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

The compensatory amino acid substitution R31G in NP7 improves viral growth and genome packaging.(a) Positions of Bat NP-specific amino acids in the head domain and R31G body domain of NP7 using 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 Fig. 3b. (b) MDCKII cells were infected at an MOI of 0.001 with wt SC35M, rNP7 or rNP7-R31G. At the indicated time post infection (p.i.), viral titres were determined by plaque assay. *P<0.05; **P<0.01; ****P<0.0001. (c) Relative ratio of genome segments identified in viral particles preparations of SC35M and rNP7-R31G. 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. 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

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

f4: The compensatory amino acid substitution R31G in NP7 improves viral growth and genome packaging.(a) Positions of Bat NP-specific amino acids in the head domain and R31G body domain of NP7 using 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 Fig. 3b. (b) MDCKII cells were infected at an MOI of 0.001 with wt SC35M, rNP7 or rNP7-R31G. At the indicated time post infection (p.i.), viral titres were determined by plaque assay. *P<0.05; **P<0.01; ****P<0.0001. (c) Relative ratio of genome segments identified in viral particles preparations of SC35M and rNP7-R31G. 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. 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: Serial passage of rNP7 in MDCKII cells resulted in a virus strain that replicated almost as efficiently as wt SC35M. Sequencing of the NP ORF revealed that the passaged rNP7 virus contained a single point mutation in the body domain of NP at position 31 (R31G) (Fig. 4a), suggesting that this mutation improved viral growth. In agreement with this hypothesis, a recombinant rNP7 harbouring this additional mutation (rNP7-R31G) replicated in MDCKII cells to markedly higher viral titres than rNP7 (Fig. 4b). We speculated that this mutation might also restore the packaging of the vRNA segments. Indeed, using equal numbers of infectious SC35M and rNP7-R31G viruses, a similar proportion of viral genome segments was detected in rNP7-R31G and wt S35M (Fig. 4c).


A conserved influenza A virus nucleoprotein code controls specific viral genome packaging
The compensatory amino acid substitution R31G in NP7 improves viral growth and genome packaging.(a) Positions of Bat NP-specific amino acids in the head domain and R31G body domain of NP7 using 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 Fig. 3b. (b) MDCKII cells were infected at an MOI of 0.001 with wt SC35M, rNP7 or rNP7-R31G. At the indicated time post infection (p.i.), viral titres were determined by plaque assay. *P<0.05; **P<0.01; ****P<0.0001. (c) Relative ratio of genome segments identified in viral particles preparations of SC35M and rNP7-R31G. 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. 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

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

f4: The compensatory amino acid substitution R31G in NP7 improves viral growth and genome packaging.(a) Positions of Bat NP-specific amino acids in the head domain and R31G body domain of NP7 using 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 Fig. 3b. (b) MDCKII cells were infected at an MOI of 0.001 with wt SC35M, rNP7 or rNP7-R31G. At the indicated time post infection (p.i.), viral titres were determined by plaque assay. *P<0.05; **P<0.01; ****P<0.0001. (c) Relative ratio of genome segments identified in viral particles preparations of SC35M and rNP7-R31G. 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. 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: Serial passage of rNP7 in MDCKII cells resulted in a virus strain that replicated almost as efficiently as wt SC35M. Sequencing of the NP ORF revealed that the passaged rNP7 virus contained a single point mutation in the body domain of NP at position 31 (R31G) (Fig. 4a), suggesting that this mutation improved viral growth. In agreement with this hypothesis, a recombinant rNP7 harbouring this additional mutation (rNP7-R31G) replicated in MDCKII cells to markedly higher viral titres than rNP7 (Fig. 4b). We speculated that this mutation might also restore the packaging of the vRNA segments. Indeed, using equal numbers of infectious SC35M and rNP7-R31G viruses, a similar proportion of viral genome segments was detected in rNP7-R31G and wt S35M (Fig. 4c).

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