| 1. |
- Westergren Jakobsson, Amanda, et al.
(författare)
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The Human Adenovirus 2 Transcriptome : An Amazing Complexity of Alternatively Spliced mRNAs
- 2021
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Ingår i: Journal of Virology. - : American Society for Microbiology. - 0022-538X .- 1098-5514. ; 95:4
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Tidskriftsartikel (refereegranskat)abstract
- We have used the Nanopore long-read sequencing platform to demonstrate how amazingly complex the human adenovirus type 2 (Ad2) transcriptome is with a flexible splicing machinery producing a range of novel mRNAs both from the early and late transcription units. In total we report more than 900 alternatively spliced mRNAs produced from the Ad2 transcriptome whereof more than 850 are novel mRNAs. A surprising finding was that more than 50% of all E1A transcripts extended upstream of the previously defined transcriptional start site. The novel start sites mapped close to the inverted terminal repeat (ITR) and within the E1A enhancer region. We speculate that novel promoters or enhancer driven transcription, so-called eRNA transcription, is responsible for producing these novel mRNAs. Their existence was verified by a peptide in the Ad2 proteome that was unique for the E1A ITR mRNA. Although we show a high complexity of alternative splicing from most early and late regions, the E3 region was by far the most complex when expressed at late times of infection. More than 400 alternatively spliced mRNAs were observed in this region alone. These mRNAs included extended L4 mRNAs containing E3 and L5 sequences and readthrough mRNAs combining E3 and L5 sequences. Our findings demonstrate that the virus has a remarkable capacity to produce novel exon combinations, which will offer the virus an evolutionary advantage to change the gene expression repertoire and protein production in an evolving environment.IMPORTANCE Work in the adenovirus system led to the groundbreaking discovery of RNA splicing and alternative RNA splicing in 1977. These mechanisms are essential in mammalian evolution by increasing the coding capacity of a genome. Here, we have used a long-read sequencing technology to characterize the complexity of human adenovirus pre-mRNA splicing in detail. It is mindboggling that the viral genome, which only houses around 36,000 bp, not being much larger than a single cellular gene, generates more than 900 alternatively spliced mRNAs. Recently, adenoviruses have been used as the backbone in several promising SARS-CoV-2 vaccines. Further improvement of adenovirus-based vaccines demands that the virus can be tamed into an innocent carrier of foreign genes. This requires a full understanding of the components that govern adenovirus replication and gene expression.
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| 2. |
- Valdés, Alberto, et al.
(författare)
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Phosphorylation Time-Course Study of the Response during Adenovirus Type 2 Infection.
- 2020
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Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 20:7
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Tidskriftsartikel (refereegranskat)abstract
- PTMs such as phosphorylations are usually involved in signal transduction pathways. To investigate the temporal dynamics of phosphoproteome changes upon viral infection, a model system of IMR-90 cells infected with human adenovirus type 2 (Ad2) is used in a time-course quantitative analysis combining titanium dioxide (TiO2 ) particle enrichment and SILAC-MS. Quantitative data from 1552 phosphorylated sites clustered the highly altered phosphorylated sites to the signaling by rho family GTPases, the actin cytoskeleton signaling, and the cAMP-dependent protein kinase A signaling pathways. Their activation is especially pronounced at early time post-infection. Changes of several phosphorylated sites involved in the glycolysis pathway, related to the activation of the Warburg effect, point at virus-induced energy production. For Ad2 proteins, 32 novel phosphorylation sites are identified and as many as 52 phosphorylated sites on 17 different Ad2 proteins are quantified, most of them at late time post-infection. Kinase predictions highlighted activation of PKA, CDK1/2, MAPK, and CKII. Overlaps of kinase motif sequences for viral and human proteins are observed, stressing the importance of phosphorylation during Ad2 infection.
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| 3. |
- Zhao, Hongxing, et al.
(författare)
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Adenovirus in the omics era - a multi-pronged strategy.
- 2020
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 594:12, s. 1879-1890
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Tidskriftsartikel (refereegranskat)abstract
- Human adenoviruses (HAdVs) are common pathogens associated with a wide variety of respiratory, ocular and gastrointestinal diseases. To achieve its effective lytic mode of replication, HAdVs have to reprogram host-cell gene expression and fine-tune viral gene expression in a temporal manner. In two decades, omics revolution has advanced our knowledge about the HAdV and host cell interplay at the RNA and protein levels. This review summarizes the current knowledge from large-scale datasets how HAdV infections adjust coding and non-coding RNA expression, as well as how they reprogram host-cell proteome during the lytic course of infection.
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| 4. |
- Zhao, Hongxing, et al.
(författare)
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Transcriptomic and proteomic analyses reveal new insights into the regulation of immune pathways during adenovirus type 2 infection
- 2019
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Ingår i: BMC Microbiology. - : BMC. - 1471-2180. ; 19
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Tidskriftsartikel (refereegranskat)abstract
- Background: Human adenovirus (Ad) infection leads to the changes of host cell gene expression and biosynthetic processes. Transcriptomics in adenovirus type 2 (Ad2)-infected lung fibroblasts (IMR-90) cells has previously been studied using RNA sequencing. However, this study included only two time points (12 and 24 hpi) using constrained 76bp long sequencing reads. Therefore, a more detailed study of transcription at different phases of infection using an up-graded sequencing technique is recalled. Furthermore, the correlation between transcription and protein expression needs to be addressed.Results: In total, 3556 unique cellular genes were identified as differentially expressed at the transcriptional level with more than 2-fold changes in Ad2-infected cells as compared to non-infected cells by using paired-end sequencing. Based on the kinetics of the gene expression changes at different times after infection, these RNAs fell into 20 clusters. Among them, cellular genes involved in immune response were highly up-regulated in the early phase before becoming down-regulated in the late phase. Comparison of differentially expressed genes at transcriptional and posttranscriptional levels revealed low correlation. Particularly genes involved in cellular immune pathways showed a negative correlation. Here, we highlight the genes which expose inconsistent expression profiles with an emphasis on key factors in cellular immune pathways including NFB, JAK/STAT, caspases and MAVS. Different from their transcriptional profiles with up- and down-regulation in the early and late phase, respectively, these proteins were up-regulated in the early phase and were sustained in the late phase. A surprising finding was that the target genes of the sustained activators failed to show response.Conclusion: There were features common to genes which play important roles in cellular immune pathways. Their expression was stimulated at both RNA and protein levels during the early phase. In the late phase however, their transcription was suppressed while protein levels remained stable. These results indicate that Ad2 and the host cell use different strategies to regulate cellular immune pathways. A control mechanism at the post-translational level must thus exist which is under the control of Ad2.
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| 5. |
- Valdés, Alberto, et al.
(författare)
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Time-resolved proteomics of adenovirus infected cells
- 2018
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Ingår i: PLOS ONE. - : PUBLIC LIBRARY SCIENCE. - 1932-6203. ; 13:9
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Tidskriftsartikel (refereegranskat)abstract
- Viral infections cause large problems in the world and deeper understanding of the disease mechanisms is needed. Here we present an analytical strategy to investigate the host cell protein changes during human adenovirus type 2 (HAdV-C2 or Ad2) infection of lung fibro-blasts by stable isotope labelling of amino acids in cell culture (SILAC) and nanoLC-MS/MS. This work focuses on early phase of infection (6 and 12 h post-infection (hpi)) but the data is combined with previously published late phase (24 and 36 hpi) proteomics data to produce a time series covering the complete infection. As many as 2169 proteins were quantitatively monitored from 6 to 36 hpi, while some proteins were time-specific. After applying different filter criteria, 2027 and 2150 proteins were quantified at 6 and 12 hpi and among them, 431 and 544 were significantly altered at the two time points. Pathway analysis showed that the De novo purine and pyrimidine biosynthesis, Glycolysis and Cytoskeletal regulation by Rho GTPase pathways were activated early during infection while inactivation of the Integrin signalling pathway started between 6 and 12 hpi. Moreover, upstream regulator analysis predicted MYC to be activated with time of infection and protein and RNA data for genes controlled by this transcription factor showed good correlation, which validated the use of protein data for this prediction. Among the identified phosphorylation sites, a group related to glycolysis and cytoskeletal reorganization were up-regulated during infection. The results show specific aspects on how the host cell proteins, the final products in the genetic information flow, are influenced by Ad2 infection, which would be overlooked if only knowledge derived from mRNA data is considered.
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| 6. |
- Gromova, Arina, et al.
(författare)
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Identification of the adenovirus type 2 C-168 protein
- 2017
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Ingår i: Virus Research. - : Elsevier BV. - 0168-1702 .- 1872-7492. ; 238, s. 110-113
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Tidskriftsartikel (refereegranskat)abstract
- 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 17.7 kDa 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.
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| 7. |
- Källsten, Malin, et al.
(författare)
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Temporal characterization of the non-structural Adenovirus type 2 proteome and phosphoproteome using high-resolving mass spectrometry
- 2017
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Ingår i: Virology. - Uppsala : ACADEMIC PRESS INC ELSEVIER SCIENCE. - 0042-6822 .- 1096-0341. ; 511, s. 240-248
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Tidskriftsartikel (refereegranskat)abstract
- The proteome and phosphoproteome of non-structural proteins of Adenovirus type 2 (Ad2) were time resolved using a developed mass spectrometry approach. These proteins are expressed by the viral genome and important for the infection process, but not part of the virus particle. We unambiguously confirm the existence of 95% of the viral proteins predicted to be encoded by the viral genome. Most non-structural proteins peaked in expression at late time post infection. We identified 27 non-redundant sites of phosphorylation on seven different non-structural proteins. The most heavily phosphorylated protein was the DNA binding protein (DBP) with 15 different sites. The phosphorylation occupancy rate could be calculated and monitored with time post infection for 15 phosphorylated sites on various proteins. In the DBP, phosphorylations with time-dependent relation were observed. The findings show the complexity of the Ad2 non-structural proteins and opens up a discussion for potential new drug targets.
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| 8. |
- Zhao, Hongxing, et al.
(författare)
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Posttranscriptional regulation in adenovirus infected cells
- 2017
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 16:2, s. 872-888
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Tidskriftsartikel (refereegranskat)abstract
- A deeper understanding of how viruses reprogramtheir hosts for production of progeny is needed to combatinfections. Most knowledge on the regulation of cellular geneexpression during adenovirus infection is derived from mRNAstudies. Here, we investigated the changes in protein expressionduring the late phase of adenovirus type 2 (Ad2) infection of theIMR-90 cell line by stable isotope labeling in cell culture withsubsequent liquid chromatography−high resolution tandemmass spectrometric analysis. Two biological replicates of samplescollected at 24 and 36 h post-infection (hpi) were investigated using swapped labeling. In total, 2648 and 2394 proteins werequantified at 24 and 36 hpi, respectively. Among them, 659 and 645 were deregulated >1.6-fold at the two time points. Theprotein 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 metabolismwere up-regulated at the protein level but unchanged at the RNA level, whereas histone proteins were down-regulated at theprotein level but up-regulated at the RNA level. The deregulation of cellular gene expression by adenovirus is mediated atmultiple levels and more complex than hitherto believed.
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| 9. |
- Chen, Moashan, et al.
(författare)
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Data on the expression of cellular lncRNAs in human adenovirus infected cells
- 2016
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Ingår i: Data in Brief. - Elsevier : Elsevier BV. - 2352-3409. ; 8, s. 1263-1279
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
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