Patterns of Ad12-specific gene transcription.
Ad12-specific transcripts were detected by DNA macroarray analyses using nylon membranes carrying single-stranded Ad12 DNA sequences from regions of the viral genome, as indicated for the controls in Fig. . In mock-infected HeLa cells (Fig. ), none of the Ad12-specific transcripts could be found. It was ascertained that all spots on the microarray carried Ad12 DNA sequences, since they all hybridized with 32
P-labeled Ad12 virion DNA (data not shown). At 14 (31
) and 24 (Fig. ) h p.i., transcripts from all early genes (E1 to E4) were detectable. Particularly high expression was observed from E1B and for RNAs corresponding to the 12.1- and 14.7-kDa E3 proteins. Although some of the late gene transcripts were detected at 14 h p.i. (fiber, endoprotease, and major core), most were found only at 24 h p.i. However, transcription of the genes for the 52-kDa L1 and the 100- and 33-kDa L4 proteins was very low. At 32 h p.i. (Fig. ), RNA corresponding to the Ad12 terminal protein, DNA polymerase, and most of the E4 genes was no longer present. However, transcripts of the 12.1- and 14.7-kDa E3 genes and the pIVa2, minor core, 11-kDa, endoprotease, and fiber genes were abundant. The hexon and penton gene transcripts were almost equally transcribed at 24 and 32 h p.i. Obviously, at 32 h p.i. most of the assembly functions, pIVa2, L1, and the endoprotease as well as the fiber, hexon, penton, and 11-kDa proteins were strongly transcribed. Similarly, the transcripts of the genes for the precursor proteins pIVa2, pVI, and pVIII were abundantly represented at 24 and 32 h p.i., as well as the transcript of the gene for the endoprotease which was responsible for the maturation of the precursor proteins. The viral proteins IIIa, VI, VIII, and IX play a double role early during infection in that they stabilize and disassemble the capsid (22
FIG. 1. DNA array analyses of Ad12 genes expressed in Ad12-infected HeLa cells. Cytoplasmic RNAs were isolated from mock-infected HeLa cells (A) or Ad12-infected HeLa cells at 24 h p.i. (B) or 32 h p.i. (C). The RNAs were reverse transcribed into cDNAs which (more ...)
Even as late as 32 h p.i. (Fig. ), some of the genes for the E3 proteins, which were, at least in part, dispensable for the replication of adenoviruses in cell culture but antagonize cellular and immune defense functions, showed predominant transcription. Lastly, at 48 h p.i., the Ad12 transcription profile was not significantly different from that at 32 h p.i. (data not shown).
In Ad12-infected cells, the 14-h time point just marks the transition from the early to the late phase of the infection cycle. Significant changes in the late expression profile occur between 14 and 24 h p.i., with continued early gene expression up to 48 h p.i. These very late transcripts might be related to the observation of up-regulated cellular gene activity late in the infection cycle.
Overview of cellular gene transcription in Ad12-infected human HeLa cells at 24, 32, and 48 h p.i.
As detailed in Materials and Methods, we initially chose nylon membranes carrying 8,400 cellular genes in duplicate to obtain an overall survey of cellular transcription patterns following Ad12 infection. About 6,000 of these genes were known human genes. Late in the infection cycle, the results shown in Fig. documented the gradual and almost general switchoff of cellular gene expression. At 32 h p.i. (Fig. ) and at 48 h p.i. (Fig. ), >90% of the cellular genes on the membrane showed down-regulated transcription or no transcription at all in comparison to mock-infected HeLa cells (Fig. ) or to Ad12-infected HeLa cells during the first 24 h p.i. (Fig. ). Surprisingly, at 32 h p.i., about 1.3% of the genes on the membrane exhibited enhanced transcription by a factor of at least 3. At 48 h p.i. (Fig. ), the transcription of about 2% of the genes was up-regulated. The products of some of these genes might be required for efficient viral replication or for safeguarding against cellular apoptosis or had played a role in countering cellular mechanisms active in curtailing viral replication. Moreover, expression of some of the genes showing increased transcription at 32 h p.i. continued to increase even at 48 h p.i. In light of the sustained transcription of Ad12 genes at very late time points, we conclude that the Ad12-infected cell retains active transcription regulatory mechanisms for both viral and cellular genes as late as 48 h p.i. As an example of an up-regulated gene (the eucaryotic translation initiation factor 4 gene) and a down-regulated gene (the major histocompatibility complex [MHC] gene), an enlargement of membrane sections in Fig. is shown (Fig. ). E1A-mediated down-regulation of the class I MHC genes was demonstrated earlier in Ad12-transformed cells (1
FIG. 2. Overview of the time course of human cellular gene transcription in mock-infected or in Ad12-infected HeLa cells at various times after infection. Cellular transcription patterns were determined by using membrane arrays containing 8,600 cellular genes. (more ...)
FIG. 3. Enlarged view of two selected segments from the arrays shown in Fig. (mock infection) and C (32 h p.i.). Two examples for differentially regulated genes were presented: up-regulation of the eucaryotic translation initiation factor 4 and (more ...)
As controls, HeLa cells at 24 h after mock infection were used for comparisons to all time points of viral infection. This time point after mock infection was chosen as a compromise, since cells stop growing approximately 24 h p.i. Hence, cells overgrown at later times after mock infection could not have served as appropriate controls.
Quantitative analyses of alterations in cellular gene transcription in HeLa cells at 12, 24, 32, and 48 h after infection with Ad12.
Although the use of nylon membrane DNA macroarrays allowed for a qualitative visual overview of cellular transcription patterns after the infection of HeLa cells with Ad12, it proved difficult to quantify these data reliably. For the more detailed identification and quantitative analysis of specific changes in cellular gene transcription, we therefore turned to glass slide cDNA microarray analyses. cDNA preparations from reverse transcribed RNA from mock-infected or Ad12-infected cells, which were labeled with two different chromophores, were simultaneously hybridized to microarrays containing 7,500 cDNA clones. Three different sets of RNA samples from three independent time course experiments of mock- and Ad12-infected cells were prepared. Each RNA preparation was analyzed on two arrays in which the dye labeling was reversed (dye swap). Hence, the data for each time point were derived from six different microarrays. In the data presented here, the two complementary hybridization experiments yielded congruent results.
The glass slide DNA microarray data were evaluated by using the software significance analysis of microarrays (27
). Table presents a number of cellular genes with significantly altered transcription in HeLa cells at 12, 24, 32, or 48 h after infection with Ad12 in comparison to mock-infected HeLa cells. In the first comparison, we analyzed transcription profiles at a time when the late phase of infection was clearly established (Fig. ). This significance analysis included all genes which yielded signals in at least four of the six arrays at each time point. After the analysis, only genes with a false discovery rate lower than 4% were included in the final gene list. At 24 h p.i., 15 genes were negatively regulated by a factor of at least 2 relative to their transcription in uninfected HeLa cells. The same genes, as well as a few additional ones, were also down-regulated by the same criteria at 32 and 48 h p.i., many of them by factors similar to or even higher than those at 24 h p.i. (Table ). The down-regulated genes included those for the transcription factors JUNB, NFKBA1, and NR4A1, the cell cycle regulator CCND1, and several cell surface receptors and apoptosis and immune response regulators. The strongest down-regulation of the cell adhesion molecule CYR61 together with the down-regulation of a number of the cytoskeletal genes, such as the SDCBP gene, were consistent with the severely compromised cellular organization of cells late in Ad12 infection.
Changes in transcriptional activities upon the infection of HeLa cells with Ad12 as determined by the DNA microarray technique and in comparison to mock-infected HeLa cellsa
In addition to this finding, and functionally important, the transcription of several cellular genes was up-regulated at 32 and 48 h p.i. The transcription of the RIS, ATF4, DSIPI, ANPEP, SCD, ALDH3A1, CYP2S1, and CTSF genes (Table ) was significantly enhanced at 32 and 48 h p.i. Importantly, these effects were essentially absent at 24 h p.i., and this finding supports the late up-regulation of specific cellular genes which was observed in the nylon filter cDNA macroarray experiments (Fig. ). The identities of these cellular genes placed them among the signal transduction or transcriptional regulator classes (RIS, DSIPI, and ATF4) and genes for protein degradation (CTSF and ANPEP genes) and lipid metabolism (SCD and CYP2S1 genes).
In the second comparison, we analyzed cells at 12 h p.i., which corresponded to the time in the viral infection cycle when viral DNA replication commenced but when late viral gene expression had not yet begun. The cellular transcription profile at 12 h p.i. was found to be significantly different from that at the late phase of infection. Approximately 30% of the identified genes were found to be up-regulated. In particular, the expression of three cellular immune response genes, the G1P2, IFIT1, and IFIT2 genes, was increased between ~10- and 30-fold (Table ). A strong induction was also observed for the RIG-I helicase gene and the MMP15 metalloproteinase gene. The KRT14 and KRT19 cytoskeleton genes were induced at 12 h p.i. but progressively repressed at later time points. Among the cellular genes that were down-regulated at 12 h p.i., repression rarely exceeded twofold. Adenovirus infection was previously shown to inhibit the transcription of histone genes (4
Validation of the alterations in cellular gene transcription by Q-RTPCR.
The significance of the results obtained by DNA microarray analyses was validated by Q-RTPCR. Generally, the same RNA preparation was used for both the Q-RTPCR and the cDNA microarray analyses. The transcriptional activity was normalized to the activity of the β-actin gene, whose expression was not noticeably affected by Ad12 infection. The results obtained for several cellular genes are summarized in Table , which also presents a quantitative comparison to the data obtained from the microarray system. For all genes analyzed, except for the late expression of the G1P2 gene, the results obtained by the two independent methods proved satisfactorily congruent. Notably, the relative change in expression of IL-6 was more pronounced by Q-RTPCR analysis than by the microarray data. The MCC gene was not present on the cDNA microarrays but was included in the results obtained from the nylon membrane experiments. Upon Ad12 infection of HeLa cells, the cellular MCC, CTSF, and ANPEP genes were markedly up-regulated, as documented by both the array and Q-RTPCR analyses (Tables and ). The majority of cellular genes was down-regulated after Ad12 infection, and this finding was corroborated by Q-RTPCR for the cellular JUNB, ATF2, IL-6, c-MYC, CYR61, and FUBP genes (Tables and ).
Cellular gene expression as analyzed by Q-RTPCR compared to microarray analyses
Western blot analysis.
In order to investigate and confirm the changes in transcription activities, the protein levels of one up-regulated gene (the CTSF gene) and one down-regulated gene (the CYR61 gene) were analyzed by the Western blotting technique (Fig. ). There were no major differences in the amount of CTSF protein between mock-infected cells and HeLa cells infected with Ad12 at 24 or 32 h p.i. At 48 h p.i., however, the CTSF protein levels increased by a factor of 5 compared to mock-infected cell protein levels. For CYR61, the opposite result was obtained (Fig. ). Up to 48 h p.i., the CYR61 protein level remained constant and comparable to that in mock-infected cells. However, at 48 h p.i., the amount of CYR61 protein dropped by a factor of around 4 compared to mock-infected cells. For both genes, the amounts of protein corresponded well to the results obtained by microarray and Q-RTPCR analyses (Table ). In conclusion, the increased transcriptional activity of the CTSF gene at 48 h p.i. also led to a marked overproduction of the gene product. A similar congruence between the decreased transcriptional and translational activities was also confirmed for the CYR61 gene. In further analyses of the Ad12 system, it will be important to address changes in translational levels in more detail.
FIG. 4. Western blot analyses for two of the differentially expressed cellular genes. As described in Materials and Methods, total cellular protein was prepared from HeLa cells at 24, 32, and 48 h p.i. with Ad12. Mock-infected HeLa cells were also analyzed. By (more ...)