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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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Mutating the 5′- or 3′-mivaRNAI seed sequence does not impair virus growth in HEK293 cells. (A) HEK293 cells were infected with the indicated viruses (5 FFU/cell) followed by a 35S-methionine pulse labeling at 24 hpi. Total protein extracts were separated on an SDS–PAGE, and protein synthesis was visualized by autoradiography. (B) Total late viral protein accumulation was also detected on the same samples by western blot analysis using an anti-Ad5 capsid antibody. Equal loading of protein samples was confirmed by western blot using an anti-actin antibody. The position of major viral proteins is indicated to the right of each panel.
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gkt172-F5: Mutating the 5′- or 3′-mivaRNAI seed sequence does not impair virus growth in HEK293 cells. (A) HEK293 cells were infected with the indicated viruses (5 FFU/cell) followed by a 35S-methionine pulse labeling at 24 hpi. Total protein extracts were separated on an SDS–PAGE, and protein synthesis was visualized by autoradiography. (B) Total late viral protein accumulation was also detected on the same samples by western blot analysis using an anti-Ad5 capsid antibody. Equal loading of protein samples was confirmed by western blot using an anti-actin antibody. The position of major viral proteins is indicated to the right of each panel.

Mentions: The fact that the VA RNAs are processed into small RNAs is an interesting observation that leads to the more important question whether the mivaRNAs have an essential function for lytic virus growth. By a microarray approach, it has been shown that several hundred cellular mRNAs are up- or downregulated in response to VA RNA overexpression (11). Among them, the T-cell-restricted intracellular antigen-1 (TIA-1) mRNA and protein expression was reported to be reduced by the 3′-mivaRNAI known as mivaRNAI-138 (11). As shown in Figure 4, the steady-state level of TIA-1 mRNA was reduced in cells infected with the VA RNAI wild-type virus at both 16 and 20 hour post-infection (hpi) (compare lane 1 with 2 and 7). Similarly TIA-1 mRNA expression was repressed in the 5′-seed sequence mutated virus (lanes 4 and 7). Interestingly, the mutations destroying the 3′-mivaRNAI-138 seed sequence failed to block TIA-1 mRNA accumulation (lanes 3 and 6). Taken together, these results suggest that mivaRNAI-138 has an miRNA function and is able to regulate cellular gene expression during a lytic infection. However, this does not necessarily prove that this miRNA function is required for the lytic life cycle of the virus. Therefore, to determine whether mutations in the mivaRNAI seed sequences have an impact on a lytic infection, HEK293 cells were infected with the Ad-VAI wt or the Ad-VAI seed sequence mutated viruses. After 20 hpi, the cells were 35S pulse-labeled for 2 h, proteins separated on an SDS–PAGE, and late viral protein synthesis visualized either by autoradiography (Figure 5A) or western blot analysis detecting adenovirus capsid proteins (Figure 5B). The results from both assays show that the synthesis rate of proteins (Figure 5A) and the steady-state level of late protein accumulation (Figure 5B) were essentially identical in Ad-VAI 3′-mut (lane 4), Ad-VAI-5′-mut (lane 5) and Ad-VAI wt (lane 3)-infected cells. In addition, the recombinant viruses all seemed to accumulate late proteins to the same extent as the Ad5 virus (lane 2). Similar results were observed in 911 cells (data not shown). Also, as expected from previous results (1), the absence of VA RNA expression effectively abolished synthesis and accumulation of late viral proteins (Ad-ΔVA-GFP; lane 6).Figure 4.


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)

Mutating the 5′- or 3′-mivaRNAI seed sequence does not impair virus growth in HEK293 cells. (A) HEK293 cells were infected with the indicated viruses (5 FFU/cell) followed by a 35S-methionine pulse labeling at 24 hpi. Total protein extracts were separated on an SDS–PAGE, and protein synthesis was visualized by autoradiography. (B) Total late viral protein accumulation was also detected on the same samples by western blot analysis using an anti-Ad5 capsid antibody. Equal loading of protein samples was confirmed by western blot using an anti-actin antibody. The position of major viral proteins is indicated to the right of each panel.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt172-F5: Mutating the 5′- or 3′-mivaRNAI seed sequence does not impair virus growth in HEK293 cells. (A) HEK293 cells were infected with the indicated viruses (5 FFU/cell) followed by a 35S-methionine pulse labeling at 24 hpi. Total protein extracts were separated on an SDS–PAGE, and protein synthesis was visualized by autoradiography. (B) Total late viral protein accumulation was also detected on the same samples by western blot analysis using an anti-Ad5 capsid antibody. Equal loading of protein samples was confirmed by western blot using an anti-actin antibody. The position of major viral proteins is indicated to the right of each panel.
Mentions: The fact that the VA RNAs are processed into small RNAs is an interesting observation that leads to the more important question whether the mivaRNAs have an essential function for lytic virus growth. By a microarray approach, it has been shown that several hundred cellular mRNAs are up- or downregulated in response to VA RNA overexpression (11). Among them, the T-cell-restricted intracellular antigen-1 (TIA-1) mRNA and protein expression was reported to be reduced by the 3′-mivaRNAI known as mivaRNAI-138 (11). As shown in Figure 4, the steady-state level of TIA-1 mRNA was reduced in cells infected with the VA RNAI wild-type virus at both 16 and 20 hour post-infection (hpi) (compare lane 1 with 2 and 7). Similarly TIA-1 mRNA expression was repressed in the 5′-seed sequence mutated virus (lanes 4 and 7). Interestingly, the mutations destroying the 3′-mivaRNAI-138 seed sequence failed to block TIA-1 mRNA accumulation (lanes 3 and 6). Taken together, these results suggest that mivaRNAI-138 has an miRNA function and is able to regulate cellular gene expression during a lytic infection. However, this does not necessarily prove that this miRNA function is required for the lytic life cycle of the virus. Therefore, to determine whether mutations in the mivaRNAI seed sequences have an impact on a lytic infection, HEK293 cells were infected with the Ad-VAI wt or the Ad-VAI seed sequence mutated viruses. After 20 hpi, the cells were 35S pulse-labeled for 2 h, proteins separated on an SDS–PAGE, and late viral protein synthesis visualized either by autoradiography (Figure 5A) or western blot analysis detecting adenovirus capsid proteins (Figure 5B). The results from both assays show that the synthesis rate of proteins (Figure 5A) and the steady-state level of late protein accumulation (Figure 5B) were essentially identical in Ad-VAI 3′-mut (lane 4), Ad-VAI-5′-mut (lane 5) and Ad-VAI wt (lane 3)-infected cells. In addition, the recombinant viruses all seemed to accumulate late proteins to the same extent as the Ad5 virus (lane 2). Similar results were observed in 911 cells (data not shown). Also, as expected from previous results (1), the absence of VA RNA expression effectively abolished synthesis and accumulation of late viral proteins (Ad-ΔVA-GFP; lane 6).Figure 4.

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