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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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Rescue of the dl720 (VA RNAI−/VA RNAII−) mutant phenotype by PKR knockdown. HEK293 cells were transfected with the indicated siRNAs for 24 h, followed by infection with mutant dl720 or Ad5 wild-type (5 FFU/cell). Cells were 35S-methionine pulse-labeled 22 hpi, and protein extracts were prepared and separated on an SDS–PAGE. Late viral protein synthesis was visualized by autoradiography. The position of major viral capsid proteins is indicated to the right of the panel. The efficiency of siRNA knockdown was confirmed by immunoblotting of cell extract with antibodies directed against actin, Dicer and PKR. Scr, scrambled siRNA.
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gkt172-F8: Rescue of the dl720 (VA RNAI−/VA RNAII−) mutant phenotype by PKR knockdown. HEK293 cells were transfected with the indicated siRNAs for 24 h, followed by infection with mutant dl720 or Ad5 wild-type (5 FFU/cell). Cells were 35S-methionine pulse-labeled 22 hpi, and protein extracts were prepared and separated on an SDS–PAGE. Late viral protein synthesis was visualized by autoradiography. The position of major viral capsid proteins is indicated to the right of the panel. The efficiency of siRNA knockdown was confirmed by immunoblotting of cell extract with antibodies directed against actin, Dicer and PKR. Scr, scrambled siRNA.

Mentions: To test whether the PKR or RNAi/miRNA pathways are most critical for the establishment of a lytic adenovirus infection, we used an siRNA approach to knockdown PKR or Dicer expression in VA RNA double mutant virus (dl720)-infected cells. In this experiment, HEK293 cells were pre-treated with siRNA for 24 or 48 h, followed by infection with the dl720 virus. Infected cells were subsequently pulse-labeled between 20 and 22 hpi with 35S-methionine, and total protein extracts were prepared and analyzed by SDS–PAGE. As shown in Figure 8, loss of the Dicer protein did not have any visible impact on late viral protein synthesis (lane 2). Similar results were obtained with an siRNA treatment for 48 h before infection (data not shown). This result would be in agreement with the hypothesis that mivaRNAI production might be required for virus multiplication. However, a knockdown of PKR almost completely rescued late viral protein synthesis (compare lanes 3 and 4). Collectively, these results suggest that, under conditions where no mivaRNAI are produced, the removal of PKR expression is sufficient to almost fully rescue viral late gene expression. The implication of these results for mivaRNAI function is further discussed later in the text.Figure 8.


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)

Rescue of the dl720 (VA RNAI−/VA RNAII−) mutant phenotype by PKR knockdown. HEK293 cells were transfected with the indicated siRNAs for 24 h, followed by infection with mutant dl720 or Ad5 wild-type (5 FFU/cell). Cells were 35S-methionine pulse-labeled 22 hpi, and protein extracts were prepared and separated on an SDS–PAGE. Late viral protein synthesis was visualized by autoradiography. The position of major viral capsid proteins is indicated to the right of the panel. The efficiency of siRNA knockdown was confirmed by immunoblotting of cell extract with antibodies directed against actin, Dicer and PKR. Scr, scrambled siRNA.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt172-F8: Rescue of the dl720 (VA RNAI−/VA RNAII−) mutant phenotype by PKR knockdown. HEK293 cells were transfected with the indicated siRNAs for 24 h, followed by infection with mutant dl720 or Ad5 wild-type (5 FFU/cell). Cells were 35S-methionine pulse-labeled 22 hpi, and protein extracts were prepared and separated on an SDS–PAGE. Late viral protein synthesis was visualized by autoradiography. The position of major viral capsid proteins is indicated to the right of the panel. The efficiency of siRNA knockdown was confirmed by immunoblotting of cell extract with antibodies directed against actin, Dicer and PKR. Scr, scrambled siRNA.
Mentions: To test whether the PKR or RNAi/miRNA pathways are most critical for the establishment of a lytic adenovirus infection, we used an siRNA approach to knockdown PKR or Dicer expression in VA RNA double mutant virus (dl720)-infected cells. In this experiment, HEK293 cells were pre-treated with siRNA for 24 or 48 h, followed by infection with the dl720 virus. Infected cells were subsequently pulse-labeled between 20 and 22 hpi with 35S-methionine, and total protein extracts were prepared and analyzed by SDS–PAGE. As shown in Figure 8, loss of the Dicer protein did not have any visible impact on late viral protein synthesis (lane 2). Similar results were obtained with an siRNA treatment for 48 h before infection (data not shown). This result would be in agreement with the hypothesis that mivaRNAI production might be required for virus multiplication. However, a knockdown of PKR almost completely rescued late viral protein synthesis (compare lanes 3 and 4). Collectively, these results suggest that, under conditions where no mivaRNAI are produced, the removal of PKR expression is sufficient to almost fully rescue viral late gene expression. The implication of these results for mivaRNAI function is further discussed later in the text.Figure 8.

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