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

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.


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Bat NP-specific amino acids in SC35M NP cause impaired packaging.(a,b) Packaging efficiency of a single minigenome supported by SC35M NP (NP), CH2, NP7 or NP7-R31G in a VLP-based packaging assay. Reconstitutions of SC35M VLPs were carried out in the presence a GFP-encoding reporter segment flanked by IS of the NP segment (IS−GFP) or in addition with the bundling signals (BS) in the 5′ and 3′ NP ORF (IS+BS−GFP). After infection of MDCKII cells and subsequent superinfection with wt SC35M, GFP-positive cells were quantified by FACS. NS, not significant; *P<0.05; **P<0.01. (c,d) Packaging efficiency mediated by SC35M NP (NP), NP7, CH2 or NP7-R31G in the presence of the reporter segment IS+GFP (R) (c) or IS+BS−GFP (R) (d) and, if indicated, in the presence of the remaining seven wild-type genome segments (+7). NS, not significant; *P<0.05; **P<0.01; ****P<0.0001. Student's t test was used for two-group comparisons. Error bars indicate the mean and s.d. of at least three independent experiments.
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f5: Bat NP-specific amino acids in SC35M NP cause impaired packaging.(a,b) Packaging efficiency of a single minigenome supported by SC35M NP (NP), CH2, NP7 or NP7-R31G in a VLP-based packaging assay. Reconstitutions of SC35M VLPs were carried out in the presence a GFP-encoding reporter segment flanked by IS of the NP segment (IS−GFP) or in addition with the bundling signals (BS) in the 5′ and 3′ NP ORF (IS+BS−GFP). After infection of MDCKII cells and subsequent superinfection with wt SC35M, GFP-positive cells were quantified by FACS. NS, not significant; *P<0.05; **P<0.01. (c,d) Packaging efficiency mediated by SC35M NP (NP), NP7, CH2 or NP7-R31G in the presence of the reporter segment IS+GFP (R) (c) or IS+BS−GFP (R) (d) and, if indicated, in the presence of the remaining seven wild-type genome segments (+7). NS, not significant; *P<0.05; **P<0.01; ****P<0.0001. Student's t test was used for two-group comparisons. Error bars indicate the mean and s.d. of at least three independent experiments.

Mentions: We constructed a GFP reporter minigenome containing either the IS of the SC35M NP segment (IS−GFP) or both the IS and BS elements (IS+BS−GFP) and compared the efficacy of CH2 and NP7 NP proteins to package these reporter minigenomes into viral particles in the absence of the other seven segments. For this purpose, SC35M VLPs were generated in human 293 T cells in the presence of the reporter minigenome (IS−GFP or IS+BS−GFP) and NP protein, either wt SC35M NP or mutant CH2 or NP7. The incorporation of the GFP reporter minigenomes into VLPs was subsequently quantified by co-infection of MDCKII cells with VLPs and wt SC35M virus. No differences were observed in the amount of VLPs released from 293 T cells as evidenced by the number of GFP-positive MDCKII cells (Fig. 5a,b). This finding indicates that the incorporation of a single genome segment into viral particles is not affected, irrespective of the presence of the bundling sequence.


A conserved influenza A virus nucleoprotein code controls specific viral genome packaging
Bat NP-specific amino acids in SC35M NP cause impaired packaging.(a,b) Packaging efficiency of a single minigenome supported by SC35M NP (NP), CH2, NP7 or NP7-R31G in a VLP-based packaging assay. Reconstitutions of SC35M VLPs were carried out in the presence a GFP-encoding reporter segment flanked by IS of the NP segment (IS−GFP) or in addition with the bundling signals (BS) in the 5′ and 3′ NP ORF (IS+BS−GFP). After infection of MDCKII cells and subsequent superinfection with wt SC35M, GFP-positive cells were quantified by FACS. NS, not significant; *P<0.05; **P<0.01. (c,d) Packaging efficiency mediated by SC35M NP (NP), NP7, CH2 or NP7-R31G in the presence of the reporter segment IS+GFP (R) (c) or IS+BS−GFP (R) (d) and, if indicated, in the presence of the remaining seven wild-type genome segments (+7). NS, not significant; *P<0.05; **P<0.01; ****P<0.0001. Student's t test was used for two-group comparisons. 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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getmorefigures.php?uid=PMC5035998&req=5

f5: Bat NP-specific amino acids in SC35M NP cause impaired packaging.(a,b) Packaging efficiency of a single minigenome supported by SC35M NP (NP), CH2, NP7 or NP7-R31G in a VLP-based packaging assay. Reconstitutions of SC35M VLPs were carried out in the presence a GFP-encoding reporter segment flanked by IS of the NP segment (IS−GFP) or in addition with the bundling signals (BS) in the 5′ and 3′ NP ORF (IS+BS−GFP). After infection of MDCKII cells and subsequent superinfection with wt SC35M, GFP-positive cells were quantified by FACS. NS, not significant; *P<0.05; **P<0.01. (c,d) Packaging efficiency mediated by SC35M NP (NP), NP7, CH2 or NP7-R31G in the presence of the reporter segment IS+GFP (R) (c) or IS+BS−GFP (R) (d) and, if indicated, in the presence of the remaining seven wild-type genome segments (+7). NS, not significant; *P<0.05; **P<0.01; ****P<0.0001. Student's t test was used for two-group comparisons. Error bars indicate the mean and s.d. of at least three independent experiments.
Mentions: We constructed a GFP reporter minigenome containing either the IS of the SC35M NP segment (IS−GFP) or both the IS and BS elements (IS+BS−GFP) and compared the efficacy of CH2 and NP7 NP proteins to package these reporter minigenomes into viral particles in the absence of the other seven segments. For this purpose, SC35M VLPs were generated in human 293 T cells in the presence of the reporter minigenome (IS−GFP or IS+BS−GFP) and NP protein, either wt SC35M NP or mutant CH2 or NP7. The incorporation of the GFP reporter minigenomes into VLPs was subsequently quantified by co-infection of MDCKII cells with VLPs and wt SC35M virus. No differences were observed in the amount of VLPs released from 293 T cells as evidenced by the number of GFP-positive MDCKII cells (Fig. 5a,b). This finding indicates that the incorporation of a single genome segment into viral particles is not affected, irrespective of the presence of the bundling sequence.

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