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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 silent mutations S71 and R73 on viral replication and on incorporation of vRNA 7. (A) MDCK cells were infected at a m.o.i. of 10−4 with wild-type recombinant A/Moscow/10/99 (H3N2) virus or a virus bearing silent mutations at M2 codons 71 and 73. The release of viral progeny into the supernatant was monitored by determining the tissue culture infective dose (TCID50). Points correspond to the mean of two experiments; the data ranges are smaller than the symbol size. (B) Strategy and output of the competition experiment.
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gkr985-F7: Effect of silent mutations S71 and R73 on viral replication and on incorporation of vRNA 7. (A) MDCK cells were infected at a m.o.i. of 10−4 with wild-type recombinant A/Moscow/10/99 (H3N2) virus or a virus bearing silent mutations at M2 codons 71 and 73. The release of viral progeny into the supernatant was monitored by determining the tissue culture infective dose (TCID50). Points correspond to the mean of two experiments; the data ranges are smaller than the symbol size. (B) Strategy and output of the competition experiment.

Mentions: As the importance of codons S71 and R73 in genome packaging has only been demonstrated for a laboratory adapted strain of H1N1 influenza A virus (5), we introduced the same silent mutations in recombinant A/Moscow/10/99 (H3N2), which is a strain that recently circulated in the human population. As compared to the wild-type virus, mutant H3N2 virus produced 5- to 10-fold less infectious virions (Figure 7A). We checked that S71–R73 mutations have no effect on vRNA synthesis or viral gene expression 8-h post-infection (data not shown). Expression of proteins M1 and M2 was analysed by Western blot using mouse monoclonal antibodies after infection at a m.o.i. of 4. To prove that they affect packaging of vRNA 7, we produced recombinant viruses from 293T cells transfected with equimolar amounts of the eight wild-type reverse genetics cDNA clones plus the reverse genetic cDNA clone of segment 7 containing the S71–R73 mutation (Figure 7B). This set-up allows competition between wild-type and mutant vRNA 7 for incorporation into viral particles. To determine the incorporation rate of the mutated segment 7 vRNPs into infectious viral particles, plaque purification was carried out from the supernatant of the transfected cells. A total of 72 randomly chosen plaque-purified viruses were analysed by sequencing: all incorporated wild-type segment 7, demonstrating that codons S71 and R73 have a major impact on the selective incorporation of segment 7 (Figure 7B).Figure 7.


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 silent mutations S71 and R73 on viral replication and on incorporation of vRNA 7. (A) MDCK cells were infected at a m.o.i. of 10−4 with wild-type recombinant A/Moscow/10/99 (H3N2) virus or a virus bearing silent mutations at M2 codons 71 and 73. The release of viral progeny into the supernatant was monitored by determining the tissue culture infective dose (TCID50). Points correspond to the mean of two experiments; the data ranges are smaller than the symbol size. (B) Strategy and output of the competition experiment.
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Related In: Results  -  Collection

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gkr985-F7: Effect of silent mutations S71 and R73 on viral replication and on incorporation of vRNA 7. (A) MDCK cells were infected at a m.o.i. of 10−4 with wild-type recombinant A/Moscow/10/99 (H3N2) virus or a virus bearing silent mutations at M2 codons 71 and 73. The release of viral progeny into the supernatant was monitored by determining the tissue culture infective dose (TCID50). Points correspond to the mean of two experiments; the data ranges are smaller than the symbol size. (B) Strategy and output of the competition experiment.
Mentions: As the importance of codons S71 and R73 in genome packaging has only been demonstrated for a laboratory adapted strain of H1N1 influenza A virus (5), we introduced the same silent mutations in recombinant A/Moscow/10/99 (H3N2), which is a strain that recently circulated in the human population. As compared to the wild-type virus, mutant H3N2 virus produced 5- to 10-fold less infectious virions (Figure 7A). We checked that S71–R73 mutations have no effect on vRNA synthesis or viral gene expression 8-h post-infection (data not shown). Expression of proteins M1 and M2 was analysed by Western blot using mouse monoclonal antibodies after infection at a m.o.i. of 4. To prove that they affect packaging of vRNA 7, we produced recombinant viruses from 293T cells transfected with equimolar amounts of the eight wild-type reverse genetics cDNA clones plus the reverse genetic cDNA clone of segment 7 containing the S71–R73 mutation (Figure 7B). This set-up allows competition between wild-type and mutant vRNA 7 for incorporation into viral particles. To determine the incorporation rate of the mutated segment 7 vRNPs into infectious viral particles, plaque purification was carried out from the supernatant of the transfected cells. A total of 72 randomly chosen plaque-purified viruses were analysed by sequencing: all incorporated wild-type segment 7, demonstrating that codons S71 and R73 have a major impact on the selective incorporation of segment 7 (Figure 7B).Figure 7.

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