Giuseppe
Emeritus
J Virol. 2008 Sep 24. [Epub ahead of print]
Mutational analysis of cis-acting RNA signals in segment 7 of influenza A virus.
Hutchinson EC, Curran MD, Read EK, Gog JR, Digard P. - Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP; Health Protection Agency, Clinical Microbiology and Public Health Laboratory, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QW, UK; DAMTP, Centre for Mathematical Sciences, University of Cambridge, Wilberforce Road, Cambridge CB3 0WA, UK.
The genomic vRNA segments of influenza A virus contain specific packaging signals at their termini that overlap the coding regions.
To further characterise cis-acting signals in segment 7, we introduced synonymous mutations into the terminal coding regions.
Mutation of codons that are normally highly conserved reduced virus growth in embryonated eggs and MDCK cells between 10 and 1000-fold compared to the wild-type virus, whereas similar alterations to non-conserved codons had little effect.
In all cases, the growth-impaired viruses showed defects in virion assembly and genome packaging.
In eggs, near normal numbers of virus particles were formed that in aggregate contained apparently equimolar quantities of the eight segments, but with around 4-fold less overall vRNA content than wild type virions, suggesting that on average, less than 8 segments per particle were packaged.
Concomitantly, the particle
FU and segmentFU ratios of the mutant viruses showed relative increases of up to 300-fold, with the behaviour of the most defective viruses approaching that predicted for random segment packaging.
Fluorescent staining of infected cells for the nucleoprotein and specific vRNAs confirmed that most mutant virus particles did not contain a full genome complement.
The specific infectivity of the mutant viruses produced by MDCK cells was also reduced, but in this system, the mutations also dramatically reduced virion production.
Overall, we conclude that segment 7 plays a key role in the influenza A genome packaging process since mutation of as few as 4 nucleotides can dramatically inhibit infectious virus production through disrupting vRNA packaging.
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PMID: 18815307 [PubMed - as supplied by publisher
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Mutational analysis of cis-acting RNA signals in segment 7 of influenza A virus.
Hutchinson EC, Curran MD, Read EK, Gog JR, Digard P. - Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP; Health Protection Agency, Clinical Microbiology and Public Health Laboratory, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QW, UK; DAMTP, Centre for Mathematical Sciences, University of Cambridge, Wilberforce Road, Cambridge CB3 0WA, UK.
The genomic vRNA segments of influenza A virus contain specific packaging signals at their termini that overlap the coding regions.
To further characterise cis-acting signals in segment 7, we introduced synonymous mutations into the terminal coding regions.
Mutation of codons that are normally highly conserved reduced virus growth in embryonated eggs and MDCK cells between 10 and 1000-fold compared to the wild-type virus, whereas similar alterations to non-conserved codons had little effect.
In all cases, the growth-impaired viruses showed defects in virion assembly and genome packaging.
In eggs, near normal numbers of virus particles were formed that in aggregate contained apparently equimolar quantities of the eight segments, but with around 4-fold less overall vRNA content than wild type virions, suggesting that on average, less than 8 segments per particle were packaged.
Concomitantly, the particle
FU and segmentFU ratios of the mutant viruses showed relative increases of up to 300-fold, with the behaviour of the most defective viruses approaching that predicted for random segment packaging. Fluorescent staining of infected cells for the nucleoprotein and specific vRNAs confirmed that most mutant virus particles did not contain a full genome complement.
The specific infectivity of the mutant viruses produced by MDCK cells was also reduced, but in this system, the mutations also dramatically reduced virion production.
Overall, we conclude that segment 7 plays a key role in the influenza A genome packaging process since mutation of as few as 4 nucleotides can dramatically inhibit infectious virus production through disrupting vRNA packaging.
-
PMID: 18815307 [PubMed - as supplied by publisher
------
