Insights into Adenovirus Uncoating from Interactions with Integrins and Mediators of Host Immunity

Nemerow, Glen R.; Stewart, Phoebe L.

doi:10.3390/v8120337

Cited by 25 publications

(13 citation statements)

References 102 publications

(146 reference statements)

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“…Single-cell, single-particle analyses show that different steps of entry occur 5 with different efficiencies in individual infected cells and thus the number of vDNAs delivered into the nucleus varies between cells [21,25,27]. AdV enters cells by receptor-mediated endocytosis, and receives chemical and mechanical cues which initiate the stepwise uncoating process of the viral DNA (vDNA) genome, and delivers vDNA into the nucleus (reviewed in [29][30][31][32]). The vDNA is imported into the nucleus in association with protein VII and the protein VII-complexed vDNA is the template for early virus transcription [25,[33][34][35][36][37][38], although partial replacement of protein VII by histones cannot be excluded [39].…”

Section: Introductionmentioning

confidence: 99%

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

Suomalainen

Prasad

Kannan

et al. 2020

Preprint

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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...

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Section: Introductionmentioning

confidence: 99%

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

Suomalainen

Prasad

Kannan

et al. 2020

Preprint

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“…The copyright holder for this preprint this version posted March 5, 2020. ; https://doi.org/10.1101/2020.02.27.967471 doi: bioRxiv preprint 5 with different efficiencies in individual infected cells and thus the number of vDNAs delivered into the nucleus varies between cells [21,25,27]. AdV enters cells by receptor-mediated endocytosis, and receives chemical and mechanical cues which initiate the stepwise uncoating process of the viral DNA (vDNA) genome, and delivers vDNA into the nucleus (reviewed in [29][30][31][32]). The vDNA is imported into the nucleus in association with protein VII and the protein VII-complexed vDNA is the template for early virus transcription [25,[33][34][35][36][37][38], although partial replacement of protein VII by histones cannot be excluded [39].…”

Section: Introductionmentioning

confidence: 99%

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

Suomalainen

Bartenschlager

Kannan

et al. 2020

Journal of Cell Science

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In clonal cultures, not all cells are equally susceptible to virus infection and the underlying mechanisms are poorly understood. Here, we developed image-based 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 single-molecule RNA fluorescence in situ hybridization (FISH), and the incoming ethynyl-deoxy-cytidine (EdC)-tagged viral genomes by copper(I)-catalyzed azide-alkyne cycloaddition (click) reaction. Surprisingly, the expression of the immediate early gene E1A only moderately correlated with the number of viral genomes in the cell nucleus. Intranuclear genome-to-genome heterogeneity was found at the level of viral transcription and, in accordance, individual genomes exhibited heterogeneous replication activity. By analyzing the cell cycle state, we found that G1 cells exhibited the highest E1A expression and displayed increased correlation between E1A expression and viral genome copy numbers. The combined image-based single molecule procedures described here are ideally suited to explore the cell-to-cell variability in viral gene expression in a range of different settings, including innate immune response.

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“…However, engineered versions are preferred vehicles for vaccine delivery and therapeutic gene transfer [ 13 ]. Adenovirus enters target cells by receptor-mediated endocytosis [ 14 ]. Following endosomal escape, Ad particles travel along microtubules (MT), and then dock at nuclear pore complexes (NPC) to deliver their DNA genome into the nucleus [ 4 , 15 , 16 ].…”

Section: Introductionmentioning

confidence: 99%

Role of kinesins in directed adenovirus transport and cytoplasmic exploration

Zhou

Scherer

et al. 2018

PLoS Pathog

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Many viruses, including adenovirus, exhibit bidirectional transport along microtubules following cell entry. Cytoplasmic dynein is responsible for microtubule minus end transport of adenovirus capsids after endosomal escape. However, the identity and roles of the opposing plus end-directed motor(s) remain unknown. We performed an RNAi screen of 38 kinesins, which implicated Kif5B (kinesin-1 family) and additional minor kinesins in adenovirus 5 (Ad5) capsid translocation. Kif5B RNAi markedly increased centrosome accumulation of incoming Ad5 capsids in human A549 pulmonary epithelial cells within the first 30 min post infection, an effect dramatically enhanced by blocking Ad5 nuclear pore targeting using leptomycin B. The Kif5B RNAi phenotype was rescued by expression of RNAi-resistant Kif5A, B, or C, and Kif4A. Kif5B RNAi also inhibited a novel form of microtubule-based “assisted-diffusion” behavior which was apparent between 30 and 60 min p.i. We found the major capsid protein penton base (PB) to recruit kinesin-1, distinct from the hexon role we previously identified for cytoplasmic dynein binding. We propose that adenovirus uses independently recruited kinesin and dynein for directed transport and for a more random microtubule-based assisted diffusion behavior to fully explore the cytoplasm before docking at the nucleus, a mechanism of potential importance for physiological cargoes as well.

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Insights into Adenovirus Uncoating from Interactions with Integrins and Mediators of Host Immunity

Cited by 25 publications

References 102 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

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

Role of kinesins in directed adenovirus transport and cytoplasmic exploration

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