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Viral Vectors for Gene Therapy: Methods and Protocols (Methods in Molecular Medicine) by Curtis A. Whether to assess the function of new genes identified from the Human Genome Project or to apply gene therapy successfully, it is often necessary to deliver genes to specific cells. T?t c? sach s? hoa gi?i thi?u t?i di?n dan du?c cac thanh vien suu t?m t? nhi?u ngu?n mi?n phi tren m?ng, vi v?y r?t co th? chua co s? d?ng y t? tac gi?. Dave Hawkes receives funding from the NHMRC and from the Name the Virus crowdfunding campaign.
The Conversation is funded by CSIRO, Melbourne, Monash, RMIT, UTS, UWA, ACU, ANSTO, ANU, Canberra, CDU, CQU, Curtin, Deakin, ECU, Flinders, Griffith, the Harry Perkins Institute, JCU, La Trobe, Murdoch, Newcastle, UQ, QUT, Swinburne, Sydney, UNDA, UNE, UniSA, UNSW, USC, USQ, UTAS, UWS, VU and Wollongong.
Viruses have traditionally been mankind’s enemies, causing disease and often mutating out of the reach of our medicines.
Viral vectors show great promise so expect to hear a lot more about them in the future, starting with this brief introduction. They’re basically viruses modified to not cause disease that are then used to introduce genetic material into cells.
Viral vectors are already being used by scientists to understand how our genes influence our bodies – from how we form memories through to why blood pressure increases when we’re stressed.
A recent short film tells the story of a young girl called Emma, who had leukaemia for the second time. In Emma’s case, the viral vector changed her blood cells into a specific type of cell researchers called a “Serial Killer” cell. Viral vectors can be based on any number of different viruses, including those that cause serious illness such as HIV, which was used in Emma’s case. But one thing all viral vectors have in common is that, unlike normal viruses, they don’t cause disease.
In addition to treating cancer, viral vectors are at the forefront of many other areas of medical research – from gene therapies to understanding the brain.
Gene therapies involve introducing normal genes into a person’s cells in place of missing or defective ones in order to correct genetic disorders. While gene therapies have been in clinical trials for many years, it was only in October 2012 that the first gene therapy product, Glybera, was given marketing approval in the European Union where it will launched this year.
Viral vectors are being used for a wide range of scientific research but one of the most interesting is in understanding how the brain works. The human brain is made up of over 100 billion cells and these cells are divided up into over 1,000 different regions, which contain make different types of cells. Viral vectors provide a way to investigate the function of specific cells or even certain proteins in different areas of the brain. Modified viruses are used to infect cells within an area of the brain and change them, by, for instance stopping the cells producing a specific protein or making them unresponsive to normal stimuli.
One of the advantages of viral-based delivery over other methods of vaccine production is that they can produce good levels of immunity but are much safer than some existing vaccines because they can’t cause infection.
Humans have feared viruses for thousands of years but, with the arrival of viral vectors, we may have discovered the next great tool in the fight against disease.
A virus is essentially an information system (encoded in DNA or RNA) surrounded by a protective coat. A researcher looking at E.coli bacteria strain at the Institute of Food Safety, Animal Health and Environment in Latvia.
Receptors and Signal TransductionChodosh, JamesBiology and evolution of human adenoviruses. Martins I, Cabral L, Pinto A, Wilson SP, Lima D and Tavares I (2011) Reversal of inflammatory pain by HSV-1-mediated overexpression of enkephalin in the caudal ventrolateral medulla.A  European Journal of Pain 15, 1008-1014.
Walder RY, Gautam M, Wilson SP, Benson CJ and Sluka KA (2011) Selective targeting of ASIC3 using artificial miRNAs inhibits primary and secondary hyperalgesia following muscle inflammation.A  Pain 152, 2348-2356. Paranjape SA, Chan O, Zhu W, Horblitt AM, Grillo CA, Wilson S, Reagan L and Sherwin RS (2011) Chronic Reduction of Insulin Receptors in the Ventromedial Hypothalamus Produces Glucose Intolerance and Islet Dysfunction in the Absence of Weight Gain.A  American Journal of Physiology - Endocrinology and Metabolism 301, E978-E983.
Krause EG, de Kloet AD, Scott KA, Flak JN, Jones K, Smeltzer MD, Ulrich-Lai YM, Woods SC, Wilson SP, Reagan LP, Herman JP and Sakai RR (2011) Blood-borne angiotensin II acts in the brain to influence behavioral and endocrine responses to psychogenic stress.
Tomasiewicz HC, Jacobs MM, Wilkinson MB, Wilson SP, Nestler EJ and Hurd YL (2012) Proenkephalin mediates the enduring effects of adolescent cannabis exposure associated with adult opiate vulnerability.A  Biological Psychiatry, in press. The department provides expert instruction and conducts innovative research in neuroscience, physiology and pharmacology. Following a lytic infection of the peripheral epithelium, HSV-1 enters sensory neurons and is trafficked by retrograde axonal transport to the sensory ganglia.
It may not display this or other websites correctly.You should upgrade or use an alternative browser. In Gene Delivery to Mammalian Cells, highly experienced researchers describe in great detail methods that have proven most useful in delivering genes to mammalian cells.
Cho nen, qui tac gi? khong cho dang tac ph?m c?a minh vui long lien h? BQT di?n dan d? du?c g? b?. We use a Creative Commons Attribution NoDerivatives licence, so you can republish our articles for free, online or in print. These genes can alter the behaviour of the cells to cure disease or allow scientists to learn about the nature of the cells.
Doctors treated Emma by removing some of her own blood cells, infecting them with a special type of human immunodeficiency virus (HIV), and injecting the infected cells back into her body. When these infected cells were injected back into Emma’s bloodstream, they grew and began to kill the cancer cells in her body. The most common way these normally dangerous, or at least unpleasant, viruses are made safe is by removing their ability to reproduce. Other popular targets include diseases caused by changes in single genes, such as cystic fibrosis, heart disease, infectious disease and diseases of the brain or eye. Until recently, this has made studying the specific function of cells in the brain very difficult.
Changes like these can help researchers understand the cells' contribution to complex behaviour such as sleep, feeding or emotional stress. Vaccines produced from viral vectors are currently being trialled for a wide variety of diseases from HIV to bird flu in ducks. Department faculty members teach students of medicine, nurse anesthesia and biomedical science in the classroom and the laboratory. Chapter cont- butions provide perspective in the use of viral vectors for applications in the brain and in the central nervous system. A latent infection then ensues where gene expression from the viral genome is limited to the LATs. We are developing models that identify a threshold of modified cells that is necessary to eliminate HIV infected cells. Volume 2: Viral Gene Transfer Techniques details procedures for delivering genes to cells in vitro and in vivo, including the use of lentiviral vectors, adenovirus, adeno-associated viruses, alphavirus, herpes simplex virus, baculovirus, and retrovirus.
Research activities focus on the roles of specific molecules in cellular function and their importance to organismal physiology and behavior. Although a PNS neuron is depicted, it is generally accepted that the events surrounding the formation of a latent infection in neurons of the PNS and the CNS (the focus of this paper) are similar. Many of these techniques have only been in practice for a few years and are still being refined and updated. Each protocol contains step-by-step instructions, along with background notes, equipment and reagent lists, and tips on troubleshooting and avoiding known pitfalls. The tegument is surrounded by a lipid membrane containing many viral glycoproteins that are important for attachment and penetration (2) of cell membranes.
Introductory chapters review the delivery methods presented, discussing their advantages and disadvantages, how they have been used successfully for gene delivery, and the future of their technology. After penetration of the virion into the cell cytoplasm it has been seen in EM studies that the tegument can drift away from the capsids. 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.ResultsIn 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.
An accompanying volume, Volume 1: Nonviral Gene Transfer Techniques, focuses on gene delivery by a variety of chemical and physical methods. 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. Alternatively a cellular protein (Zhangfei) may inhibit the ability of Vp16 to activate the IE genes.44 With lack of gene expression or DNA replication the viral genome becomes progressively covered with nucleosomes and forms stable chromatin (6).
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 EP0 and IE180 mRNAs and their antisense partners.ConclusionMost (if not all) 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. Long-term HSV vectors utilize this promoter by inserting a complementary DNA for the gene to be expressed in front of the LAT promoter.
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.
The PRV is a valuable model organism in herpesvirus research [2] and also a powerful tool in neuroscience, employed to map neural circuits [[3-5], and [6]] and gene delivery [7].
First, the IE genes are expressed, independently of de novo protein synthesis from the virus. PRV IE180 protein [homologous to herpes simplex virus (HSV) ICP4 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. Finally, the L genes are transcribed, encoding mainly structural proteins involved in virion assembly.
Herpesviruses are capable of two types of infections: lytic (or productive) and latent [2]. 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 (LAT region) 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 ICP27 protein, which helps in the recognition of the internal polyA signals, resulting in differential transcript lengths [16].
The gene expression of α-herpesviruses, such as HSV types 1 and 2, Varicella-zoster virus, bovine herpesvirus type 1 and PRV has been investigated earlier by traditional methods including Northern blot analysis, ribonuclease protection assay, and end-point RT-PCR analysis. 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. 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. 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.
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 [27] 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. 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 (Rs) 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.ResultsExperimental designFor each gene, a minimum of 3 independent replicates were carried out for statistical confidence, and the median of these values along with the standard error was calculated.
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 (Ct) values for PRV genes at different time points than infecting with a higher MOI (data not shown).
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 (data not shown), and therefore we restricted our analysis to 6 h post-infection (pi). Prior to PRV infection, cells were either untreated or treated with cycloheximide (CHX), a protein synthesis blocker, or phosphonoacetic acid (PAA), 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 (this issue will be described elsewhere). Another reason was that it produced a much higher amount of specific cDNAs than oligo-dT priming (data not shown), which was especially important under our experimental conditions with a low MOI. 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 (maximal gene expression occurred at 6 h pi for all genes except us3 and llt2, which peaked at 4 h and at 2 h pi, respectively).
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 (formation of a single product with the appropriate molecular weight was accepted); 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 [see Additional file 1]. 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. In general, inhibitory drugs are applied to infected cells for a prolonged incubation period (24, 48 or 72 h; [[31,32] and [33]].
At the indicated time points, RNA was isolated and converted to cDNA, which was subsequently analyzed by qRT2-PCR.
We explain this phenomenon in that the IE180 proteins exert an inhibitory effect on their own synthesis (upon the binding of their own promoters; [34]), which is resolved by CHX blocking protein synthesis from the IE180 mRNAs. The repression of llt1 expression at 2 h pi is relatively low (Ri-CHX-2h = 0.281), but this antisense transcript is significantly blocked by CHX at 4 and 6 h pi. Overall, our CHX analysis indicated that the only true IE protein-encoding gene of PRV is ie180.
While the icp27 and icp0 genes in the HSV are IE genes, their PRV homologs ul54 [11] and ep0 [10] genes were earlier described as E genes, which was confirmed by our CHX analysis.
The icp22 gene of HSV is expressed with IE kinetics; our analysis revealed that its homologous counterpart, the us1 gene of PRV, is significantly blocked by CHX, and hence it is not an IE gene. As a result of CHX treatment, LLT2 exhibits elevated levels at 2 and 4 h pi, which indicates that IE180 transcription factor exerts a negative effect on its expression. However, llt2 displays fairly low relative expression at 1 h pi [see Additional file 2], and therefore it cannot be regarded as an IE gene beyond doubt.Figure 3Effects of the CHX on the expression of ie180, llt1, llt2 and ep0 genes. 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. 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 (at least in our system). The PRV genes were ranked from the least inhibited (highest Ri-PAA) to the most inhibited (lowest Ri-PAA). 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.
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 (ul52) of the HSV as the last member of the E group, and a typical L gene (ul32) of the HSV as the first member of the L group. 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 strong inhibitory effect of PAA on the us1 expression is surprising because it is an IE gene in the HSV. Furthermore, ul13 is an L gene in the HSV, but its transcripts were detected at 2 h pi in the PRV. Peaking of the us3 gene expression at 4 h can be explained by the important role of US3 protein kinase in blocking the apoptotic pathway of the compromised cell in the E phase of infection [61].
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. Horizontal bars show the PRV genes ranked on the basis of Ri-PAA values in an increasing sequence. The data from these experiments are presented in detail in the text [see Additional file 2 and 4]. The us3 PRV gene peaks at 4 h, llt2 at 2 h, while the other genes reach their maximum at 6 h within the 0-6 h examination period.0-6 h incubation periodThe 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 (RΔ) in each time interval for the comparison [see Additional file 4b].
Surprisingly, genes belonging in a particular group very rarely give similarly high values as any of the genes belonging in other groups.
The only exception was ul13 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 our system, therefore, most of the data (except the relatively high R2h for us1), suggest that it is an L gene. However, this gene is characterized as an E gene in the PRV [39], which was confirmed by our PAA analysis.
Pearson's correlation analysis was performed to group the PRV genes into clusters on the basis of similar expression dynamics (using pairwise RΔ values) over the four time intervals.
The dendogram depicts the whole-genome profiling of protein-coding PRV genes, following de novo infection of PK-15 cells.
Nested 3' co-terminal genes were found to belong in the same kinetic classes, indicating the existence of shared regulatory mechanisms of gene expression.
An exception is the late ul27 gene, which forms a cluster overlapping in parallel with the ul28 and ul29 genes expressed with E kinetics. Of the 11 divergent pairs, 6 genes were found to belong in the same and 5 in different kinetic classes.
PRV genes are represented as arrows, which indicate their relative lengths, orientations and kinetic classes determined by PAA analysis.
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 ul5 and ul6 are divergent genes and do not overlap with ul1, ul2 and ul3. It should e borne in mind that ul26 and ul27, 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.
Several genes (us2, us6, us8 and us9) in the US region of the PRV genome belong in this group, too. The ul17 and ul19 genes, which are oriented parallelly, but non-overlapping, likewise belong in group L1. The ul32 and ul33 genes are divergently oriented, while ul4 and ul3.5 are convergent genes.
Group E3: Two (ul11 and ul12) of the 4 members of this group are divergently oriented with the third member, ul9. The ul49.5 and ul51 are L genes oriented in parallel and separated by the divergent early ul50 gene. Genes with similar expression profiles appear to be under a common regulation, which cannot be explained solely by the nested localization (these genes also have common promoters) of genes. Different authors have often assigned the same genes to different kinetic groups, which may be explained by the poor resolution of the applied techniques. We employed a novel qRT2-PCR technique using (1) strand-specific primers for the RT to obtain a higher yield and to eliminate the effects of the potential antisense transcripts; and (2) the average maximal ECt values as the controls for the calculation of relative expression ratios (Rs). 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. The explanation of this phenomenon lies in the fact that the ie180 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.
The ep0 and ul54 genes (IE genes in the HSV) were shown to be E genes by others [[10] and [11]], and this was confirmed by our analyses.
Furthermore, together with a set of L genes, us1 belongs in group L2 according to the clustering of the genes on the basis of pairwise Pearson correlation coefficients. The moderately high relative amount of us1 transcripts at 2 h pi is the only E-like characteristic of this gene. However, us1 displays a high Ct value, suggesting a low absolute amount of transcripts at 2 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 us1 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 3 PRV genes as early as 1 h pi [see Additional file 4]. 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 1, 2 and 4 h, as indicated by their higher R values at these time points [see Additional file 4a]. The E gene products proved to display a higher net increase (RΔ) than the L genes within the 0-1 h, 1-2 h and 2-4 h pi periods. Unexpectedly, the ratio of increase (Ra) between 1 and 2 h was higher for a number of L genes than any of the E genes [see Additional file 4c]. 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.
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. Theoretically, downstream genes could also be translated from a polycistronic RNA if alternative splicing removed upstream gene(s) from the pre-mRNA, or if potential IRES-like sequences helped recruit ribosomes, thereby initiating a cap-independent translation from the downstream gene (we have found no data in the literature concerning these possibilities). 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 (high pairwise r values) were placed in the same group.
Genes in the same kinetic class (identified by PAA analysis) are generally situated in the same group generated by the use of Person's correlation. Moreover, members of a particular group (with high r values) rarely display high correlation coefficients with genes belonging in different groups.
The CHX analysis suggested that IE180 protein facilitates LAP (latency-associated transcript promoter) activity, and inhibits ASP (putative antisense promoter activity). The PAA treatment resulted in a significantly elevated antisense transcript level at 3 of the 4 time points in LLTs, which indicates the existence of another regulatory level besides IE180 protein action. We assume that transcription from one DNA strand negatively influences the expression of transcripts from the complementary DNA strand. As an example, PAA has a negative effect on the transcription of ie180 (the level of IE180 mRNA is reduced to a quarter) at 4 h pi, which results in a lower rate of transcription from ie180 (thereby facilitating the expression of LLT2); and a lower amount of inhibitory IE180 proteins, which also facilitates LLT2 expression. Furthermore, genes with the same kinetic properties exhibit a distinctive distribution pattern along the PRV genome. Additionally, convergent genes and gene clusters in most (3 out of 11) 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. 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 (chromatin?) scale.ConclusionAlthough 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 (1 h), 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 (the ie180 gene) and that the us1 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 6 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.



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