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A supramolecular assembly formed by influenza A virus genomic RNA segments.

Fournier E, Moules V, Essere B, Paillart JC, Sirbat JD, Isel C, Cavalier A, Rolland JP, Thomas D, Lina B, Marquet R - Nucleic Acids Res. (2011)

Bottom Line: The regions involved in the strongest interactions were identified and correspond to known packaging signals.A limited set of nucleotides in the 5' region of vRNA 7 was shown to interact with vRNA 6 and to be crucial for packaging of the former vRNA.Collectively, our findings support a model in which the eight genomic RNA segments are selected and packaged as an organized supramolecular complex held together by direct base pairing of the packaging signals.

View Article: PubMed Central - PubMed

Affiliation: Architecture et Réactivité de l'ARN, Université de Strasbourg, CNRS, IBMC, 15 rue René Descartes, 67084 Strasbourg, France.

ABSTRACT
The influenza A virus genome consists of eight viral RNAs (vRNAs) that form viral ribonucleoproteins (vRNPs). Even though evidence supporting segment-specific packaging of vRNAs is accumulating, the mechanism ensuring selective packaging of one copy of each vRNA into the viral particles remains largely unknown. We used electron tomography to show that the eight vRNPs emerge from a common 'transition zone' located underneath the matrix layer at the budding tip of the virions, where they appear to be interconnected and often form a star-like structure. This zone appears as a platform in 3D surface rendering and is thick enough to contain all known packaging signals. In vitro, all vRNA segments are involved in a single network of intermolecular interactions. The regions involved in the strongest interactions were identified and correspond to known packaging signals. A limited set of nucleotides in the 5' region of vRNA 7 was shown to interact with vRNA 6 and to be crucial for packaging of the former vRNA. Collectively, our findings support a model in which the eight genomic RNA segments are selected and packaged as an organized supramolecular complex held together by direct base pairing of the packaging signals.

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Related in: MedlinePlus

Interaction of wild-type vRNA 7 and vRNA 7Δ100 5′ with vRNA 6 in the presence of NP. vRNA 7 without or with a 100 nt deletion at the 5′-end of the coding sequence was modified to include a 3′ overhang complementary to a biotinylated DNA oligonucleotide. Modified vRNA 7 and vRNA 6 were first transcribed and incubated separately with saturating amounts of NP, then incubated together. The biotinylated DNA oligonucleotide was used to retain the complexes on magnetic beads, and segment-specific PCR was used to detect vRNA 7 (A) and vRNA 6 (B) retained on the beads in the absence (−NP) or in the presence of NP (+NP).
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gkr985-F5: Interaction of wild-type vRNA 7 and vRNA 7Δ100 5′ with vRNA 6 in the presence of NP. vRNA 7 without or with a 100 nt deletion at the 5′-end of the coding sequence was modified to include a 3′ overhang complementary to a biotinylated DNA oligonucleotide. Modified vRNA 7 and vRNA 6 were first transcribed and incubated separately with saturating amounts of NP, then incubated together. The biotinylated DNA oligonucleotide was used to retain the complexes on magnetic beads, and segment-specific PCR was used to detect vRNA 7 (A) and vRNA 6 (B) retained on the beads in the absence (−NP) or in the presence of NP (+NP).

Mentions: To corroborate the idea that packaging signals mediate interactions between vRNPs via direct vRNA-vRNA interaction, we next focused on vRNA 7 and its interaction with vRNA 6. The analysis of the RNA complexes formed in vitro described above did not allow the study of the intermolecular interactions taking place in the presence of NP: this protein inhibited in vitro transcription, and if using purified vRNAs, the vRNA/NP complexes could not be resolved on agarose gels due to aggregation (data not shown). We therefore developed an alternative protocol in which a modified vRNA 7 without or with a 100 nt deletion at the 5′-end of the coding sequence was modified to include a 3′ overhang complementary to a biotinylated DNA oligonucleotide. Modified vRNA 7 and vRNA 6 were first transcribed and incubated separately with saturating amounts of NP. They were then incubated together, and the NP-covered vRNA 7 and vRNA 7/vRNA 6 complexes were retained on streptavidin-coated beads. Finally, vRNAs retained on the beads were detected by segment-specific RT-PCR. Even though some background binding of vRNA 7 and vRNA 7Δ100 5′ to the beads was detected when the biotinylated DNA oligonucleotide was omitted, the PCR signal was stronger when it was included in the reaction mixture (Figure 5A). Binding of vRNA 6 to the beads was detected in the presence of vRNA 7, but not in its absence or in the presence vRNA 7Δ100 5′ (Figure 5B) in agreement with the analysis by native gel electrophoresis (Figure 4B). Importantly, similar results were observed with ‘naked’ vRNAs and NP-covered RNAs (Figure 5), indicating that NP does not prevent the interaction between vRNPs 6 and 7 and that it does not affect its specificity.Figure 5.


A supramolecular assembly formed by influenza A virus genomic RNA segments.

Fournier E, Moules V, Essere B, Paillart JC, Sirbat JD, Isel C, Cavalier A, Rolland JP, Thomas D, Lina B, Marquet R - Nucleic Acids Res. (2011)

Interaction of wild-type vRNA 7 and vRNA 7Δ100 5′ with vRNA 6 in the presence of NP. vRNA 7 without or with a 100 nt deletion at the 5′-end of the coding sequence was modified to include a 3′ overhang complementary to a biotinylated DNA oligonucleotide. Modified vRNA 7 and vRNA 6 were first transcribed and incubated separately with saturating amounts of NP, then incubated together. The biotinylated DNA oligonucleotide was used to retain the complexes on magnetic beads, and segment-specific PCR was used to detect vRNA 7 (A) and vRNA 6 (B) retained on the beads in the absence (−NP) or in the presence of NP (+NP).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3300030&req=5

gkr985-F5: Interaction of wild-type vRNA 7 and vRNA 7Δ100 5′ with vRNA 6 in the presence of NP. vRNA 7 without or with a 100 nt deletion at the 5′-end of the coding sequence was modified to include a 3′ overhang complementary to a biotinylated DNA oligonucleotide. Modified vRNA 7 and vRNA 6 were first transcribed and incubated separately with saturating amounts of NP, then incubated together. The biotinylated DNA oligonucleotide was used to retain the complexes on magnetic beads, and segment-specific PCR was used to detect vRNA 7 (A) and vRNA 6 (B) retained on the beads in the absence (−NP) or in the presence of NP (+NP).
Mentions: To corroborate the idea that packaging signals mediate interactions between vRNPs via direct vRNA-vRNA interaction, we next focused on vRNA 7 and its interaction with vRNA 6. The analysis of the RNA complexes formed in vitro described above did not allow the study of the intermolecular interactions taking place in the presence of NP: this protein inhibited in vitro transcription, and if using purified vRNAs, the vRNA/NP complexes could not be resolved on agarose gels due to aggregation (data not shown). We therefore developed an alternative protocol in which a modified vRNA 7 without or with a 100 nt deletion at the 5′-end of the coding sequence was modified to include a 3′ overhang complementary to a biotinylated DNA oligonucleotide. Modified vRNA 7 and vRNA 6 were first transcribed and incubated separately with saturating amounts of NP. They were then incubated together, and the NP-covered vRNA 7 and vRNA 7/vRNA 6 complexes were retained on streptavidin-coated beads. Finally, vRNAs retained on the beads were detected by segment-specific RT-PCR. Even though some background binding of vRNA 7 and vRNA 7Δ100 5′ to the beads was detected when the biotinylated DNA oligonucleotide was omitted, the PCR signal was stronger when it was included in the reaction mixture (Figure 5A). Binding of vRNA 6 to the beads was detected in the presence of vRNA 7, but not in its absence or in the presence vRNA 7Δ100 5′ (Figure 5B) in agreement with the analysis by native gel electrophoresis (Figure 4B). Importantly, similar results were observed with ‘naked’ vRNAs and NP-covered RNAs (Figure 5), indicating that NP does not prevent the interaction between vRNPs 6 and 7 and that it does not affect its specificity.Figure 5.

Bottom Line: The regions involved in the strongest interactions were identified and correspond to known packaging signals.A limited set of nucleotides in the 5' region of vRNA 7 was shown to interact with vRNA 6 and to be crucial for packaging of the former vRNA.Collectively, our findings support a model in which the eight genomic RNA segments are selected and packaged as an organized supramolecular complex held together by direct base pairing of the packaging signals.

View Article: PubMed Central - PubMed

Affiliation: Architecture et Réactivité de l'ARN, Université de Strasbourg, CNRS, IBMC, 15 rue René Descartes, 67084 Strasbourg, France.

ABSTRACT
The influenza A virus genome consists of eight viral RNAs (vRNAs) that form viral ribonucleoproteins (vRNPs). Even though evidence supporting segment-specific packaging of vRNAs is accumulating, the mechanism ensuring selective packaging of one copy of each vRNA into the viral particles remains largely unknown. We used electron tomography to show that the eight vRNPs emerge from a common 'transition zone' located underneath the matrix layer at the budding tip of the virions, where they appear to be interconnected and often form a star-like structure. This zone appears as a platform in 3D surface rendering and is thick enough to contain all known packaging signals. In vitro, all vRNA segments are involved in a single network of intermolecular interactions. The regions involved in the strongest interactions were identified and correspond to known packaging signals. A limited set of nucleotides in the 5' region of vRNA 7 was shown to interact with vRNA 6 and to be crucial for packaging of the former vRNA. Collectively, our findings support a model in which the eight genomic RNA segments are selected and packaged as an organized supramolecular complex held together by direct base pairing of the packaging signals.

Show MeSH
Related in: MedlinePlus