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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 co-infection with the VA RNAI seed sequence mutated viruses. (A) HeLa cells were infected with the indicated combinations of viruses (5 FFU/cell) and 35 S-methionine pulse-labeled at 48 hpi. In the case of co-infections (lanes 8–11), cells were infected with both viruses at 2.5 FFU/cell to achieve the final multiplicity of 5 FFU/cell. Total proteins were resolved on an SDS–PAGE, and new protein synthesis was visualized by autoradiography. To confirm an equal loading of protein samples, the abundance of actin was visualized by western blot analysis using an anti-actin antibody. The position of the major viral capsid proteins is indicated to the right of the panel. (B) The expression of full-length VA RNAI in the same infections was tested by northern blot using a 32P-labeled oligonucleotide probe complementary to the apical stem of VA RNAI (upper panel). The equal loading of RNA on the gel was verified by northern blot analysis detecting the 7SL RNA (lower panel).
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gkt172-F7: Rescue of the dl720 (VA RNAI−/VA RNAII−) mutant phenotype by co-infection with the VA RNAI seed sequence mutated viruses. (A) HeLa cells were infected with the indicated combinations of viruses (5 FFU/cell) and 35 S-methionine pulse-labeled at 48 hpi. In the case of co-infections (lanes 8–11), cells were infected with both viruses at 2.5 FFU/cell to achieve the final multiplicity of 5 FFU/cell. Total proteins were resolved on an SDS–PAGE, and new protein synthesis was visualized by autoradiography. To confirm an equal loading of protein samples, the abundance of actin was visualized by western blot analysis using an anti-actin antibody. The position of the major viral capsid proteins is indicated to the right of the panel. (B) The expression of full-length VA RNAI in the same infections was tested by northern blot using a 32P-labeled oligonucleotide probe complementary to the apical stem of VA RNAI (upper panel). The equal loading of RNA on the gel was verified by northern blot analysis detecting the 7SL RNA (lower panel).

Mentions: To determine whether the lack of a phenotype for the mivaRNAI seed sequence mutant viruses also could be observed in another standard human cell line used in adenovirus research, we tested the significance of the seed paring interactions in a HeLa cell infection. However, as the recombinant viruses we constructed based on the AdEasy system that lacks most of the Ad5 E1 region, they cannot replicate in non-E1 expressing cell lines, like HeLa cells. To overcome this problem, we resorted to a trans-complementation assay. In this experiment, HeLa cells were co-infected with the seed sequence mutant viruses and the Ad5 mutant dl720, which has a wild-type E1 region but is defective in both VA RNAI and VA RNAII expression (16). The rationale for this experiment is that dl720 will provide the E1 functions, whereas the recombinant viruses will provide the VA RNAI function (wt or seed mutant). In this experiment, HeLa cells were infected with combinations of the viruses, and infected cells were pulse-labeled 46 hpi with 35S-methionine. Protein extracts and total cytoplasmic RNA were prepared and analyzed by SDS–PAGE and by northern blotting. As shown in Figure 7A, infection of HeLa cells with the recombinant viruses alone (lanes 4–7) did, as expected, not result in an inhibition of host cell gene expression or an induction of efficient late viral protein synthesis. In contrast, co-infection of dl720 and the recombinant viruses resulted in an efficient shut-down of host cell gene expression and an essentially complete rescue of late viral protein synthesis (compare lane 2 and lanes 8–10). The rescue of late viral protein synthesis requires VA RNAI expression (Figure 7A, lane 11) and correlates with a dramatic increase in VA RNAI accumulation (Figure 7B, lanes 8–10), most likely resulting from viral DNA replication causing an increase in the copy number of the VA RNA gene. Collectively, our results suggest that seed sequence interaction of the 5′ or the 3′ mivaRNAI is not essential for the establishment of an efficient lytic adenovirus infection in HeLa, 911 or HEK293 cells.Figure 7.


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 co-infection with the VA RNAI seed sequence mutated viruses. (A) HeLa cells were infected with the indicated combinations of viruses (5 FFU/cell) and 35 S-methionine pulse-labeled at 48 hpi. In the case of co-infections (lanes 8–11), cells were infected with both viruses at 2.5 FFU/cell to achieve the final multiplicity of 5 FFU/cell. Total proteins were resolved on an SDS–PAGE, and new protein synthesis was visualized by autoradiography. To confirm an equal loading of protein samples, the abundance of actin was visualized by western blot analysis using an anti-actin antibody. The position of the major viral capsid proteins is indicated to the right of the panel. (B) The expression of full-length VA RNAI in the same infections was tested by northern blot using a 32P-labeled oligonucleotide probe complementary to the apical stem of VA RNAI (upper panel). The equal loading of RNA on the gel was verified by northern blot analysis detecting the 7SL RNA (lower panel).
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Related In: Results  -  Collection

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Show All Figures
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gkt172-F7: Rescue of the dl720 (VA RNAI−/VA RNAII−) mutant phenotype by co-infection with the VA RNAI seed sequence mutated viruses. (A) HeLa cells were infected with the indicated combinations of viruses (5 FFU/cell) and 35 S-methionine pulse-labeled at 48 hpi. In the case of co-infections (lanes 8–11), cells were infected with both viruses at 2.5 FFU/cell to achieve the final multiplicity of 5 FFU/cell. Total proteins were resolved on an SDS–PAGE, and new protein synthesis was visualized by autoradiography. To confirm an equal loading of protein samples, the abundance of actin was visualized by western blot analysis using an anti-actin antibody. The position of the major viral capsid proteins is indicated to the right of the panel. (B) The expression of full-length VA RNAI in the same infections was tested by northern blot using a 32P-labeled oligonucleotide probe complementary to the apical stem of VA RNAI (upper panel). The equal loading of RNA on the gel was verified by northern blot analysis detecting the 7SL RNA (lower panel).
Mentions: To determine whether the lack of a phenotype for the mivaRNAI seed sequence mutant viruses also could be observed in another standard human cell line used in adenovirus research, we tested the significance of the seed paring interactions in a HeLa cell infection. However, as the recombinant viruses we constructed based on the AdEasy system that lacks most of the Ad5 E1 region, they cannot replicate in non-E1 expressing cell lines, like HeLa cells. To overcome this problem, we resorted to a trans-complementation assay. In this experiment, HeLa cells were co-infected with the seed sequence mutant viruses and the Ad5 mutant dl720, which has a wild-type E1 region but is defective in both VA RNAI and VA RNAII expression (16). The rationale for this experiment is that dl720 will provide the E1 functions, whereas the recombinant viruses will provide the VA RNAI function (wt or seed mutant). In this experiment, HeLa cells were infected with combinations of the viruses, and infected cells were pulse-labeled 46 hpi with 35S-methionine. Protein extracts and total cytoplasmic RNA were prepared and analyzed by SDS–PAGE and by northern blotting. As shown in Figure 7A, infection of HeLa cells with the recombinant viruses alone (lanes 4–7) did, as expected, not result in an inhibition of host cell gene expression or an induction of efficient late viral protein synthesis. In contrast, co-infection of dl720 and the recombinant viruses resulted in an efficient shut-down of host cell gene expression and an essentially complete rescue of late viral protein synthesis (compare lane 2 and lanes 8–10). The rescue of late viral protein synthesis requires VA RNAI expression (Figure 7A, lane 11) and correlates with a dramatic increase in VA RNAI accumulation (Figure 7B, lanes 8–10), most likely resulting from viral DNA replication causing an increase in the copy number of the VA RNA gene. Collectively, our results suggest that seed sequence interaction of the 5′ or the 3′ mivaRNAI is not essential for the establishment of an efficient lytic adenovirus infection in HeLa, 911 or HEK293 cells.Figure 7.

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