Temporal dynamics of adenovirus 5 gene expression in normal human cells

Crisostomo, Leandro; Soriano, Andrea Michelle; Mendez, Megan; Graves, Drayson; Pelka, Peter

doi:10.1371/journal.pone.0211192

Cited by 39 publications

(43 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The data show that in some cell nuclei but not others, the vDNAs have non-uniform transcriptional activity and progress asynchronously into the replication phase. Specifically, our results indicate that the E1A, E1B-55K and VI viral transcripts accumulate in high numbers in infected cancer cells and non-transformed cells, such as HDF-TERT cells, typically reaching levels >200 transcripts per cell, in agreement with population-based assays [42,58,90]. In comparison, only about 5% of HeLa cell mRNAs have mean transcript numbers larger than 200 per cell [56].…”

Section: Discussionsupporting

confidence: 80%

Cell-to-cell and genome-to-genome variability of Adenovirus transcription tuned by the cell cycle

Suomalainen

Prasad

Kannan

et al. 2020

Preprint

View full text Add to dashboard Cite

These authors contributed equally to this work. Abstract In clonal cultures, not all cells are equally susceptible to virus infection. Underlying mechanisms of infection variability are poorly understood. Here, we developed imagebased single cell measurements to scrutinize the heterogeneity of adenovirus (AdV) infection. AdV delivers, transcribes and replicates a linear double-stranded DNA genome in the nucleus. We measured the abundance of viral transcripts by singlemolecule RNA fluorescence in situ hybridization (FISH), and the incoming ethynyldeoxy-cytidine (EdC)-tagged viral genome by copper(I)-catalyzed azide-alkyne cycloaddition (click) reaction. The early transcripts increased from 2-12 hours, the late ones from 12-23 hours post infection (pi), indicating distinct accumulation kinetics. Surprisingly, the expression of the immediate early transactivator gene E1A only moderately correlated with the number of viral genomes in the cell nucleus, although the incoming viral DNA remained largely intact until 7 hours pi. Genome-to-genome heterogeneity was found at the level of viral transcription, as indicated by colocalization with the large intron containing early region E4 transcripts, uncorrelated to the multiplicity of incoming genomes in the nucleus. In accordance, individual genomes exhibited heterogeneous replication activity, as shown by single-strand DNA-FISH and immunocytochemistry. These results indicate that the variability in viral gene expression and replication are not due to defective genomes but due to host cell heterogeneity. By analyzing the cell cycle state, we found that G1 cells exhibited the highest E1A expression, and significantly increased the correlation between E1A expression and viral genome copy numbers. This combined imagebased single molecule procedure is ideally suited to explore the cell-to-cell variability in viral infection, including transcriptional activators and repressors, RNA splicing mechanisms, and the impact of the 3-dimensional nuclear topology on gene regulation. 3 Author SummaryAdenoviruses (AdV) are ubiquitous pathogens in vertebrates. They persist in infected people, and cause unpredictable outbreaks, morbidity and mortality across the globe.Here we report that the common human AdV type C5 (AdV-C5) gives rise to considerable infection variability at the level of single cells in culture, and that a major underlying reason is the cell-to-cell heterogeneity. By combining sensitive single molecule in situ technology for detecting the incoming viral DNA and newly synthesized viral transcripts we show that viral gene expression is heterogeneous between infected human cells, as well as individual genomes. We report a moderate correlation between the number of viral genomes in the nucleus and immediate early E1A transcripts. This correlation is increased in the G1 phase of the cell cycle, where the E1A transcripts were found to be more abundant than in any other cell cycle phase. Our results demonstrate the importance of cell-to-cell variability measurements for understand...

show abstract

Section: Discussionsupporting

confidence: 80%

Cell-to-cell and genome-to-genome variability of Adenovirus transcription tuned by the cell cycle

Suomalainen

Prasad

Kannan

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…Moreover, a broad overview ( Supplementary Table 1) or a protein by protein breakdown (Fig. 2) of transcripts expressing known adenovirus ORFs confirmed the expected pattern of increasing dominance of late protein expression as the infection progresses 13 .…”

Section: Resultssupporting

confidence: 58%

Deep splicing plasticity of the human adenovirus type 5 transcriptome drives virus evolution

et al. 2020

View full text Add to dashboard Cite

Viral genomes have high gene densities and complex transcription strategies rendering transcriptome analysis through short-read RNA-seq approaches problematic. Adenovirus transcription and splicing is especially complex. We used long-read direct RNA sequencing to study adenovirus transcription and splicing during infection. This revealed a previously unappreciated complexity of alternative splicing and potential for secondary initiating codon usage. Moreover, we find that most viral transcripts tend to shorten polyadenylation lengths as infection progresses. Development of an open reading frame centric bioinformatics analysis pipeline provided a deeper quantitative and qualitative understanding of adenovirus's genetic potential. Across the viral genome adenovirus makes multiple distinctly spliced transcripts that code for the same protein. Over 11,000 different splicing patterns were recorded across the viral genome, most occurring at low levels. This low-level use of alternative splicing patterns potentially enables the virus to maximise its coding potential over evolutionary timescales.

show abstract

“…At 12 hours post infection (hpi) the majority of viral transcripts detected were early RNAs, particularly E1A-large and E1A-small, E1B-19K and E1B-55K, early promoter DBP, and E4orf3 ( Figure 3A ). However, at this time point we still detected low-level viral late transcripts that progressed beyond the L1 polyadenylation site, corroborating recent work [54]. At 24 hpi, however, viral gene expression shifted to be dominated by late gene expression, as well as early transcripts derived from the E1B locus ( Figure 3B ).…”

Section: Resultssupporting

confidence: 91%

“…However, the constraints of short-read sequencing precluded proper assembly of these sites into full-length transcripts [43,53,70]. Furthermore, targeted expression analysis over a time-course of infection was limited to already known transcripts [54]. Using direct RNA sequencing we have been able to confirm that these RNAs exist (e.g., the various x, y, and z leaders preceding some molecules of Fiber transcripts), as well as show regulated expression over a time-course of infection.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Novel splicing and open reading frames revealed by long-read direct RNA sequencing of adenovirus transcripts

Hayer²,

Depledge

et al. 2019

Preprint

View full text Add to dashboard Cite

20Adenovirus is a common human pathogen that relies on host cell processes for production 21 and processing of viral RNA. Although adenoviral promoters, splice junctions, and cleavage and 22 proteins by linking separate gene transcription units. Our work highlights how long-read 33 sequencing technologies can reveal further complexity within viral transcriptomes. : bioRxiv preprint another multifunctional protein that can cooperate with E1A to alter cellular gene expression 61 downstream of p53 as well as form the targeting component of a viral ubiquitin ligase [18][19][20][21][22][23]. 62The remaining early transcription units are all transcriptionally activated by E1A and encode for 63 products of related function. The E2 region on the reverse strand of the AdV genome has both an 64 early and a late promoter, as well as two distinct polyadenylation sites, leading to upstream E2A 65 and downstream E2B transcripts [24]. E2A encodes for the viral DNA-binding protein (DBP), while 66 alternative splicing to E2B encodes for the protein-priming terminal protein (pTP) as well as the 67AdV DNA polymerase (AdPol) [25][26][27]. The E3 region encoded on the top strand also has two 68

show abstract

Temporal dynamics of adenovirus 5 gene expression in normal human cells

Cited by 39 publications

References 38 publications

Cell-to-cell and genome-to-genome variability of Adenovirus transcription tuned by the cell cycle

Cell-to-cell and genome-to-genome variability of Adenovirus transcription tuned by the cell cycle

Deep splicing plasticity of the human adenovirus type 5 transcriptome drives virus evolution

Novel splicing and open reading frames revealed by long-read direct RNA sequencing of adenovirus transcripts

Contact Info

Product

Resources

About