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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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Possible arrangements of the vRNPs within budding H3N2 influenza A virions. Top views of the possible arrangements of the vRNPs in P2 (A and B) and P3 (C and D) based on tomography data alone (a and c) or incorporating the in vitro interaction data (B and D). The intermolecular RNA interactions identified in vitro are indicated by thick grey line. The two thin lines correspond to the interaction between vRNAs 3 and 4 and are mutually exclusive, depending on the actual location of vRNAs 2 and 3.
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gkr985-F8: Possible arrangements of the vRNPs within budding H3N2 influenza A virions. Top views of the possible arrangements of the vRNPs in P2 (A and B) and P3 (C and D) based on tomography data alone (a and c) or incorporating the in vitro interaction data (B and D). The intermolecular RNA interactions identified in vitro are indicated by thick grey line. The two thin lines correspond to the interaction between vRNAs 3 and 4 and are mutually exclusive, depending on the actual location of vRNAs 2 and 3.

Mentions: As our results suggest that the intermolecular vRNAs interactions we detected in vitro are relevant to vRNP packaging, we combined these data with our tomography data to analyse the relative disposition of vRNPs within human H3N2 influenza virions (Figure 8). According to Figure 2D, it is possible to discriminate vRNAs 1, 2 and 3 from the other vRNAs, based on the length of the vRNPs, but not between them. Similarly, it is possible to discriminate vRNAs 4 and 5, on one hand, and vRNAs 6–8 (for P2) or 6 and 7 (for P3), on the other hand, from the other vRNAs. In all four 3D reconstructions, two of the three longest vRNPs are contiguous and were used as starting points for anticlockwise labelling of the vRNPs, while the third one is opposite to them (Figure 2; Supplementary Figure S2 and Supplementary Movies S1A, S1B, S2A and S2B). Even though there is little difference between the length of the fourth and fifth vRNPs, the fourth one was always the central one, strongly suggesting that vRNP h contains vRNA 4 (Figure 1B, panels P1-1, P1-2 and P3-2; Figure 2A–D; Supplementary Figure S2 and Supplementary Movies S1A, S1B, S2A and S2B). The central location of vRNA 4 fits with the observation that this vRNA interacts with three different partners in vitro, whereas vRNA 5 has only one partner (Figures 3, 8B and D). This in turn allows identification of vRNA 5 in the 3D reconstructions of P2 and P3. In P3, vRNA 8 can be identified as the shortest vRNP (Figures 2B, C and 8C), and it is located next to vRNA 5 in agreement with the in vitro interaction data (Figures 3 and 8D). Noticeably, the tomography data indicate that all influenza A H3N2 virions do not share the same internal organization, as vRNA 5 has different locations in P2 and P3 (Figure 8A and C). Note that the interaction network defines a block of four external vRNPs comprising vRNAs 1, 7, 6 and 2 or 3, but that the orientation of this block can be deduced neither from tomography nor from the in vitro interaction network. Nevertheless, by combining the tomography data with the in vitro interaction network, and assuming that, in virions, interactions preferentially take place between close neighbours, the number of possible arrangements of the vRNPs in P2 and P3 is reduced from 5 760 to 4 (Figure 8C and D). As there are two vRNAs that are each involved in three intermolecular interactions while not interacting together (vRNAs 4 and 6), there will always be at least one interaction linking non-adjacent vRNAs, and we selected arrangements with exactly one such interaction (Figure 8C and D). Measuring the length of the vRNPs in P4 was difficult, as their upper end could not always be identified precisely. However, our data suggest that the internal organization of the vRNAs in P1 and P4 is similar to P3.Figure 8.


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)

Possible arrangements of the vRNPs within budding H3N2 influenza A virions. Top views of the possible arrangements of the vRNPs in P2 (A and B) and P3 (C and D) based on tomography data alone (a and c) or incorporating the in vitro interaction data (B and D). The intermolecular RNA interactions identified in vitro are indicated by thick grey line. The two thin lines correspond to the interaction between vRNAs 3 and 4 and are mutually exclusive, depending on the actual location of vRNAs 2 and 3.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr985-F8: Possible arrangements of the vRNPs within budding H3N2 influenza A virions. Top views of the possible arrangements of the vRNPs in P2 (A and B) and P3 (C and D) based on tomography data alone (a and c) or incorporating the in vitro interaction data (B and D). The intermolecular RNA interactions identified in vitro are indicated by thick grey line. The two thin lines correspond to the interaction between vRNAs 3 and 4 and are mutually exclusive, depending on the actual location of vRNAs 2 and 3.
Mentions: As our results suggest that the intermolecular vRNAs interactions we detected in vitro are relevant to vRNP packaging, we combined these data with our tomography data to analyse the relative disposition of vRNPs within human H3N2 influenza virions (Figure 8). According to Figure 2D, it is possible to discriminate vRNAs 1, 2 and 3 from the other vRNAs, based on the length of the vRNPs, but not between them. Similarly, it is possible to discriminate vRNAs 4 and 5, on one hand, and vRNAs 6–8 (for P2) or 6 and 7 (for P3), on the other hand, from the other vRNAs. In all four 3D reconstructions, two of the three longest vRNPs are contiguous and were used as starting points for anticlockwise labelling of the vRNPs, while the third one is opposite to them (Figure 2; Supplementary Figure S2 and Supplementary Movies S1A, S1B, S2A and S2B). Even though there is little difference between the length of the fourth and fifth vRNPs, the fourth one was always the central one, strongly suggesting that vRNP h contains vRNA 4 (Figure 1B, panels P1-1, P1-2 and P3-2; Figure 2A–D; Supplementary Figure S2 and Supplementary Movies S1A, S1B, S2A and S2B). The central location of vRNA 4 fits with the observation that this vRNA interacts with three different partners in vitro, whereas vRNA 5 has only one partner (Figures 3, 8B and D). This in turn allows identification of vRNA 5 in the 3D reconstructions of P2 and P3. In P3, vRNA 8 can be identified as the shortest vRNP (Figures 2B, C and 8C), and it is located next to vRNA 5 in agreement with the in vitro interaction data (Figures 3 and 8D). Noticeably, the tomography data indicate that all influenza A H3N2 virions do not share the same internal organization, as vRNA 5 has different locations in P2 and P3 (Figure 8A and C). Note that the interaction network defines a block of four external vRNPs comprising vRNAs 1, 7, 6 and 2 or 3, but that the orientation of this block can be deduced neither from tomography nor from the in vitro interaction network. Nevertheless, by combining the tomography data with the in vitro interaction network, and assuming that, in virions, interactions preferentially take place between close neighbours, the number of possible arrangements of the vRNPs in P2 and P3 is reduced from 5 760 to 4 (Figure 8C and D). As there are two vRNAs that are each involved in three intermolecular interactions while not interacting together (vRNAs 4 and 6), there will always be at least one interaction linking non-adjacent vRNAs, and we selected arrangements with exactly one such interaction (Figure 8C and D). Measuring the length of the vRNPs in P4 was difficult, as their upper end could not always be identified precisely. However, our data suggest that the internal organization of the vRNAs in P1 and P4 is similar to P3.Figure 8.

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