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A conserved influenza A virus nucleoprotein code controls specific viral genome packaging

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ABSTRACT

Packaging of the eight genomic RNA segments of influenza A viruses (IAV) into viral particles is coordinated by segment-specific packaging sequences. How the packaging signals regulate the specific incorporation of each RNA segment into virions and whether other viral or host factors are involved in this process is unknown. Here, we show that distinct amino acids of the viral nucleoprotein (NP) are required for packaging of specific RNA segments. This was determined by studying the NP of a bat influenza A-like virus, HL17NL10, in the context of a conventional IAV (SC35M). Replacement of conserved SC35M NP residues by those of HL17NL10 NP resulted in RNA packaging defective IAV. Surprisingly, substitution of these conserved SC35M amino acids with HL17NL10 NP residues led to IAV with altered packaging efficiencies for specific subsets of RNA segments. This suggests that NP harbours an amino acid code that dictates genome packaging into infectious virions.

No MeSH data available.


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Substitution of 14 Bat NP-specific amino acids in CH4 is required to rescue SC35M.(a) Alignment of SC35M NP and CH4 variants. Bat NP-specific amino acids are indicated in red. (b) The 19 Bat NP-specific amino acids in CH4 (highlighted in black) were mutated to SC35M-specific amino acids leaving either 14 (CH4.14), 4 (CH4.4) or no Bat NP-specific amino acids (CH4.0) and 100, 86 and 80 Bat NP-specific nucleotides, respectively. Relative polymerase activities of the indicated CH4 variants were determined by polymerase reconstitution assays in HEK293T cells. Mean and s.d. of three independent experiments are indicated in parenthesis. +successful rescue; −no rescue of recombinant viruses. (c) MDCKII cells were infected at an MOI of 0.001 with either wt SC35M, rCH4.4 or SC35M-CH4.0. At the indicated time points post infection (p.i.) virus titres were determined by plaque assay. Error bars indicate the mean and s.d. of at least three independent experiments.
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f2: Substitution of 14 Bat NP-specific amino acids in CH4 is required to rescue SC35M.(a) Alignment of SC35M NP and CH4 variants. Bat NP-specific amino acids are indicated in red. (b) The 19 Bat NP-specific amino acids in CH4 (highlighted in black) were mutated to SC35M-specific amino acids leaving either 14 (CH4.14), 4 (CH4.4) or no Bat NP-specific amino acids (CH4.0) and 100, 86 and 80 Bat NP-specific nucleotides, respectively. Relative polymerase activities of the indicated CH4 variants were determined by polymerase reconstitution assays in HEK293T cells. Mean and s.d. of three independent experiments are indicated in parenthesis. +successful rescue; −no rescue of recombinant viruses. (c) MDCKII cells were infected at an MOI of 0.001 with either wt SC35M, rCH4.4 or SC35M-CH4.0. At the indicated time points post infection (p.i.) virus titres were determined by plaque assay. Error bars indicate the mean and s.d. of at least three independent experiments.

Mentions: To understand why rescue of SC35M with the NP chimeras CH1, CH3 and CH4 failed, we focused on CH4 because it is nested inside both CH1 and CH3, contains only 19 Bat NP-specific amino acids, and harbours the fewest number of Bat NP-specific nucleotides. Moreover, it supported SC35M polymerase activity in polymerase reconstitution assays as efficiently as SC35M NP (Fig. 1c). CH4 was further modified to contain either 14 (CH4.14), 4 (CH4.4) or 0 (CH4.0) Bat NP-specific amino acids (Fig. 2a,b). In CH4.14 the five most divergent residues of the 19 bat NP-specific amino acids with respect to charge and size were replaced with SC35M ones, while only four Bat NP-specific amino acids were left in CH4.4. All NP mutants supported the polymerase activity of SC35M; however, only recombinant SC35M encoding either CH4.4 (rCH4.4) or CH4.0 (rCH4.0) could be successfully generated (Fig. 2b). As expected, rCH4.0 replicated to similar titres as wt SC35M, while rCH4.4 replication was slightly reduced at 12 and 24 h.p.i. (Fig. 2c). These results suggest that the 10 Bat NP-specific amino acids in CH4.14 prevent successful rescue of SC35M.


A conserved influenza A virus nucleoprotein code controls specific viral genome packaging
Substitution of 14 Bat NP-specific amino acids in CH4 is required to rescue SC35M.(a) Alignment of SC35M NP and CH4 variants. Bat NP-specific amino acids are indicated in red. (b) The 19 Bat NP-specific amino acids in CH4 (highlighted in black) were mutated to SC35M-specific amino acids leaving either 14 (CH4.14), 4 (CH4.4) or no Bat NP-specific amino acids (CH4.0) and 100, 86 and 80 Bat NP-specific nucleotides, respectively. Relative polymerase activities of the indicated CH4 variants were determined by polymerase reconstitution assays in HEK293T cells. Mean and s.d. of three independent experiments are indicated in parenthesis. +successful rescue; −no rescue of recombinant viruses. (c) MDCKII cells were infected at an MOI of 0.001 with either wt SC35M, rCH4.4 or SC35M-CH4.0. At the indicated time points post infection (p.i.) virus titres were determined by plaque assay. Error bars indicate the mean and s.d. of at least three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5035998&req=5

f2: Substitution of 14 Bat NP-specific amino acids in CH4 is required to rescue SC35M.(a) Alignment of SC35M NP and CH4 variants. Bat NP-specific amino acids are indicated in red. (b) The 19 Bat NP-specific amino acids in CH4 (highlighted in black) were mutated to SC35M-specific amino acids leaving either 14 (CH4.14), 4 (CH4.4) or no Bat NP-specific amino acids (CH4.0) and 100, 86 and 80 Bat NP-specific nucleotides, respectively. Relative polymerase activities of the indicated CH4 variants were determined by polymerase reconstitution assays in HEK293T cells. Mean and s.d. of three independent experiments are indicated in parenthesis. +successful rescue; −no rescue of recombinant viruses. (c) MDCKII cells were infected at an MOI of 0.001 with either wt SC35M, rCH4.4 or SC35M-CH4.0. At the indicated time points post infection (p.i.) virus titres were determined by plaque assay. Error bars indicate the mean and s.d. of at least three independent experiments.
Mentions: To understand why rescue of SC35M with the NP chimeras CH1, CH3 and CH4 failed, we focused on CH4 because it is nested inside both CH1 and CH3, contains only 19 Bat NP-specific amino acids, and harbours the fewest number of Bat NP-specific nucleotides. Moreover, it supported SC35M polymerase activity in polymerase reconstitution assays as efficiently as SC35M NP (Fig. 1c). CH4 was further modified to contain either 14 (CH4.14), 4 (CH4.4) or 0 (CH4.0) Bat NP-specific amino acids (Fig. 2a,b). In CH4.14 the five most divergent residues of the 19 bat NP-specific amino acids with respect to charge and size were replaced with SC35M ones, while only four Bat NP-specific amino acids were left in CH4.4. All NP mutants supported the polymerase activity of SC35M; however, only recombinant SC35M encoding either CH4.4 (rCH4.4) or CH4.0 (rCH4.0) could be successfully generated (Fig. 2b). As expected, rCH4.0 replicated to similar titres as wt SC35M, while rCH4.4 replication was slightly reduced at 12 and 24 h.p.i. (Fig. 2c). These results suggest that the 10 Bat NP-specific amino acids in CH4.14 prevent successful rescue of SC35M.

View Article: PubMed Central - PubMed

ABSTRACT

Packaging of the eight genomic RNA segments of influenza A viruses (IAV) into viral particles is coordinated by segment-specific packaging sequences. How the packaging signals regulate the specific incorporation of each RNA segment into virions and whether other viral or host factors are involved in this process is unknown. Here, we show that distinct amino acids of the viral nucleoprotein (NP) are required for packaging of specific RNA segments. This was determined by studying the NP of a bat influenza A-like virus, HL17NL10, in the context of a conventional IAV (SC35M). Replacement of conserved SC35M NP residues by those of HL17NL10 NP resulted in RNA packaging defective IAV. Surprisingly, substitution of these conserved SC35M amino acids with HL17NL10 NP residues led to IAV with altered packaging efficiencies for specific subsets of RNA segments. This suggests that NP harbours an amino acid code that dictates genome packaging into infectious virions.

No MeSH data available.


Related in: MedlinePlus