependent on the primer efficiency; these data therefore cannot be reliably used to the estimate the absolute transcript amount.
if the us <dig> gene has an important function early in the lytic cycle of the virus, it might fulfill this without de novo synthesis by being released upon infection from the tegument layer of the virus where it might be incorporated.
our data do not demonstrate a clear-cut demarcation line between the groups of e and l genes, suggesting that the categorization of herpesvirus genes on the basis of the requirements for dna replication might be arbitrary.
the kinetic types of genes identified by our paa analysis resemble homologous hsv genes more than the published prv genes, which can be explained by the semiquantitative methods used for the kinetic analysis of the prv genes.
the present study revealed that temporal classification of herpesvirus genes is also possible through study of the gene expression of untreated cells in consequence of the high sensitivity of the real-time rt-pcr technique.
as a further interesting point, we detected mrnas by qrt2-pcr in all but  <dig> prv genes as early as  <dig> h pi .
this observation indicates that prv genes are either leaky or subject to regulatory mechanisms which have not yet been elucidated.
we found that e genes are generally expressed in a higher proportion than l genes at  <dig>   <dig> and  <dig> h, as indicated by their higher r values at these time points .
the e gene products proved to display a higher net increase  than the l genes within the 0- <dig> h, 1- <dig> h and 2- <dig> h pi periods.
however, by the 4- <dig> h pi interval, this trend had definitely reversed, i.e.
the l genes exhibited high r values .
unexpectedly, the ratio of increase  between  <dig> and  <dig> h was higher for a number of l genes than any of the e genes .
since the r2h values of these l genes were lower than those of the e genes, the high r value is a result of the very low r1h values of late genes.
most of the l genes also have higher r and r values than those of e genes, which is explained by the fact that the e genes reach a relatively high expression level by  <dig> h pi.
the classification of the prv genes through the chx and paa analyses led to results very similar those generated by the analysis of untreated cells via the following calculation: r/r .
the differences between the results of the two approaches can be explained in that paa analysis alone gives only a rough picture of the gene expression; a detailed kinetic analysis of the viral gene expression furnishes a more sophisticated picture.
some genes classified as e or l by paa analysis display "irregular" kinetics under untreated conditions.
the ie <dig> and perhaps us <dig> genes cannot be classified by paa analysis alone.
analysis of the transcription kinetics of overlapping transcript sets is an important issue.
it is not known whether downstream genes on polycistronic mrnas are translated, and if so, to what extent.
this makes interpretation of the mrna expression data difficult because the mrna levels cannot be correlated with the amounts of the corresponding proteins, which are the workhorse molecules in regulating cellular physiology.
in principle, a downstream gene is translated if it is transcribed from its own promoter.
theoretically, downstream genes could also be translated from a polycistronic rna if alternative splicing removed upstream gene from the pre-mrna, or if potential ires-like sequences helped recruit ribosomes, thereby initiating a cap-independent translation from the downstream gene .
thus, analysis of the downstream genes without discriminating between transcription from their own promoters or by read-through from upstream genes might result in a false categorization.
the prv genes were also classified by analysis of the gene expression throughout the entire examination period by using pearson's correlation analysis.
genes with similar expression profiles  were placed in the same group.
genes in the same kinetic class  are generally situated in the same group generated by the use of person's correlation.
moreover, members of a particular group  rarely display high correlation coefficients with genes belonging in different groups.
the prv genes conformed well to  <dig> patterns of expression.
only  <dig> viral genes  did not fit into any of the  <dig> groups.
we found an inverse expression profile of the sense/antisense partners in the ep0-llt <dig> and ie180-llt <dig> genes .
combination of the untreated data with the results of the chx and paa analyses revealed an interesting relationship for coordinated regulation of sense/antisense partners.
the chx analysis suggested that ie <dig> protein facilitates lap  activity, and inhibits asp .
the paa treatment resulted in a significantly elevated antisense transcript level at  <dig> of the  <dig> time points in llts, which indicates the existence of another regulatory level besides ie <dig> protein action.
we assume that transcription from one dna strand negatively influences the expression of transcripts from the complementary dna strand.
the interaction can occur at the level of transcription  and/or translation by forming double-stranded rnas by the sense and antisense transcripts.
as an example, paa has a negative effect on the transcription of ie <dig>  at  <dig> h pi, which results in a lower rate of transcription from ie <dig> ; and a lower amount of inhibitory ie <dig> proteins, which also facilitates llt <dig> expression.
overall, the llt <dig> level increases  <dig> -fold relative to the untreated conditions.
furthermore, genes with the same kinetic properties exhibit a distinctive distribution pattern along the prv genome.
nested genes appear to belong in the same kinetic group.
additionally, convergent genes and gene clusters in most  cases belong to different kinetic classes.
it should be remembered that the paa-based approach and the time intervals applied for kinetic analysis produce only a coarse resolution of gene expression.
it is possible that convergent genes allocated to the same kinetic classes might display different expression profiles on a finer scale.
the above genome organization principles may point to the existence of as yet unknown regulatory mechanisms.
it may be speculated whether a read-through of transcription across convergent genes is the basis of this regulation.
moreover, groups formed in terms of high pearson's correlation coefficients contain many genes localized at adjacent loci on the prv genome.
most genes with high correlation coefficients are not nested genes.
this means that the similarity of their expression profiles cannot be explained by the control of a common promoter.
further, several genes in the same group are separated by genes that display different kinetic profiles.
these results suggest the existence of a genetic mechanism that synchronizes gene expression on a higher-order  scale.
CONCLUSIONS
although the relative amounts of the transcripts between genes cannot be compared because primer efficiencies may vary both in rt and in pcr, our method allows a comparison of genes on the basis of their expression dynamics.
we detected the expression of almost all genes from the earliest time point , further, we have found that a sharp boundary cannot be drawn a between the groups of e and l genes in terms of expression profiles.
we have also found that the prv has only a single ie gene  and that the us <dig> gene is expressed in l kinetics.
our kinetic studies revealed that paa analysis alone is not sufficient for the classification of genes on the basis of their expression profiles.
the analysis of gene expression throughout the  <dig> h examination period demonstrated that these genes can be subdivided into further clusters, and that genes showing significant differences in response to paa treatment can have very similar expression characteristics in untreated conditions.
in our analysis, nested genes displayed the same expression profiles, whereas convergent genes mostly exhibited different kinetic properties.
it should be noted that a finer resolution of the gene expression might expose slight differences in the expressions of nested genes.
approximately half of the divergent genes belong in the same class, and the other half of them in different kinetic classes.
the new calculation technique that we have developed is also applicable to evaluate the loss-of-function phenotypes of mutant herpesviruses viruses and, in principle, for the analysis of gene expression in every temporally changing genetic system.

 the pseudorabies virus is a member of the alpha-herpesviruses and an important pathogen of pigs, causing aujeszky's disease. the prv is a valuable model organism in herpesvirus research and also a powerful tool in neuroscience, employed to map neural circuits and gene delivery. the prv life cycle is primarily controlled at the level of transcription. the genes of α-herpesviruses are divided into three major temporal classes, which are regulated in a coordinated, cascade-like fashion. first, the ie genes are expressed, independently of de novo protein synthesis from the virus. the products of these genes are transcription factors and other regulatory proteins. prv ie protein is the major regulator of the transcription of the e genes, which in turn are mainly involved in replication of the viral dna. blockers of dna synthesis inhibit the e gene expression to a much lesser extent than the l gene expression. on a finer scale, the e genes can be subdivided into β and β genes. finally, the l genes are transcribed, encoding mainly structural proteins involved in virion assembly. the expression of the l genes is partially or completely dependent on the viral dna replication. hsv encodes ie genes: icp icp icp icp and icp by contrast, prv has been reported to express ep proteins in the e kinetics, and it lacks the icp gene. the ie gene of prv has been shown to be a true ie gene in various experimental systems; however, there is no consensus as to whether the us protein is expressed in the ie or the e kinetics. herpesviruses are capable of two types of infections: lytic and latent. in the lytic pathway, the entire transcription machinery of the herpesvirus is initiated, and the progress of infection eventually leads to the production of new virions and the lysis of infected cells. in contrast, in latency, only a limited segment of the herpesvirus genome is transcriptionally active, no new virus particles are produced and the cells survive the infection. a characteristic feature of the organization of the herpesvirus genome is the presence of nested genes producing 3'-coterminal transcripts. the read-through of overlapping genes is regulated by the icp protein, which helps in the recognition of the internal polya signals, resulting in differential transcript lengths. the gene expression of α-herpesviruses, such as hsv types and varicella-zoster virus, bovine herpesvirus type and prv has been investigated earlier by traditional methods including northern blot analysis, ribonuclease protection assay, and end-point rt-pcr analysis. however, each of these techniques is associated with a number of disadvantages. for example, northern blot analysis is labor-intensive, allows only semiquantitative determination of the mrna level and is unsuitable for multiple mrna analysis. moreover, hybridization-based membrane arrays profile changes in a nonlinear fashion, tending to overemphasize large alterations, and they are insensitive to smaller variations. ribonuclease protection assay-based methods require the use of polyacrylamide gel electrophoresis and typically utilize radioactively labeled probes. the limitations of endpoint pcr technique are the time-consuming procedure, the poor precision and the variable endpoints between samples. over the past few years, microarray techniques have revolutionized practically all disciplines of molecular biology, including herpesvirus research. as compared with traditional methods, microarray analysis is superior in that it is applicable for the simultaneous analysis of a large number of genes, and even whole genomes. the disadvantage of dna chip technology is associated with the uncertain quality control: it is impossible to assess the identity of dna immobilized on any microarray. further, there are many artifacts associated with image and data analysis. real-time rt-pcr is an alternative to microarray techniques for the analysis of transcription from multiple genes. the main advantage of real-time pcr is that it is more sensitive to low-fold changes than other high-throughput assays. in addition, the real-time pcr technique provides a reproducible quantitation of dna copies, and has a large dynamic range, and various controls can be included to ensure accuracy, such as a loading control to verify equal cdna loading, a no-primer control to prove a measure of non-amplification-related background, a no-template control to screen for contamination of reagents or false amplification, and a no-rt control with confirm the absence of dna contamination. in contrast to microarray techniques, in real-time pcr the parameters for each gene can be optimized individually. moreover, the identity of pcr products can be confirmed through melting curve analysis, restriction endonuclease analysis, or dna sequencing. in fact, real-time pcr is often used to verify gene expression data obtained by microarrays. nevertheless, considerable pitfalls may be associated with this technique. the major limitations of real-time rt-pcr relative to microarray techniques are the higher cost and labor intensiveness for a large number of samples. another disadvantage of real-time pcr as compared with blotting techniques is that only the accumulation, but not the size of the transcripts can be monitored. microarray techniques have recently been applied to investigate herpesvirus gene expression; to analyze the effects of the deletion of particular viral genes or of the specific experimental conditions on whole-genome viral gene expression; and to analyze the impact of virus infection on the expression of cellular genes. the expression of prv genes has been studied by traditional methods, but many its genes have not yet been characterized at all. flori and co-workers investigated the dialog between prv and epithelial cells, but obtained poor resolution for viral transcripts that did not provide conclusive data on the temporal expression of the prv genes. to date, real-time rt-pcr has not been frequently utilized in herpesvirus research for global gene expression analysis. with this technique oster and höllsberg carried out a kinetic analysis of genes of human herpesvirus 6b, a β-herpesvirus, and dittmer and colleagues performed a whole-genome profiling of the rhesus monkey rhadinovirus, a γ-herpesvirus. as far as we are aware, no genome-wide expression data obtained by qrt2-pcr have been published so far on α-herpesviruses. in the present study, we describe the development and utilization of a real-time rt-pcr assay for the global analysis of prv gene expression. we applied a novel method that allows the kinetic characterization of individual prv genes, and also the comparison of the expression dynamics of different viral genes. expression profiles were constructed on the basis of relative expression ratios calculated as described in the materials section. this method is applicable for the analysis of gene expression in any genetic system that progressively changes in time.  experimental design for each gene, a minimum of independent replicates were carried out for statistical confidence, and the median of these values along with the standard error was calculated. in these experiments, porcine kidney - epithelial cells were infected with prv with a low multiplicity of infection. the reason for this was that our preliminary experiments indicated that infecting cells with a low dose of the virus produced a much better resolution of the cycle threshold values for prv genes at different time points than infecting with a higher moi. however, the use of a low moi led to a larger proportion of the cells remaining uninfected; accordingly, in order to avoid the initiation of a new infection cycle we chose a relatively short maximal incubation period. initially, we attempted an 8-h investigation period, but observed a second infection wave of the virus, and therefore we restricted our analysis to h post-infection. the transcription of prv genes was monitored at time points: and h. prior to prv infection, cells were either untreated or treated with cycloheximide, a protein synthesis blocker, or phosphonoacetic acid, an inhibitor of dna synthesis. we used strand-specific primers for the reverse transcription, one of the reasons for this being that we intended to exclude the distorting effects of potential overlapping antisense rnas transcribed from the antiparallel dna strands, which cannot be distinguished by oligo-dt- or random priming-based rt. another reason was that it produced a much higher amount of specific cdnas than oligo-dt priming, which was especially important under our experimental conditions with a low moi. we utilized the mathematical model for relative quantification described by soong et al.; however, we calculated the r values in a different way: we used the average of the maximal ect-sample values for each gene as a control, which was normalized with the average of the corresponding 28s values. thus, in our calculation, a particular r value indicates the ratio of the mrna level of a gene at a given time point to the maximal rna level of this gene. the relative amounts of the transcripts of different genes cannot be compared because the primer efficiencies may vary both in rt and in pcr. even so, use of the maximal expression as the control value allows a comparison of the expression dynamics of viral genes both at individual time points and across the entire infection period. the specificity of the pcr products was confirmed by quality control experiments, including tm analysis and page; if some doubts remained, the amplification products were subjected to dna sequencing or restriction endonuclease analysis using amplicons containing recognition sites for a particular restriction endonuclease. the accuracy of sampling and qrt2-pcr analysis were ensured by using loading controls, as well as no-rt, no-primer and no-template controls. reproducibility of the internal control a major concern regarding the choice of internal reference gene control in viral gene expression studies is the constancy of the reference gene products throughout the entire examination period. we tested whether the 28s ribosomal rna of the pig fulfilled this requirement, and found that this gene was suitable as reference gene for the study of prv infection, at least in our 6-h examination period. the 28s rrna values were highly reproducible: the mean value ± sd of all measurements the experiments was ± cycles throughout. classification of prv genes in terms of dependence on de novo protein synthesis and dna replication traditionally, lytic herpesvirus genes are classified into distinct kinetic groups on the basis of the effects of protein and dna synthesis inhibition on the gene expression. in general, inhibitory drugs are applied to infected cells for a prolonged incubation period and compared the inhibitory effects of these drugs by calculating the r value ratios for the treated and untreated samples. chx analysis to test the requirement of de novo protein synthesis for prv mrna production, pk- cells were untreated or treated with μg/ml chx prior to the infection of cells with the virus. at the indicated time points, rna was isolated and converted to cdna, which was subsequently analyzed by qrt2-pcr. the degree of inhibition was found to range between and 100% for all but one protein encoding prv gene and two antisense transcripts, and therefore it cannot be regarded as an ie gene beyond doubt. paa analysis for examination of the dependence of the prv genes on dna replication, pk- cells were infected with the virus in the absence or presence of μg/ml paa, an inhibitor of dna polymerase. it was expected that paa would exert a more drastic effect on the l genes because the expressions of these genes are highly dependent on dna replication. however, paa must affect the e gene expression, too. the reason for this is that the gene expression is dependent not only on the promoter activity, but also on the copy numbers of the genes, which are higher after dna replication than in the initial phase of infection when the prv dna is represented in a single copy in a cell. the inhibitory effect of paa on gene expression was calculated by using the ratio of the r values for the paa-treated/untreated samples at h pi: a low ratio indicates a strong inhibitory effect and vice versa. the prv genes were ranked from the least inhibited to the most inhibited. we expected that such a ranking of the prv genes on the basis of the inhibitory effect would provide natural clusters of e and l genes. indeed, the results obtained conformed fairly well to the data published earlier on hsv and prv genes, though there is a certain degree of disagreement between the data published by the different authors on the hsv genes, and only incomplete expression data are available for the prv genes. moreover, in many cases a prv gene is characterized without comparative analysis, e.g. "it appears h pi". for comparison of the hsv and prv genes, we used five main sources for the hsv data: microarray data published by wagner; various expression data compiled by roizman; and the data collected by mettenleiter. for the prv genes, we used the data compiled by mettenleiter. we classified the analyzed but uncategorized prv genes via the following principles: if the mrna was detected between and h pi,, between and h pi, or h pi, the particular gene was classified as e, e/l or l, respectively. in the upper part of the ranking list,, we mostly find typical e genes, with typical l genes in the bottom part of the list. however, we could not draw a clear-cut line between the e and l genes; instead, we put the genes with intermediate values in the e/l group. the precise locations of borderlines between kinetic classes were drawn arbitrarily, because we found smooth transitions between the ri-paa values. we chose a typical e gene of the hsv as the last member of the e group, and a typical l gene of the hsv as the first member of the l group. the protein-encoding genes with the highest ri-paa values were ul, ul and us. these high ri-paa values indicate that paa does not exert a significant cytotoxic effect on the gene expression apart from blocking dna replication. the llt transcripts display a surprising response to paa treatment: the level of llt increases to -fold at h, and drops to -fold at h pi relative to the untreated sample; while the level of llt increases markedly close to 40-fold at h, and 3-fold at h pi. the genes with the lowest ri-paa values are all but one l genes in the hsv: ul, ul and ul. the strong inhibitory effect of paa on the us expression is surprising because it is an ie gene in the hsv. the ie gene expression is also significantly inhibited by paa; however, we found that ie and to a certain extent us were expressed in an "irregular" manner in other analyses, too. ul and ul which are l genes in hsv, do not appear to belong in the e kinetic class. both genes were characterized as l genes in the prv by de wind et al. using northern blot analysis. furthermore, ul is an l gene in the hsv, but its transcripts were detected at h pi in the prv. the ul and ul genes are e genes in the hsv, but were characterized as e/l and l genes, respectively, in the prv by dean and cheung, respectively, which is in agreement with our results. we classified the kinetically still uncharacterized prv genes as follows; e genes: us ul and ul l genes: ul ul ul ul ul ul ul and ul peaking of the us gene expression at h can be explained by the important role of us protein kinase in blocking the apoptotic pathway of the compromised cell in the e phase of infection. our kinetic classifications accorded more strongly with those published on the hsv than with those on the prv. the reason may be that the expression profiling of prv genes was performed by others with low resolution techniques. our data allow the following conclusions: there is no sharp boundary between the e and l genes; classification of the prv genes on the basis of the ri-paa values yielded similar results those for the homologous hsv genes. a noteworthy exception is the us gene, which according to our paa analysis is expressed in l kinetics; the ie and llt genes cannot be classified on the basis of the ri-paa data alone, because they display unique expression kinetics; llt and llt respond differently to chx and paa treatment, indicating that these antisense transcripts are, at least partially, under different regulation. we assume that the putative antisense promoter controls the expression of llt this table demonstrates the function and kinetic classification of prv genes in comparison with available data on hsv and prv genes. a prv genes were classified on the bases of the ratio of the paa-treated and untreated samples, at h pi. b gene functions in italics rely on studies of the hsv- homologs. kinetics of prv gene expression in untreated cells real-time rt-pcr is a high-resolution technique, which allows a detailed analysis of gene expression dynamics and a reliable temporal classification of viral mrnas without drug treatment. the data from these experiments are presented in detail in the text. the us prv gene peaks at h, llt at h, while the other genes reach their maximum at h within the 0- h examination period. 0- h incubation period the accurate and reproducible data generated by real-time pcr allow the analysis of gene expression throughout the entire time course of infection. in order to classify genes on the basis of their expression kinetics, we performed pearson's correlation analysis for each gene pair of the prv, using the net increase in each time interval for the comparison. genes were clustered into the same group if their pairwise correlation coefficients were high, ranging from r ≥ to depending on the group. surprisingly, genes belonging in a particular group very rarely give similarly high values as any of the genes belonging in other groups. albeit pearson's coefficient expresses the kinetic properties of a gene in single numbers, the expression curves of genes belonging in the same group proved to be very similar. we distinguished gene sets. group l of prv genes contains genes, of which were identified as l and two as e/l genes by our paa analysis. the only exception was ul which although an l gene in the hsv, behaved as an e gene in all of our analyses. the profile curves of the genes clustered on the basis of pearson's correlation of pairwise rΔ values, gave very similar patterns of expression within the groups. in each period of time, the expression curves can also be characterized by the formula: rΔ - rΔt. for example, group l was characterized by hardly any change in the first h, a moderate rise in mrna level between and h, and a sudden increase in mrna copy number from to h. group l contains one e/l and l genes. the expression profiles of these genes are characterized by a continuous intensive increase from to h. our paa analysis revealed that all of the members of group l are l genes, except for us which is an ie gene in the hsv. in our system, therefore, most of the data, suggest that it is an l gene. this group is characterized by the expression profile 0+/0-/+++. group e and group e comprise e genes, except for ul which is an l gene in the hsv. however, this gene is characterized as an e gene in the prv, which was confirmed by our paa analysis. these genes are characterized by the expression profile: 0+/+/-. group e, expression profile: +/+/0+; group e, profile: 0/+/ group m is composed of members, shown by our paa analysis to exhibit either e or l kinetics. the expression profiles of these genes are very similar, which draws attention to the fact that paa analysis alone is not sufficient for the grouping of genes with a similar expression profile. the members of group m exhibit a very high correlation and similar expression profiles. group llt contains llt genes that have an unusual expression profile. the following genes cannot be clustered on the basis of pearson's coefficients: ie, ul, ul and us, which are likewise unusual indeed, these genes have peculiar expression profiles. the expression profile can also be visualized by heatmap presentation, using the expression data generated with the rΔtvalues. gene expression profiles of prv gene clusters were characterized by plus and minus signs at various time intervals. the numbers of plus or minus signs indicate the relative rate of increase or decrease of transcripts. co-regulated gene clusters we utilized our data to analyze whether the expression properties of prv genes localized in adjacent genomic loci display similarities. paa analysis pearson's correlation analysis of the gene clusters generated by using pearson's correlation coefficients revealed that genes within the same clusters are often located in neighboring genomic positions. group l is composed of members. the adjacent ul ul ul ul and ul genes are members of this group. the high pairwise correlation coefficients and the similar profile curves of the rΔ values of these genes cannot be explained by the nested co-localization alone, because ul and ul are divergent genes and do not overlap with ul ul and ul the ul gene is convergent with ul and ul26; and ul is divergent with ul moreover, ul is convergent with ul and ul it should e borne in mind that ul and ul an adjacent gene pair in the hsv, are relocalized into a remote position in the prv, due to a large inversion, but retain their expression properties. this is also true for ul ul and ul. several genes in the us region of the prv genome belong in this group, too. the ul and ul genes, which are oriented parallelly, but non-overlapping, likewise belong in group l the ul and ul genes are in a divergent position with ul group l is composed of members. the ul and ul genes are divergently oriented, while ul and ul are convergent genes. group l is a small group with members. the ul and ul genes are arranged in a divergent orientation. group e2: two of the members of this group, ul and ul are overlapping genes. group e3: two of the members of this group are divergently oriented with the third member, ul group m1: ul and ul are a convergent gene pair. the ul and ul are l genes oriented in parallel and separated by the divergent early ul gene. furthermore, ul and ul are a convergent adjacent gene pair. genes with similar expression profiles appear to be under a common regulation, which cannot be explained solely by the nested localization of genes. group llt: the llt and llt expression patterns show a high degree of similarity. however, there is noteworthy inverse relationship in the expression profiles of llt and llt with those of their complementary partners, ep and ie mrnas, respectively. discussion gene expressions of α-herpesviruses have already been analyzed by various techniques including northern-blot and microarray analysis. different authors have often assigned the same genes to different kinetic groups, which may be explained by the poor resolution of the applied techniques. only partial data are available on the expression of prv genes. flori et al. carried out genome-wide gene expression analysis via a microarray technique, but this approach did not produce evaluable data on the kinetic properties of the prv genes. we employed a novel qrt2-pcr technique using strand-specific primers for the rt to obtain a higher yield and to eliminate the effects of the potential antisense transcripts; and the average maximal ect values as the controls for the calculation of relative expression ratios. other approaches, such as the models of pfaffl's and souazé et al.,, and the Δct and ΔΔct models are simpler than the model we used; however, they all neglect the variation in the amplification efficiencies between cdna samples, which was present in our system. traditionally, herpesvirus genes are grouped into kinetic classes on the basis of gene expression inhibitory effects of protein and dna synthesis-blocking reagents. the ie genes should not be significantly inhibited by any of the above substances, the e genes are inhibited only by dna synthesis blockers, and the expression of the l genes is substantially inhibited by both protein and dna synthesis blockade. we grouped the prv genes into ie, e, e/l and l categories on the basis of the results of chx and paa treatment,. our findings conformed well with those published for the hsv genes. the expression of the ie gene is enhanced by chx treatment. the explanation of this phenomenon lies in the fact that the ie protein normally inhibits its own expression a short time after the onset of virus infection, a feature which is absent in chx-treated cells due to novel protein synthesis being blocked by the drug. the other prv genes were inhibited to a significant extent by chx at every examined time point. consequently, ie is the only ie gene of the prv. the ep and ul genes were shown to be e genes by others, and this was confirmed by our analyses. interestingly, us which is an ie gene in the hsv, appeared to be an l gene in most of our analyses: its expression was significantly inhibited by paa; it afforded a low amount of mrnas at h pi, and a high rate of increase of transcript level at 4- h pi, all of these being l characteristics. furthermore, together with a set of l genes, us belongs in group l according to the clustering of the genes on the basis of pairwise pearson correlation coefficients. the moderately high relative amount of us transcripts at h pi is the only e-like characteristic of this gene. however, us displays a high ct value, suggesting a low absolute amount of transcripts at h pi. we may mention here that the ct value of a transcript is dependent on the primer efficiency; these data therefore cannot be reliably used to the estimate the absolute transcript amount. if the us gene has an important function early in the lytic cycle of the virus, it might fulfill this without de novo synthesis by being released upon infection from the tegument layer of the virus where it might be incorporated. our data do not demonstrate a clear-cut demarcation line between the groups of e and l genes, suggesting that the categorization of herpesvirus genes on the basis of the requirements for dna replication might be arbitrary. the kinetic types of genes identified by our paa analysis resemble homologous hsv genes more than the published prv genes, which can be explained by the semiquantitative methods used for the kinetic analysis of the prv genes. the present study revealed that temporal classification of herpesvirus genes is also possible through study of the gene expression of untreated cells in consequence of the high sensitivity of the real-time rt-pcr technique. as a further interesting point, we detected mrnas by qrt2-pcr in all but prv genes as early as h pi. this observation indicates that prv genes are either leaky or subject to regulatory mechanisms which have not yet been elucidated. we found that e genes are generally expressed in a higher proportion than l genes at and h, as indicated by their higher r values at these time points. the e gene products proved to display a higher net increase than the l genes within the 0- h, 1- h and 2- h pi periods. however, by the 4- h pi interval, this trend had definitely reversed, i.e. the l genes exhibited high r values. unexpectedly, the ratio of increase between and h was higher for a number of l genes than any of the e genes. since the r2h values of these l genes were lower than those of the e genes, the high r value is a result of the very low r1h values of late genes. most of the l genes also have higher r and r values than those of e genes, which is explained by the fact that the e genes reach a relatively high expression level by h pi. the classification of the prv genes through the chx and paa analyses led to results very similar those generated by the analysis of untreated cells via the following calculation: r/r. the differences between the results of the two approaches can be explained in that paa analysis alone gives only a rough picture of the gene expression; a detailed kinetic analysis of the viral gene expression furnishes a more sophisticated picture. some genes classified as e or l by paa analysis display "irregular" kinetics under untreated conditions. the ie and perhaps us genes cannot be classified by paa analysis alone. analysis of the transcription kinetics of overlapping transcript sets is an important issue. it is not known whether downstream genes on polycistronic mrnas are translated, and if so, to what extent. this makes interpretation of the mrna expression data difficult because the mrna levels cannot be correlated with the amounts of the corresponding proteins, which are the workhorse molecules in regulating cellular physiology. in principle, a downstream gene is translated if it is transcribed from its own promoter. theoretically, downstream genes could also be translated from a polycistronic rna if alternative splicing removed upstream gene from the pre-mrna, or if potential ires-like sequences helped recruit ribosomes, thereby initiating a cap-independent translation from the downstream gene. thus, analysis of the downstream genes without discriminating between transcription from their own promoters or by read-through from upstream genes might result in a false categorization. the prv genes were also classified by analysis of the gene expression throughout the entire examination period by using pearson's correlation analysis. genes with similar expression profiles were placed in the same group. genes in the same kinetic class are generally situated in the same group generated by the use of person's correlation. moreover, members of a particular group rarely display high correlation coefficients with genes belonging in different groups. the prv genes conformed well to patterns of expression. only viral genes did not fit into any of the groups. we found an inverse expression profile of the sense/antisense partners in the ep0-llt and ie180-llt genes. combination of the untreated data with the results of the chx and paa analyses revealed an interesting relationship for coordinated regulation of sense/antisense partners. the chx analysis suggested that ie protein facilitates lap activity, and inhibits asp. the paa treatment resulted in a significantly elevated antisense transcript level at of the time points in llts, which indicates the existence of another regulatory level besides ie protein action. we assume that transcription from one dna strand negatively influences the expression of transcripts from the complementary dna strand. the interaction can occur at the level of transcription and/or translation by forming double-stranded rnas by the sense and antisense transcripts. as an example, paa has a negative effect on the transcription of ie at h pi, which results in a lower rate of transcription from ie; and a lower amount of inhibitory ie proteins, which also facilitates llt expression. overall, the llt level increases -fold relative to the untreated conditions. furthermore, genes with the same kinetic properties exhibit a distinctive distribution pattern along the prv genome. nested genes appear to belong in the same kinetic group. additionally, convergent genes and gene clusters in most cases belong to different kinetic classes. it should be remembered that the paa-based approach and the time intervals applied for kinetic analysis produce only a coarse resolution of gene expression. it is possible that convergent genes allocated to the same kinetic classes might display different expression profiles on a finer scale. the above genome organization principles may point to the existence of as yet unknown regulatory mechanisms. it may be speculated whether a read-through of transcription across convergent genes is the basis of this regulation. moreover, groups formed in terms of high pearson's correlation coefficients contain many genes localized at adjacent loci on the prv genome. most genes with high correlation coefficients are not nested genes. this means that the similarity of their expression profiles cannot be explained by the control of a common promoter. further, several genes in the same group are separated by genes that display different kinetic profiles. these results suggest the existence of a genetic mechanism that synchronizes gene expression on a higher-order scale.  although the relative amounts of the transcripts between genes cannot be compared because primer efficiencies may vary both in rt and in pcr, our method allows a comparison of genes on the basis of their expression dynamics. we detected the expression of almost all genes from the earliest time point, further, we have found that a sharp boundary cannot be drawn a between the groups of e and l genes in terms of expression profiles. we have also found that the prv has only a single ie gene and that the us gene is expressed in l kinetics. our kinetic studies revealed that paa analysis alone is not sufficient for the classification of genes on the basis of their expression profiles. the analysis of gene expression throughout the h examination period demonstrated that these genes can be subdivided into further clusters, and that genes showing significant differences in response to paa treatment can have very similar expression characteristics in untreated conditions. in our analysis, nested genes displayed the same expression profiles, whereas convergent genes mostly exhibited different kinetic properties. it should be noted that a finer resolution of the gene expression might expose slight differences in the expressions of nested genes. approximately half of the divergent genes belong in the same class, and the other half of them in different kinetic classes. the new calculation technique that we have developed is also applicable to evaluate the loss-of-function phenotypes of mutant herpesviruses viruses and, in principle, for the analysis of gene expression in every temporally changing genetic system. 
our investigations revealed that genes with the same expression properties form clusters on the prv genome: nested overlapping genes belong in the same kinetic class, while most convergent genes belong in different kinetic classes. we calculated the relative expression ratios in a novel way, which allowed us to compare different prv genes with respect to their expression dynamics, and to divide the prv genes into distinct kinetic classes. no sharp boundary was found between the groups of early and late genes classified on the basis of their requirement for viral dna synthesis. in principle, this new calculation technique is applicable for the analysis of gene expression in all temporally changing genetic systems. we additionally established the kinetic properties of uncharacterized prv genes and revised or confirmed data on prv genes earlier examined by traditional methods such as northern blot analysis. this is the first publication on the whole-genome analysis of the gene expression of an alpha-herpesvirus by qrt2-pcr. the expressions of the prv genes were analyzed, categorized and compared by qrt2-pcr assay, with the average of the minimum cycle threshold used as a control for the calculation of a particular r value. real-time rt-pcr is a very sensitive, accurate and reproducible technique that can be used to detect very small amounts of rna molecules, and it can therefore be applied for analysis of the expression of herpesvirus genes from the very early period of infection. most prv genes begin to be expressed from the onset of viral expression. in this study, we have developed and applied a quantitative reverse transcriptase-based real-time pcr technique in order to profile transcription from the whole genome of prv after lytic infection in porcine kidney cells. pseudorabies virus, a neurotropic herpesvirus of pigs, serves as an excellent model system with which to investigate the herpesvirus life cycle both in cultured cells and in vivo.
 pseudorabies virus, a neurotropic herpesvirus of pigs, serves as an excellent model system with which to investigate the herpesvirus life cycle both in cultured cells and in vivo. real-time rt-pcr is a very sensitive, accurate and reproducible technique that can be used to detect very small amounts of rna molecules, and it can therefore be applied for analysis of the expression of herpesvirus genes from the very early period of infection.  in this study, we have developed and applied a quantitative reverse transcriptase-based real-time pcr technique in order to profile transcription from the whole genome of prv after lytic infection in porcine kidney cells. we calculated the relative expression ratios in a novel way, which allowed us to compare different prv genes with respect to their expression dynamics, and to divide the prv genes into distinct kinetic classes. this is the first publication on the whole-genome analysis of the gene expression of an alpha-herpesvirus by qrt2-pcr. we additionally established the kinetic properties of uncharacterized prv genes and revised or confirmed data on prv genes earlier examined by traditional methods such as northern blot analysis. our investigations revealed that genes with the same expression properties form clusters on the prv genome: nested overlapping genes belong in the same kinetic class, while most convergent genes belong in different kinetic classes. further, we detected inverse relationships as concerns the expressions of ep and ie mrnas and their antisense partners.  most prv genes begin to be expressed from the onset of viral expression. no sharp boundary was found between the groups of early and late genes classified on the basis of their requirement for viral dna synthesis. the expressions of the prv genes were analyzed, categorized and compared by qrt2-pcr assay, with the average of the minimum cycle threshold used as a control for the calculation of a particular r value. in principle, this new calculation technique is applicable for the analysis of gene expression in all temporally changing genetic systems. 