RNA packaging of flu virus

[Anne]

[SIZE=-1]
http://jvi.asm.org/cgi/content/abstract/82/5/2295


journal of Virology, March 2008, p. 2295-2304, Vol. 82, No. 5
0022-538X/08/$08.00+0 doi:10.1128/JVI.02267-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.[/SIZE]
Highly Conserved Regions of Influenza A Virus Polymerase Gene Segments Are Critical for Efficient Viral RNA Packaging<sup>

</sup>

Glenn A. Marsh,¹<sup>,

</sup>^,# Raúl Rabadán,³<sup>,

</sup> Arnold J. Levine,³ and Peter Palese¹^,2^* Departments of Microbiology,¹ Medicine, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, New York 10029,² Institute for Advanced Study, Einstein Dr., Princeton, New Jersey 08540³
Received 18 October 2007/ Accepted 7 December 2007
<!-- ABS --> The genome of the influenza A virus is composed of eight differentsegments of negative-sense RNA. These eight segments are incorporatedinto budding virions in an equimolar ratio through a mechanismthat is not fully understood. Two different models have beenproposed for packaging the viral ribonucleoproteins into newlyassembling virus particles: the random-incorporation model andthe selective-incorporation model. In the last few years, increasingevidence from many different laboratories that supports theselective-incorporation model has been accumulated. In particular,different groups have shown that some large viral RNA regionswithin the coding sequences at both the 5' and 3' ends of almostevery segment are sufficient for packaging foreign RNA sequences.If the packaging regions are crucial for the viability of thevirus, we would expect them to be conserved. Using large-scaleanalysis of influenza A virus sequences, we developed a methodof identifying conserved RNA regions whose conservation cannotbe explained by population structure or amino acid conservation.Interestingly, the conserved sequences are located within theregions identified as important for efficient packaging. Byutilizing influenza virus reverse genetics, we have rescuedmutant viruses containing synonymous mutations within thesehighly conserved regions. Packaging of viral RNAs in these viruseswas analyzed by reverse transcription using a universal primerand quantitative PCR for individual segments. Employing thisapproach, we have identified regions in the polymerase genesegments that, if mutated, result in reductions of more than90% in the packaging of that particular polymerase viral RNA.Reductions in the level of packaging of a polymerase viral RNAfrequently resulted in reductions of other viral RNAs as well,and the results form a pattern of hierarchy of segment interactions.This work provides further evidence for a selective packagingmechanism for influenza A viruses, demonstrating that thesehighly conserved regions are important for efficient packaging.

 

[gsgs]

Re: RNA packaging of flu virus

what's the "random incorporation model" ?
I've read recently that segments are selected at random and that
8-11 random segments are encapsulated and packaged in a virus
and that many viruses are defect because they lack some segment.
They may still be able to reassort with other viruses

see this thread:
http://67.210.96.104/forum/showthread.php?t=50277

 

[gsgs]

Re: RNA packaging of flu virus

I didn't realize, how uncertified that theory was :



http://jvi.asm.org/cgi/content/full/76/14/7133
[2002,full text available]
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...Our results nevertheless add to the preponderance of evidence that influenza virus packages its segments nonspecifically. The recent finding that critical packaging signals map to within the UTR sequences common to all influenza virus vRNAs lends particularly strong support to this view (28). Though seemingly inefficient, a random packaging mechanism could in theory suffice if each influenza virus virion incorporates, on average, more than eight vRNAs. Other authors have calculated, for example, that random packaging of 10 to 12 vRNAs per particle would enable 2.8 to 9.3% of virions to acquire the full complement of eight (7, 26); those values are compatible with the estimated 5 to 10% ratio of infectious to noninfectious particles found in actual influenza virus populations (2, 14). ...
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This article has been cited by other articles:
* Marsh, G. A., Rabadan, R., Levine, A. J., Palese, P. (2008). Highly Conserved Regions of Influenza A Virus Polymerase Gene Segments Are Critical for Efficient Viral RNA Packaging. J. Virol. 82: 2295-2304
[only abstract is available]
* Marsh, G. A., Hatami, R., Palese, P. (2007). Specific Residues of the Influenza A Virus Hemagglutinin Viral RNA Are Important for Efficient Packaging into Budding Virions. J. Virol. 81: 9727-9736
* Gog, J. R., Afonso, E. D. S., Dalton, R. M., Leclercq, I., Tiley, L., Elton, D., von Kirchbach, J. C., Naffakh, N., Escriou, N., Digard, P. (2007). Codon conservation in the influenza A virus genome defines RNA packaging signals. Nucleic Acids Res 35: 1897-1907
* Regan, J. F., Liang, Y., Parslow, T. G. (2006). Defective Assembly of Influenza A Virus due to a Mutation in the Polymerase Subunit PA. J. Virol. 80: 252-261
* Lowen, A. C., Boyd, A., Fazakerley, J. K., Elliott, R. M. (2005). Attenuation of Bunyavirus Replication by Rearrangement of Viral Coding and Noncoding Sequences. J. Virol. 79: 6940-6946
* McCown, M. F., Pekosz, A. (2005). The Influenza A Virus M2 Cytoplasmic Tail Is Required for Infectious Virus Production and Efficient Genome Packaging. J. Virol. 79: 3595-3605
*Fujii, K., Fujii, Y., Noda, T., Muramoto, Y., Watanabe, T., Takada, A., Goto, H., Horimoto, T., Kawaoka, Y. (2005). Importance of both the Coding and the Segment-Specific Noncoding Regions of the Influenza A Virus NS Segment for Its Efficient Incorporation into Virions. J. Virol. 79: 3766-3774
*Escors, D., Izeta, A., Capiscol, C., Enjuanes, L. (2003). Transmissible Gastroenteritis Coronavirus Packaging Signal Is Located at the 5' End of the Virus Genome. J. Virol. 77: 7890-7902
*Fujii, Y., Goto, H., Watanabe, T., Yoshida, T., Kawaoka, Y. (2003). Selective incorporation of influenza virus RNA segments into virions. Proc. Natl. Acad. Sci. USA 100: 2002-2007

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http://www.pnas.org/cgi/content/full/100/4/2002
[2003, full text available]
...Two models have been proposed for the generation of infectious virions containing eight vRNA segments. The random-incorporation model assumes a common structural feature in all the vRNAs, enabling any combination of vRNAs to be incorporated randomly into virions. The selective-incorporation model predicts the presence of specific structures in each vRNA segment, leading to the incorporation of a set of eight vRNA segments into virions. Here we demonstrate that eight different vRNA segments must be present for efficient virion formation and that sequences within the coding region of (and thus unique to) the neuraminidase vRNA possess a signal that drives incorporation of this segment into virions. These findings indicate a unique contribution from individual vRNA segments and thus suggest a selective (rather than random) mechanism of vRNA recruitment into virions...

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...identified a key region of the open reading frame (nucleotides 1659 to 1671) that is critical for the efficient packaging of an influenza virus H1 HA segment...
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Specific packaging signals exist that extend into the coding regions of most if not all segments

Surprisingly, in most cases, single nucleotide changes dramatically reduced segment packaging. Thus our analysis identifies cis-acting sequences in the influenza virus genome at the nucleotide level. Furthermore, we propose that strain-specific differences exist in certain packaging signals, most notably the haemagglutinin gene; this finding has major implications for the evolution of pandemic viruses.
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The genome of influenza A virus consists of eight single-strand negative-sense RNA segments, each comprised of a coding region and a noncoding region. The noncoding region of the NS segment is thought to provide the signal for packaging; however, we recently showed that the coding regions located at both ends of the hemagglutinin and neuraminidase segments were important for their incorporation into virions. In an effort to improve our understanding of the mechanism of influenza virus genome packaging, we sought to identify the regions of NS viral RNA (vRNA) that are required for its efficient incorporation into virions. Deletion analysis showed that the first 30 nucleotides of the 3' coding region are critical for efficient NS vRNA incorporation and that deletion of the 3' segment-specific noncoding region drastically reduces NS vRNA incorporation into virions. Furthermore, silent mutations in the first 30 nucleotides of the 3' NS coding region reduced the incorporation efficiency of the NS segment and affected virus replication. These results suggested that segment-specific noncoding regions together with adjacent coding regions (especially at the 3' end) form a structure that is required for efficient influenza A virus vRNA packaging.
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[Anne]

Re: RNA packaging of flu virus

" Surprisingly, in most cases, single nucleotide changes dramatically reduced segment packaging "

it 's mean that assembly is not done by " chance" ( hasard ) or random, and that a synonymous mutation have a bad effect for packaging.

 

[gsgs]

Re: RNA packaging of flu virus

probably, although apparantly the last word has not been spoken yet.

And as I understand it's only for some very small special regions
of the genome. 1% or such.