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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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VA RNAI seed sequence mutations. Schematic drawing showing the secondary structure of VA RNAI with the 5′- and 3′-seed sequence mutations indicated in bold. The strand designation is illustrated for the VA RNAI wt transcript. The arrows indicate the position of the Dicer-processing site.
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gkt172-F2: VA RNAI seed sequence mutations. Schematic drawing showing the secondary structure of VA RNAI with the 5′- and 3′-seed sequence mutations indicated in bold. The strand designation is illustrated for the VA RNAI wt transcript. The arrows indicate the position of the Dicer-processing site.

Mentions: To be able to study the function of the VA RNAI-derived 5′- and 3′-mivaRNAs on virus growth, the GFP cassette in Ad-ΔVA-GFP was replaced with the wild-type VA RNAI gene or VA RNAI genes with 5′- and 3′-seed sequence mutations (Figure 2), generating recombinant viruses Ad-VAI wt, Ad-VAI 5′-mut and Ad-VAI 3′-mut, respectively. The miRNA seed sequence (nucleotides: 2–8) is the key element mediating pairing between an miRNA and a target mRNA (12). The seed mutations introduced here were selected such that they would not interfere with the A-box of the internal VA RNAI promoter but destroy seed pairing between the 5′- and 3′-mivaRNAI and their hypothetical target mRNA(s). It should be noted that VA RNAI has two start sites (24,25). A minor VA RNAI(A) start and a major VA RNAI(G) start that initiates transcription three nucleotides downstream of the A start. We have previously shown that the 5′-strand of the VA RNAI(A) start mivaRNA generates RISC complexes with significantly higher cleavage activity compared with the 5′-strand of the VA RNAI(G) start mivaRNA (8). Importantly, the 5′-seed sequence mutations introduced here will change the potential target mRNA seed paring for both the A and G start 5′-mivaRNAs. Moreover, as the VA RNAI—PKR interaction is mediated by the apical stem and central domain (26), the introduced seed sequence mutations will not interfere with the ability of VA RNAI to bind and inhibit PKR. Full-length VA RNAI expression was measured by northern blot analysis of total RNA prepared from mutant virus-infected HEK293 cells. As shown in (Figure 3A), Ad-ΔVA-GFP–infected cells showed, as expected, a complete lack of VA RNAI expression (lane 6). In contrast, Ad-VAI 3′-mut– (lane 4) and Ad-VAI 5′-mut (lane 5)–infected cells showed similar levels of VA RNAI expression compared with Ad-VAI wt-infected cells (lane 3). Taken together, these results demonstrate that introduction of the seed sequence mutations into the 5′- and 3′-strands of mivaRNAI did not adversely affect VA RNAI transcription.Figure 2.


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)

VA RNAI seed sequence mutations. Schematic drawing showing the secondary structure of VA RNAI with the 5′- and 3′-seed sequence mutations indicated in bold. The strand designation is illustrated for the VA RNAI wt transcript. The arrows indicate the position of the Dicer-processing site.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt172-F2: VA RNAI seed sequence mutations. Schematic drawing showing the secondary structure of VA RNAI with the 5′- and 3′-seed sequence mutations indicated in bold. The strand designation is illustrated for the VA RNAI wt transcript. The arrows indicate the position of the Dicer-processing site.
Mentions: To be able to study the function of the VA RNAI-derived 5′- and 3′-mivaRNAs on virus growth, the GFP cassette in Ad-ΔVA-GFP was replaced with the wild-type VA RNAI gene or VA RNAI genes with 5′- and 3′-seed sequence mutations (Figure 2), generating recombinant viruses Ad-VAI wt, Ad-VAI 5′-mut and Ad-VAI 3′-mut, respectively. The miRNA seed sequence (nucleotides: 2–8) is the key element mediating pairing between an miRNA and a target mRNA (12). The seed mutations introduced here were selected such that they would not interfere with the A-box of the internal VA RNAI promoter but destroy seed pairing between the 5′- and 3′-mivaRNAI and their hypothetical target mRNA(s). It should be noted that VA RNAI has two start sites (24,25). A minor VA RNAI(A) start and a major VA RNAI(G) start that initiates transcription three nucleotides downstream of the A start. We have previously shown that the 5′-strand of the VA RNAI(A) start mivaRNA generates RISC complexes with significantly higher cleavage activity compared with the 5′-strand of the VA RNAI(G) start mivaRNA (8). Importantly, the 5′-seed sequence mutations introduced here will change the potential target mRNA seed paring for both the A and G start 5′-mivaRNAs. Moreover, as the VA RNAI—PKR interaction is mediated by the apical stem and central domain (26), the introduced seed sequence mutations will not interfere with the ability of VA RNAI to bind and inhibit PKR. Full-length VA RNAI expression was measured by northern blot analysis of total RNA prepared from mutant virus-infected HEK293 cells. As shown in (Figure 3A), Ad-ΔVA-GFP–infected cells showed, as expected, a complete lack of VA RNAI expression (lane 6). In contrast, Ad-VAI 3′-mut– (lane 4) and Ad-VAI 5′-mut (lane 5)–infected cells showed similar levels of VA RNAI expression compared with Ad-VAI wt-infected cells (lane 3). Taken together, these results demonstrate that introduction of the seed sequence mutations into the 5′- and 3′-strands of mivaRNAI did not adversely affect VA RNAI transcription.Figure 2.

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