Limits...
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.


Related in: MedlinePlus

A subset of Bat NP-specific amino acids of CH4 causes severe attenuation and irregular packaging of genome segments.(a) Alignment of SC35M NP and NP mutant proteins harbouring 14 (NP14), 10 (NP10) or 7 (NP7) Bat NP-specific amino acids of CH4. Upper panel shows the sequence logo of the consensus sequence of NP of available IAV strains (n=27,675 strains). (b) Positions of Bat NP-specific amino acids in the modelled crystal structure of SC35M NP. The program PyMOL was used to assign the indicated positions. Bat-specific amino acids are marked in the colour code used in a. Note that amino acid 239 is not surface exposed. (c) Relative SC35M polymerase activities in the presence of the mutant NP proteins determined by polymerase reconstitution assays. Bat-specific amino acids and nucleotides are indicated. SC35M rescue experiments were performed with NP segments encoding the indicated mutant proteins. +successful rescue; −no rescue. (d) MDCKII cells were infected at an MOI of 0.001 with wt SC35M or recombinant virus rNP7. At the indicated time post infection (p.i.), virus titres were determined by plaque assay. (e) Relative ratio of the number of viral particles (counted by electron microscopy) divided by the number of infectious particles (determined by plaque assay) between wt SC35M and rNP7. Values obtained for SC35M were set to 1. *P<0.05. (f) Ratio of incorporated NP and M1 proteins in viral particles between SC35M and rNP7. Protein levels were determined by Western blot analysis of virus stocks with equal infectivity (PFU). *P<0.05; ***P<0.001. (g) Relative ratio of genome segments identified in viral particles preparations of SC35M and rNP7. RNA was prepared from virus stocks with identical infectivity (PFU) and subjected to quantitative RT-PCR. Levels of viral genome transcripts obtained with wt SC35M were set to 1. *P<0.05; **P<0.01; ***P<0.001. (h) Relative ratio of the viral transcript level in wt SC35M- or rNP7-infected cells. Steady state levels of viral transcripts (mRNA, cRNA and vRNA) and 5S ribosomal RNA (5S rRNA) were determined by primer extension analysis using total RNA from MDCKII cells infected at an MOI of 5 with wt SC35M or rCH2 for 6 h. Signal intensities were normalized to the signal intensities obtained with 5S RNA. Normalized values obtained in cells infected with wt SC35M were set to 1. *P<0.05; **P<0.01; ***P<0.001. Student's t test was used for two-group comparisons. Error bars indicate the mean and s.d. of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5035998&req=5

f3: A subset of Bat NP-specific amino acids of CH4 causes severe attenuation and irregular packaging of genome segments.(a) Alignment of SC35M NP and NP mutant proteins harbouring 14 (NP14), 10 (NP10) or 7 (NP7) Bat NP-specific amino acids of CH4. Upper panel shows the sequence logo of the consensus sequence of NP of available IAV strains (n=27,675 strains). (b) Positions of Bat NP-specific amino acids in the modelled crystal structure of SC35M NP. The program PyMOL was used to assign the indicated positions. Bat-specific amino acids are marked in the colour code used in a. Note that amino acid 239 is not surface exposed. (c) Relative SC35M polymerase activities in the presence of the mutant NP proteins determined by polymerase reconstitution assays. Bat-specific amino acids and nucleotides are indicated. SC35M rescue experiments were performed with NP segments encoding the indicated mutant proteins. +successful rescue; −no rescue. (d) MDCKII cells were infected at an MOI of 0.001 with wt SC35M or recombinant virus rNP7. At the indicated time post infection (p.i.), virus titres were determined by plaque assay. (e) Relative ratio of the number of viral particles (counted by electron microscopy) divided by the number of infectious particles (determined by plaque assay) between wt SC35M and rNP7. Values obtained for SC35M were set to 1. *P<0.05. (f) Ratio of incorporated NP and M1 proteins in viral particles between SC35M and rNP7. Protein levels were determined by Western blot analysis of virus stocks with equal infectivity (PFU). *P<0.05; ***P<0.001. (g) Relative ratio of genome segments identified in viral particles preparations of SC35M and rNP7. RNA was prepared from virus stocks with identical infectivity (PFU) and subjected to quantitative RT-PCR. Levels of viral genome transcripts obtained with wt SC35M were set to 1. *P<0.05; **P<0.01; ***P<0.001. (h) Relative ratio of the viral transcript level in wt SC35M- or rNP7-infected cells. Steady state levels of viral transcripts (mRNA, cRNA and vRNA) and 5S ribosomal RNA (5S rRNA) were determined by primer extension analysis using total RNA from MDCKII cells infected at an MOI of 5 with wt SC35M or rCH2 for 6 h. Signal intensities were normalized to the signal intensities obtained with 5S RNA. Normalized values obtained in cells infected with wt SC35M were set to 1. *P<0.05; **P<0.01; ***P<0.001. Student's t test was used for two-group comparisons. Error bars indicate the mean and s.d. of three independent experiments.

Mentions: To further analyze the effect of Bat NP-specific amino acids on the production of recombinant SC35M, we first introduced the 14 Bat NP-specific amino acids of CH4.14 into SC35M NP resulting in the SC35M NP14 mutant (Fig. 3a). NP14 was identical on amino acid level with CH4.14 but lacked the large amount of Bat NP-specific nucleotides (Fig. 2b). In addition to NP14, we generated additional SC35M NP mutants with either 10 (NP10) or 7 (NP7) Bat NP-specific amino acids (Fig. 3a). The majority of the inserted Bat NP-specific amino acids are unique and not conserved among conventional IAV strains (Fig. 3a). Most of these residues are found on the surface of the NP head domain (Fig. 3b). NP14, NP10 as well as NP7 supported viral polymerase activity, although to varying degrees (Fig. 3c). However, only recombinant SC35M encoding NP7 (rNP7) could be successfully generated (Fig. 3c). Infection of MDCKII cells with rNP7 at an MOI of 0.001 (Fig. 3d) or 5 (Supplementary Fig. 3) resulted in up to 100-fold lower virus titres compared with wt SC35M. To further determine which of the seven amino acids of NP7 contributed to impaired virus yield, a series of NP7 variants lacking either individual or clusters of Bat NP-specific amino acids was generated (Supplementary Fig. 6a). All of these mutants supported polymerase activity and in all cases SC35M viruses containing these NP mutants could be successfully generated (Supplementary Fig. 6b). SC35M virus encoding a NP7 variant with only six Bat NP-specific mutations, designated rNP7(2-7), was still attenuated in MDCKII cells and replicated to 50-fold lower virus titres than wt SC35M at 24 h.p.i. (Supplementary Fig. 6c). In contrast, replication of the other rNP7 variants was not significantly attenuated (Supplementary Fig. 6c). These findings suggest that attenuation of rNP7 is caused by several Bat NP-specific amino acids acting in a concerted manner.


A conserved influenza A virus nucleoprotein code controls specific viral genome packaging
A subset of Bat NP-specific amino acids of CH4 causes severe attenuation and irregular packaging of genome segments.(a) Alignment of SC35M NP and NP mutant proteins harbouring 14 (NP14), 10 (NP10) or 7 (NP7) Bat NP-specific amino acids of CH4. Upper panel shows the sequence logo of the consensus sequence of NP of available IAV strains (n=27,675 strains). (b) Positions of Bat NP-specific amino acids in the modelled crystal structure of SC35M NP. The program PyMOL was used to assign the indicated positions. Bat-specific amino acids are marked in the colour code used in a. Note that amino acid 239 is not surface exposed. (c) Relative SC35M polymerase activities in the presence of the mutant NP proteins determined by polymerase reconstitution assays. Bat-specific amino acids and nucleotides are indicated. SC35M rescue experiments were performed with NP segments encoding the indicated mutant proteins. +successful rescue; −no rescue. (d) MDCKII cells were infected at an MOI of 0.001 with wt SC35M or recombinant virus rNP7. At the indicated time post infection (p.i.), virus titres were determined by plaque assay. (e) Relative ratio of the number of viral particles (counted by electron microscopy) divided by the number of infectious particles (determined by plaque assay) between wt SC35M and rNP7. Values obtained for SC35M were set to 1. *P<0.05. (f) Ratio of incorporated NP and M1 proteins in viral particles between SC35M and rNP7. Protein levels were determined by Western blot analysis of virus stocks with equal infectivity (PFU). *P<0.05; ***P<0.001. (g) Relative ratio of genome segments identified in viral particles preparations of SC35M and rNP7. RNA was prepared from virus stocks with identical infectivity (PFU) and subjected to quantitative RT-PCR. Levels of viral genome transcripts obtained with wt SC35M were set to 1. *P<0.05; **P<0.01; ***P<0.001. (h) Relative ratio of the viral transcript level in wt SC35M- or rNP7-infected cells. Steady state levels of viral transcripts (mRNA, cRNA and vRNA) and 5S ribosomal RNA (5S rRNA) were determined by primer extension analysis using total RNA from MDCKII cells infected at an MOI of 5 with wt SC35M or rCH2 for 6 h. Signal intensities were normalized to the signal intensities obtained with 5S RNA. Normalized values obtained in cells infected with wt SC35M were set to 1. *P<0.05; **P<0.01; ***P<0.001. Student's t test was used for two-group comparisons. Error bars indicate the mean and s.d. of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: A subset of Bat NP-specific amino acids of CH4 causes severe attenuation and irregular packaging of genome segments.(a) Alignment of SC35M NP and NP mutant proteins harbouring 14 (NP14), 10 (NP10) or 7 (NP7) Bat NP-specific amino acids of CH4. Upper panel shows the sequence logo of the consensus sequence of NP of available IAV strains (n=27,675 strains). (b) Positions of Bat NP-specific amino acids in the modelled crystal structure of SC35M NP. The program PyMOL was used to assign the indicated positions. Bat-specific amino acids are marked in the colour code used in a. Note that amino acid 239 is not surface exposed. (c) Relative SC35M polymerase activities in the presence of the mutant NP proteins determined by polymerase reconstitution assays. Bat-specific amino acids and nucleotides are indicated. SC35M rescue experiments were performed with NP segments encoding the indicated mutant proteins. +successful rescue; −no rescue. (d) MDCKII cells were infected at an MOI of 0.001 with wt SC35M or recombinant virus rNP7. At the indicated time post infection (p.i.), virus titres were determined by plaque assay. (e) Relative ratio of the number of viral particles (counted by electron microscopy) divided by the number of infectious particles (determined by plaque assay) between wt SC35M and rNP7. Values obtained for SC35M were set to 1. *P<0.05. (f) Ratio of incorporated NP and M1 proteins in viral particles between SC35M and rNP7. Protein levels were determined by Western blot analysis of virus stocks with equal infectivity (PFU). *P<0.05; ***P<0.001. (g) Relative ratio of genome segments identified in viral particles preparations of SC35M and rNP7. RNA was prepared from virus stocks with identical infectivity (PFU) and subjected to quantitative RT-PCR. Levels of viral genome transcripts obtained with wt SC35M were set to 1. *P<0.05; **P<0.01; ***P<0.001. (h) Relative ratio of the viral transcript level in wt SC35M- or rNP7-infected cells. Steady state levels of viral transcripts (mRNA, cRNA and vRNA) and 5S ribosomal RNA (5S rRNA) were determined by primer extension analysis using total RNA from MDCKII cells infected at an MOI of 5 with wt SC35M or rCH2 for 6 h. Signal intensities were normalized to the signal intensities obtained with 5S RNA. Normalized values obtained in cells infected with wt SC35M were set to 1. *P<0.05; **P<0.01; ***P<0.001. Student's t test was used for two-group comparisons. Error bars indicate the mean and s.d. of three independent experiments.
Mentions: To further analyze the effect of Bat NP-specific amino acids on the production of recombinant SC35M, we first introduced the 14 Bat NP-specific amino acids of CH4.14 into SC35M NP resulting in the SC35M NP14 mutant (Fig. 3a). NP14 was identical on amino acid level with CH4.14 but lacked the large amount of Bat NP-specific nucleotides (Fig. 2b). In addition to NP14, we generated additional SC35M NP mutants with either 10 (NP10) or 7 (NP7) Bat NP-specific amino acids (Fig. 3a). The majority of the inserted Bat NP-specific amino acids are unique and not conserved among conventional IAV strains (Fig. 3a). Most of these residues are found on the surface of the NP head domain (Fig. 3b). NP14, NP10 as well as NP7 supported viral polymerase activity, although to varying degrees (Fig. 3c). However, only recombinant SC35M encoding NP7 (rNP7) could be successfully generated (Fig. 3c). Infection of MDCKII cells with rNP7 at an MOI of 0.001 (Fig. 3d) or 5 (Supplementary Fig. 3) resulted in up to 100-fold lower virus titres compared with wt SC35M. To further determine which of the seven amino acids of NP7 contributed to impaired virus yield, a series of NP7 variants lacking either individual or clusters of Bat NP-specific amino acids was generated (Supplementary Fig. 6a). All of these mutants supported polymerase activity and in all cases SC35M viruses containing these NP mutants could be successfully generated (Supplementary Fig. 6b). SC35M virus encoding a NP7 variant with only six Bat NP-specific mutations, designated rNP7(2-7), was still attenuated in MDCKII cells and replicated to 50-fold lower virus titres than wt SC35M at 24 h.p.i. (Supplementary Fig. 6c). In contrast, replication of the other rNP7 variants was not significantly attenuated (Supplementary Fig. 6c). These findings suggest that attenuation of rNP7 is caused by several Bat NP-specific amino acids acting in a concerted manner.

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