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The adenovirus VA RNA-derived miRNAs are not essential for lytic virus growth in tissue culture cells.

Kamel W, Segerman B, Öberg D, Punga T, Akusjärvi G - Nucleic Acids Res. (2013)

Bottom Line: Here, we constructed recombinant adenoviruses with mutations in the seed sequence of both the 5'- and the 3'-strand of the mivaRNAI duplex.The results showed that late viral protein synthesis, as well as new virus progeny formation, was essentially unaffected by the seed sequence mutations under lytic replicative conditions in HeLa or HEK293 cells.Further, by depletion of protein kinase R (PKR) in HEK293 cells, we show that the suppressive effect of VA RNAI on the interferon-induced PKR pathway is most critical for late gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University, 75123 Uppsala, Sweden.

ABSTRACT
At late times during a lytic infection human adenovirus type 5 produces ∼10(8) copies per cell of virus-associated RNA I (VA RNAI). This short highly structured RNA polymerase III transcript has previously been shown to be essential for lytic virus growth. A fraction of VA RNAI is processed by Dicer into small RNAs, so-called mivaRNAIs, which are efficiently incorporated into the RNA-induced silencing complex. Here, we constructed recombinant adenoviruses with mutations in the seed sequence of both the 5'- and the 3'-strand of the mivaRNAI duplex. The results showed that late viral protein synthesis, as well as new virus progeny formation, was essentially unaffected by the seed sequence mutations under lytic replicative conditions in HeLa or HEK293 cells. Collectively, our results suggest that either strand of the mivaRNAI duplex does not have target mRNA interactions that are critical for the establishment of virus growth under lytic conditions. Further, by depletion of protein kinase R (PKR) in HEK293 cells, we show that the suppressive effect of VA RNAI on the interferon-induced PKR pathway is most critical for late gene expression.

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Comparable cytotoxicity of VA RNAI wild-type and seed sequence mutant viruses. The indicated viruses were 5-fold serially diluted on 911 cells from a starting point of 1000 FFU/cell. At 4 and 6 days post-infection, MTS assay was performed and the EC50 value calculated. Shown is the fold increase in viral cytotoxicity compared with the VA RNA negative Ad-ΔVA-GFP virus. *P < 0.05, the data shown are the mean values of three independent experiments ± SEM.
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gkt172-F6: Comparable cytotoxicity of VA RNAI wild-type and seed sequence mutant viruses. The indicated viruses were 5-fold serially diluted on 911 cells from a starting point of 1000 FFU/cell. At 4 and 6 days post-infection, MTS assay was performed and the EC50 value calculated. Shown is the fold increase in viral cytotoxicity compared with the VA RNA negative Ad-ΔVA-GFP virus. *P < 0.05, the data shown are the mean values of three independent experiments ± SEM.

Mentions: Although late protein synthesis seemed to be normal in the mutant virus-infected cells, we tested the possibility that the mutations in the seed sequences may perturb a subsequent and essential function for efficient virus growth. For this experiment, 911 cells (19) were infected with serial dilutions of the VA RNA wild-type and seed sequence mutant viruses and the cytotoxic activity tested in a cell viability assay (MTS assay) at 4 and 6 days post-infection (Figure 6). As expected from earlier results, the Ad-ΔVA-GFP virus showed a 20-fold reduction in cytotoxicity at day 6 compared with Ad-VAI wt expressing the wild-type VA RNAI gene. Importantly, both Ad-VAI 3′-mut and Ad-VAI 5′-mut exhibited essentially the same cytotoxicity as the recombinant virus expressing the wild-type VA RNAI gene. The inhibition of the VA RNA-deleted virus was apparent at Day 4 with a 2- to 3-fold lower cytotoxicity compared with the VA RNAI wild-type and mutant expressing viruses. Taken together, our results suggest that the seed sequence interaction of the 5′- or the 3′-mivaRNAI is not essential for the establishment of an efficient lytic adenovirus infectious cycle in 911 cells.Figure 6.


The adenovirus VA RNA-derived miRNAs are not essential for lytic virus growth in tissue culture cells.

Kamel W, Segerman B, Öberg D, Punga T, Akusjärvi G - Nucleic Acids Res. (2013)

Comparable cytotoxicity of VA RNAI wild-type and seed sequence mutant viruses. The indicated viruses were 5-fold serially diluted on 911 cells from a starting point of 1000 FFU/cell. At 4 and 6 days post-infection, MTS assay was performed and the EC50 value calculated. Shown is the fold increase in viral cytotoxicity compared with the VA RNA negative Ad-ΔVA-GFP virus. *P < 0.05, the data shown are the mean values of three independent experiments ± SEM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643585&req=5

gkt172-F6: Comparable cytotoxicity of VA RNAI wild-type and seed sequence mutant viruses. The indicated viruses were 5-fold serially diluted on 911 cells from a starting point of 1000 FFU/cell. At 4 and 6 days post-infection, MTS assay was performed and the EC50 value calculated. Shown is the fold increase in viral cytotoxicity compared with the VA RNA negative Ad-ΔVA-GFP virus. *P < 0.05, the data shown are the mean values of three independent experiments ± SEM.
Mentions: Although late protein synthesis seemed to be normal in the mutant virus-infected cells, we tested the possibility that the mutations in the seed sequences may perturb a subsequent and essential function for efficient virus growth. For this experiment, 911 cells (19) were infected with serial dilutions of the VA RNA wild-type and seed sequence mutant viruses and the cytotoxic activity tested in a cell viability assay (MTS assay) at 4 and 6 days post-infection (Figure 6). As expected from earlier results, the Ad-ΔVA-GFP virus showed a 20-fold reduction in cytotoxicity at day 6 compared with Ad-VAI wt expressing the wild-type VA RNAI gene. Importantly, both Ad-VAI 3′-mut and Ad-VAI 5′-mut exhibited essentially the same cytotoxicity as the recombinant virus expressing the wild-type VA RNAI gene. The inhibition of the VA RNA-deleted virus was apparent at Day 4 with a 2- to 3-fold lower cytotoxicity compared with the VA RNAI wild-type and mutant expressing viruses. Taken together, our results suggest that the seed sequence interaction of the 5′- or the 3′-mivaRNAI is not essential for the establishment of an efficient lytic adenovirus infectious cycle in 911 cells.Figure 6.

Bottom Line: Here, we constructed recombinant adenoviruses with mutations in the seed sequence of both the 5'- and the 3'-strand of the mivaRNAI duplex.The results showed that late viral protein synthesis, as well as new virus progeny formation, was essentially unaffected by the seed sequence mutations under lytic replicative conditions in HeLa or HEK293 cells.Further, by depletion of protein kinase R (PKR) in HEK293 cells, we show that the suppressive effect of VA RNAI on the interferon-induced PKR pathway is most critical for late gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University, 75123 Uppsala, Sweden.

ABSTRACT
At late times during a lytic infection human adenovirus type 5 produces ∼10(8) copies per cell of virus-associated RNA I (VA RNAI). This short highly structured RNA polymerase III transcript has previously been shown to be essential for lytic virus growth. A fraction of VA RNAI is processed by Dicer into small RNAs, so-called mivaRNAIs, which are efficiently incorporated into the RNA-induced silencing complex. Here, we constructed recombinant adenoviruses with mutations in the seed sequence of both the 5'- and the 3'-strand of the mivaRNAI duplex. The results showed that late viral protein synthesis, as well as new virus progeny formation, was essentially unaffected by the seed sequence mutations under lytic replicative conditions in HeLa or HEK293 cells. Collectively, our results suggest that either strand of the mivaRNAI duplex does not have target mRNA interactions that are critical for the establishment of virus growth under lytic conditions. Further, by depletion of protein kinase R (PKR) in HEK293 cells, we show that the suppressive effect of VA RNAI on the interferon-induced PKR pathway is most critical for late gene expression.

Show MeSH
Related in: MedlinePlus