Hongxing Zhao

Strategic attack on host cell gene expression during adenovirus infection

ABSTRACT To understand the interaction between the virus and its host, we used three sources of cDNA microarrays to examine the expression of 12,309 unique genes at 6 h postinfection of HeLa cells with high multiplicities of adenovirus type 2. Seventy-six genes with significantly changed expression ratios were identified, suggesting that adenovirus only modulates expression of a limited set of cellular genes. Quantitative real-time PCR analyses on selected genes were performed to confirm the microarray results. Significantly, a pronounced transcriptional activation by the promiscuous E1A-289R transcriptional activator was not apparent. Instead, promoter sequences in 45% of the upregulated genes harbored a potential E2F binding site, suggesting that the ability of the amino-terminal domain of E1A to regulate E2F-dependent transcription may be a major pathway for regulation of cellular gene expression. CDC25A was the only upregulated gene directly involved in cell cycle control. In contrast, several genes implicated in cell growth arrest were repressed. The transforming growth factor beta superfamily was specifically affected in the expression of both the upstream ligand and an intracellular regulator. In agreement with previous reports, adenovirus also targeted the innate immune response by downregulating several cytokines, including CLL2, CXCL1, and interleukin-6. Finally, stress response genes encoding GADD45B, ATF3, and TP53AP1 were upregulated. Importantly, we also found a novel countermeasure—activation of the apoptosis inhibitor survivin.

The transcriptome of the adenovirus infected cell

Alternations of cellular gene expression following an adenovirus type 2 infection of human primary cells were studied by using superior sensitive cDNA sequencing. In total, 3791 cellular genes were identified as differentially expressed more than 2-fold. Genes involved in DNA replication, RNA transcription and cell cycle regulation were very abundant among the up-regulated genes. On the other hand, genes involved in various signaling pathways including TGF-β, Rho, G-protein, Map kinase, STAT and NF-κB stood out among the down-regulated genes. Binding sites for E2F, ATF/CREB and AP2 were prevalent in the up-regulated genes, whereas binding sites for SRF and NF-κB were dominant among the down-regulated genes. It is evident that the adenovirus has gained a control of the host cell cycle, growth, immune response and apoptosis at 24 h after infection. However, efforts from host cell to block the cell cycle progression and activate an antiviral response were also observed.

Identification of Specific Cellular Genes Up-Regulated Late in Adenovirus Type 12 Infection

ABSTRACT The infection of human cells by adenoviruses leads to a gradual reduction in the activity of host cell functions while viral gene expression progresses in a regulated way. We used the DNA microarray technique to determine the transcriptional activity profiles of cellular genes upon infection with adenovirus type 12 (Ad12). The microarray data were validated by quantitative real-time PCR for genes which showed significant alterations after Ad12 infection. At 12 h postinfection, there is a striking up-regulation between 10- and 30-fold in the expression of the G1P2, IFIT1, and IFIT2 cellular immune response genes compared to mock-infected cells. At later stages of infection, when the majority of regulated cellular genes has been turned down, a limited number of cellular genes exhibit increased activities by factors of 3 or less. These genes belong to the signal transduction or transcriptional regulator classes or are active in protein degradation, like ANPEP, an aminopeptidase. The SCD and CYP2S1 genes function in lipid metabolism. The eucaryotic translation initiation factor 4 is up-regulated, and one of the major histocompatibility complex genes is diminished in activity. For two of the genes, one up-regulated (CTSF gene) and one down-regulated (CYR61 gene), alterations in gene activity were confirmed at the protein level by Western blotting experiments. Increased genetic activity of cellular genes late in adenovirus infection has not been reported previously and demonstrates that Ad12 has a sustained control of host cell gene expression well into the late phase of infection.

A new look at adenovirus splicing.

Adenovirus type 2 RNA splicing events were quantitatively mapped by using deep cDNA sequencing. The majority of the previously identified splice sites were detected. The lack of complete consistency between the present and previous results is because of some sites which were incorrectly mapped in previous studies, such as the splice sites for pVII, pVIII and E3-11.6K. Several previously predicted splice sites such as that for E3-14.5K and E4ORF3/4 were not detected. In addition, several new splice sites were identified. The novel RNAs may code for hitherto undetected proteins or alternatively spliced mRNAs for known proteins. The open reading frames downstream of two novel splice sites, located in the major late transcription unit region, were shown to be highly conserved. Another interesting possibility is that some of them are non-coding RNAs. Finally, the adenovirus mRNA polyadenylation sites were accurately mapped and in some cases shown to be heterogeneous.

Activation of the interferon-induced STAT pathway during an adenovirus type 12 infection.

We have previously described a temporal regulation of host cell gene expression during adenovirus type 2 infection (Ad2) of primary human fibroblasts. Among the eleven percent of genes deregulated by Ad2, a large fraction included genes involved in cell cycle, growth control and antiviral defense, consistent with the capacity of Ad2 to efficiently master the infected cell and cause an effectively productive infection. Adenovirus type 12 (Ad12), which belongs to the highly oncogenic subgroup, is characterised by slow progression, less cytopathic effect and lower virus yield compared to the non-oncogenic Ad2. Microarray analysis of host cell gene expression in Ad12 infected human lung fibroblasts (IMR90) demonstrated a quantitatively and qualitatively less impact on host cell gene expression, compared to Ad2. Of the relatively few genes up regulated during the course of Ad12 infection only two (5%) were identified as potential E2F targets, compared to the significant activation of E2F-dependent transcription observed during an Ad2 infection. Although approximately 30% of the genes deregulated by Ad12 were previously identified in Ad2-infected cells, a distinct difference was observed in a group of interferon-stimulated genes (ISGs). G1P2, IFI6, IFI16, IFIT1, IFIT2, IFITM1 and IRF9 were activated during the very late stage of infection, and a consistent induction of IFNbeta gene expression, preceding induction of the ISGs, was demonstrated by quantitative real-time PCR analysis. An activated JAK/STAT signalling pathway was also indicated by the accumulation of all components (STAT1, STAT2 and IRF9) of the ISGF3 transcription factor. Significantly, none of these ISGs was activated in Ad2 infected IMR90 cells. Thus, the inability of Ad12 to evade the interferon response might explain its restricted virulence.

Fluctuating expression of microRNAs in adenovirus infected cells.

The changes in cellular microRNA (miRNA) expression during the course of an adenovirus type 2 infection in human lung fibroblast were studied by deep RNA sequencing. Expressions of 175 miRNAs with over 100 transcripts per million nucleotides were changed more than 1.5-fold. The expression patterns of these miRNAs changed dramatically during the course of the infection, from upregulation of the miRNAs known as tumor suppressors (such as miR-22, miR-320, let-7, miR-181b, and miR-155) and down-regulation of oncogenic miRNAs (such as miR-21 and miR-31) early to downregulation of tumor suppressor miRNAs (such as let-7 family, mir-30 family, 23/27 cluster) and upregulation of oncogenic miRNAs (include miR-125, miR-27, miR-191) late after infection. The switch in miRNA expression pattern occurred when adenovirus DNA replication started. Furthermore, deregulation of cellular miRNA expression was a step-wise and special sets of miRNAs were deregulated in different phases of infection.

Distinct temporal changes in host cell lncRNA expression during the course of an adenovirus infection.

Abstract The deregulation of cellular long non-coding RNA (lncRNA) expression during a human adenovirus infection was studied by deep sequencing. Expression of lncRNAs increased substantially following the progression of the infection. Among 645 significantly expressed lncRNAs, the expression of 398 was changed more than 2-fold. More than 80% of them were up-regulated and 80% of them were detected during the late phase. Based on the genomic locations of the deregulated lncRNAs in relation to known mRNAs and miRNAs, they were predicted to be involved in growth, structure, apoptosis and wound healing in the early phase, cell proliferation in the intermediate phase and protein synthesis, modification and transport in the late phase. The most significant functions of cellular RNA-binding proteins, previously shown to interact with the deregulated lncRNAs identified here, are involved in RNA splicing, nuclear export and translation events. We hypothesize that adenoviruses exploit the lncRNA network to optimize their reproduction.

Posttranscriptional Regulation in Adenovirus Infected Cells

A deeper understanding of how viruses reprogram their hosts for production of progeny is needed to combat infections. Most knowledge on the regulation of cellular gene expression during adenovirus infection is derived from mRNA studies. Here, we investigated the changes in protein expression during the late phase of adenovirus type 2 (Ad2) infection of the IMR-90 cell line by stable isotope labeling in cell culture with subsequent liquid chromatography-high resolution tandem mass spectrometric analysis. Two biological replicates of samples collected at 24 and 36 h post-infection (hpi) were investigated using swapped labeling. In total, 2648 and 2394 proteins were quantified at 24 and 36 hpi, respectively. Among them, 659 and 645 were deregulated >1.6-fold at the two time points. The protein expression was compared with RNA expression using cDNA sequencing data. The correlation was surprisingly low (r = 0.3), and several examples of posttranscriptional regulation were observed; e.g., proteins related to carbohydrate metabolism were up-regulated at the protein level but unchanged at the RNA level, whereas histone proteins were down-regulated at the protein level but up-regulated at the RNA level. The deregulation of cellular gene expression by adenovirus is mediated at multiple levels and more complex than hitherto believed.

Data on the expression of cellular lncRNAs in human adenovirus infected cells

Expression of cellular long non-coding RNAs (lncRNAs) in human primary lung fibroblasts (IMR-90) during the course of adenovirus type 2 (Ad2) infection was studied by strand-specific whole transcriptome sequencing. In total, 645 cellular lncRNAs were expressed at a significant level and 398 of them were changed more than 2-fold. The changes in expression followed a distinct temporal pattern. Significantly, 80% of the changes occurred at the late phase and 80% of the de-regulated lncRNAs were up-regulated. The three largest groups of deregulated lncRNAs were 125 antisense RNAs, 111 pseudogenes and 85 long intergenic non-coding RNAs (lincRNAs). Lastly, more than 36% of lncRNAs have been shown to interact with RNA binding proteins.

Identification of the adenovirus type 2 C-168 protein

A hitherto predicted but undetected protein, C-168, in adenovirus type 2 (Ad2) has been identified using mass spectrometry (MS) based proteomics. The gene of this 17.7kDa protein is located on the forward strand in the major late transcription unit between base pairs 9294 and 9797. A tryptic peptide, derived from the C-terminal part of the protein, was identified with high amino acid sequence coverage. A candidate splice site for the corresponding mRNA is also presented. The protein sequence is unusual with repeats of serine, glycine and arginine. A bioinformatics prediction of protein function and localization is presented.