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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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Construction of a VA RNAI and VA RNAII B-box double mutant virus. (A) Schematic drawing showing a simplified transcription map of the Ad5 genome with the position of the early (open arrows) and late transcription units (filled arrows) indicated. The deletions in the E1, E3 and VA RNA regions are indicated by crosses [see (14) and later in the text]. The box-labeled GFP/VA indicates the position where the modified VA RNAI genes and the GFP reporter gene were inserted. (B) Expansion of the genome position encoding the VA RNA genes with the mutations introduced into the B-box of VA RNAI and VA RNAII indicated. Dashes indicate nucleotides deleted during the construction of the variant VA RNA genes, with a HindIII cleavage site replacing the box B position. (C) Representative northern blot of VA RNA expression in the parental AdEasy virus and the VA RNAI/VARNAII-deleted virus, Ad-ΔVA-GFP. As a control, cytoplasmic RNA isolated from pVAI and pVAII (21)-transfected cells were also analyzed.
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gkt172-F1: Construction of a VA RNAI and VA RNAII B-box double mutant virus. (A) Schematic drawing showing a simplified transcription map of the Ad5 genome with the position of the early (open arrows) and late transcription units (filled arrows) indicated. The deletions in the E1, E3 and VA RNA regions are indicated by crosses [see (14) and later in the text]. The box-labeled GFP/VA indicates the position where the modified VA RNAI genes and the GFP reporter gene were inserted. (B) Expansion of the genome position encoding the VA RNA genes with the mutations introduced into the B-box of VA RNAI and VA RNAII indicated. Dashes indicate nucleotides deleted during the construction of the variant VA RNA genes, with a HindIII cleavage site replacing the box B position. (C) Representative northern blot of VA RNA expression in the parental AdEasy virus and the VA RNAI/VARNAII-deleted virus, Ad-ΔVA-GFP. As a control, cytoplasmic RNA isolated from pVAI and pVAII (21)-transfected cells were also analyzed.

Mentions: The VA RNAs are transcribed by RNA polymerase III from internal promoters consisting of two essential regulatory elements, the A-box and the B-box (20). To inactivate the expression of the endogenous VA RNA genes, we mutated the B-box in both the VA RNAI and VA RNAII promoters (Figure 1B). By this strategy, we generated a new AdEasy vector backbone devoid of functional VA RNAI and VA RNAII genes (pAd-ΔVA). To generate the control virus, Ad-ΔVA-GFP, expressing a GFP reporter gene under the transcriptional control of a CMV promoter, plasmid pShuttle-CMV (14) was reconstructed to a virus by homologous recombination with the modified Ad-ΔVA genome depicted in Figure 1A. To characterize VA RNA expression, cytoplasmic RNA was prepared from HEK293 cells transfected with plasmids expressing either VA RNAI or VA RNAII or infected with the Ad-ΔVA-GFP virus and analyzed by northern blotting using a DNA probe detecting both VA RNAs. Although the VA RNAs are identical in length, VA RNAII seems to have more compact structure migrating significantly faster than VA RNAI on a denaturing polyacrylamide gel (Figure 1C, lanes 1 and 2) (22). The absence of expression of both VA RNAs in the Ad-ΔVA-GFP virus-infected cells could, therefore, independently be confirmed (Figure 1C, lane 3). As expected from previous results (23), VA RNAI has the strongest promoter and is the predominant VA RNA species expressed in Ad-infected cells (Figure 1C, lane 4).Figure 1.


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)

Construction of a VA RNAI and VA RNAII B-box double mutant virus. (A) Schematic drawing showing a simplified transcription map of the Ad5 genome with the position of the early (open arrows) and late transcription units (filled arrows) indicated. The deletions in the E1, E3 and VA RNA regions are indicated by crosses [see (14) and later in the text]. The box-labeled GFP/VA indicates the position where the modified VA RNAI genes and the GFP reporter gene were inserted. (B) Expansion of the genome position encoding the VA RNA genes with the mutations introduced into the B-box of VA RNAI and VA RNAII indicated. Dashes indicate nucleotides deleted during the construction of the variant VA RNA genes, with a HindIII cleavage site replacing the box B position. (C) Representative northern blot of VA RNA expression in the parental AdEasy virus and the VA RNAI/VARNAII-deleted virus, Ad-ΔVA-GFP. As a control, cytoplasmic RNA isolated from pVAI and pVAII (21)-transfected cells were also analyzed.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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gkt172-F1: Construction of a VA RNAI and VA RNAII B-box double mutant virus. (A) Schematic drawing showing a simplified transcription map of the Ad5 genome with the position of the early (open arrows) and late transcription units (filled arrows) indicated. The deletions in the E1, E3 and VA RNA regions are indicated by crosses [see (14) and later in the text]. The box-labeled GFP/VA indicates the position where the modified VA RNAI genes and the GFP reporter gene were inserted. (B) Expansion of the genome position encoding the VA RNA genes with the mutations introduced into the B-box of VA RNAI and VA RNAII indicated. Dashes indicate nucleotides deleted during the construction of the variant VA RNA genes, with a HindIII cleavage site replacing the box B position. (C) Representative northern blot of VA RNA expression in the parental AdEasy virus and the VA RNAI/VARNAII-deleted virus, Ad-ΔVA-GFP. As a control, cytoplasmic RNA isolated from pVAI and pVAII (21)-transfected cells were also analyzed.
Mentions: The VA RNAs are transcribed by RNA polymerase III from internal promoters consisting of two essential regulatory elements, the A-box and the B-box (20). To inactivate the expression of the endogenous VA RNA genes, we mutated the B-box in both the VA RNAI and VA RNAII promoters (Figure 1B). By this strategy, we generated a new AdEasy vector backbone devoid of functional VA RNAI and VA RNAII genes (pAd-ΔVA). To generate the control virus, Ad-ΔVA-GFP, expressing a GFP reporter gene under the transcriptional control of a CMV promoter, plasmid pShuttle-CMV (14) was reconstructed to a virus by homologous recombination with the modified Ad-ΔVA genome depicted in Figure 1A. To characterize VA RNA expression, cytoplasmic RNA was prepared from HEK293 cells transfected with plasmids expressing either VA RNAI or VA RNAII or infected with the Ad-ΔVA-GFP virus and analyzed by northern blotting using a DNA probe detecting both VA RNAs. Although the VA RNAs are identical in length, VA RNAII seems to have more compact structure migrating significantly faster than VA RNAI on a denaturing polyacrylamide gel (Figure 1C, lanes 1 and 2) (22). The absence of expression of both VA RNAs in the Ad-ΔVA-GFP virus-infected cells could, therefore, independently be confirmed (Figure 1C, lane 3). As expected from previous results (23), VA RNAI has the strongest promoter and is the predominant VA RNA species expressed in Ad-infected cells (Figure 1C, lane 4).Figure 1.

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