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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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Effect of terminal deletions on the three strongest in vitro vRNA/vRNA interactions. (A) Schematic representation and nomenclature of the wild-type and mutant vRNAs. Deletions are represented by grey rectangles. Numbering of the genomic vRNAs is from 3′- to 5′-end. (B–D) Representative gels and quantifications are shown for interactions between wild-type or mutant vRNAs 6 and 7 (B), 4 and 8 (C) and 4 and 7 (D). Intermolecular complexes are marked by asterisks. The weight fraction (%) of the RNA mass migrating as an intermolecular complex was determined for each vRNA pair, and for each panel the intermolecular complex obtained with mutant vRNAs was normalized relative to the complex formed by the two wild-type vRNAs. Quantifications are expressed as mean ± SEM (n = 3−10).
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gkr985-F4: Effect of terminal deletions on the three strongest in vitro vRNA/vRNA interactions. (A) Schematic representation and nomenclature of the wild-type and mutant vRNAs. Deletions are represented by grey rectangles. Numbering of the genomic vRNAs is from 3′- to 5′-end. (B–D) Representative gels and quantifications are shown for interactions between wild-type or mutant vRNAs 6 and 7 (B), 4 and 8 (C) and 4 and 7 (D). Intermolecular complexes are marked by asterisks. The weight fraction (%) of the RNA mass migrating as an intermolecular complex was determined for each vRNA pair, and for each panel the intermolecular complex obtained with mutant vRNAs was normalized relative to the complex formed by the two wild-type vRNAs. Quantifications are expressed as mean ± SEM (n = 3−10).

Mentions: To test whether this network might be relevant to the selective packaging of the vRNPs, we analysed the effect of deletions in the terminal regions of the vRNAs, where the segment-specific packaging signals are located (5,15,16,19,22,23), on the three strongest interactions detected in our assay, namely the interactions between vRNAs 6 and 7, 4 and 8, and 4 and 7 (Figure 3B). Deleting 100 nt in the 5′ region of vRNA 6 reduced the interaction with vRNA 7 by 2.5-fold (Figure 4A and B). Similarly, this interaction was reduced 10-fold by a 100 nt deletion in the 5′ region of vRNA 7 (Figure 4B). On the other hand deletions in the 3′ region of either vRNA 6 or vRNA 7 had no significant effect on the interaction between these vRNAs. Concerning the interaction between vRNAs 4 and 8, deleting 97 or 100 nt at the 5′-end of the coding region of vRNA 4 or 8 reduced complex formation by 2- and 2.5-fold, respectively, whereas deletions at the 3′-end of the coding regions of vRNA 4 or 8 did not impair this interaction (Figure 4A and C). A somewhat different picture was observed with the interaction between vRNAs 4 and 7 (Figure 4A and D). Deletions in the 3′ and 5′ regions of vRNA 7 both impacted this interaction, even though the deletion in the 3′ region had a more dramatic effect (4-fold, versus a 35% reduction). On the contrary, deletions at the 3′ or the 5′-end of the coding region of vRNA 4 had no significant impact on this interaction. It is possible that the region(s) of vRNA 4 interacting with vRNA 7 is (are) located either in the UTRs or in a more central part of the coding region. However, we cannot exclude the existence of stable redundant interactions involving both terminal domains of the coding region of vRNA 4.Figure 4.


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)

Effect of terminal deletions on the three strongest in vitro vRNA/vRNA interactions. (A) Schematic representation and nomenclature of the wild-type and mutant vRNAs. Deletions are represented by grey rectangles. Numbering of the genomic vRNAs is from 3′- to 5′-end. (B–D) Representative gels and quantifications are shown for interactions between wild-type or mutant vRNAs 6 and 7 (B), 4 and 8 (C) and 4 and 7 (D). Intermolecular complexes are marked by asterisks. The weight fraction (%) of the RNA mass migrating as an intermolecular complex was determined for each vRNA pair, and for each panel the intermolecular complex obtained with mutant vRNAs was normalized relative to the complex formed by the two wild-type vRNAs. Quantifications are expressed as mean ± SEM (n = 3−10).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr985-F4: Effect of terminal deletions on the three strongest in vitro vRNA/vRNA interactions. (A) Schematic representation and nomenclature of the wild-type and mutant vRNAs. Deletions are represented by grey rectangles. Numbering of the genomic vRNAs is from 3′- to 5′-end. (B–D) Representative gels and quantifications are shown for interactions between wild-type or mutant vRNAs 6 and 7 (B), 4 and 8 (C) and 4 and 7 (D). Intermolecular complexes are marked by asterisks. The weight fraction (%) of the RNA mass migrating as an intermolecular complex was determined for each vRNA pair, and for each panel the intermolecular complex obtained with mutant vRNAs was normalized relative to the complex formed by the two wild-type vRNAs. Quantifications are expressed as mean ± SEM (n = 3−10).
Mentions: To test whether this network might be relevant to the selective packaging of the vRNPs, we analysed the effect of deletions in the terminal regions of the vRNAs, where the segment-specific packaging signals are located (5,15,16,19,22,23), on the three strongest interactions detected in our assay, namely the interactions between vRNAs 6 and 7, 4 and 8, and 4 and 7 (Figure 3B). Deleting 100 nt in the 5′ region of vRNA 6 reduced the interaction with vRNA 7 by 2.5-fold (Figure 4A and B). Similarly, this interaction was reduced 10-fold by a 100 nt deletion in the 5′ region of vRNA 7 (Figure 4B). On the other hand deletions in the 3′ region of either vRNA 6 or vRNA 7 had no significant effect on the interaction between these vRNAs. Concerning the interaction between vRNAs 4 and 8, deleting 97 or 100 nt at the 5′-end of the coding region of vRNA 4 or 8 reduced complex formation by 2- and 2.5-fold, respectively, whereas deletions at the 3′-end of the coding regions of vRNA 4 or 8 did not impair this interaction (Figure 4A and C). A somewhat different picture was observed with the interaction between vRNAs 4 and 7 (Figure 4A and D). Deletions in the 3′ and 5′ regions of vRNA 7 both impacted this interaction, even though the deletion in the 3′ region had a more dramatic effect (4-fold, versus a 35% reduction). On the contrary, deletions at the 3′ or the 5′-end of the coding region of vRNA 4 had no significant impact on this interaction. It is possible that the region(s) of vRNA 4 interacting with vRNA 7 is (are) located either in the UTRs or in a more central part of the coding region. However, we cannot exclude the existence of stable redundant interactions involving both terminal domains of the coding region of vRNA 4.Figure 4.

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