Reverse genetics studies on the filamentous morphology of influenza A virus

[Anne]

[SIZE=-1]J Gen Virol 84 (2003), 517-527; DOI 10.1099/vir.0.18803-0

http://vir.sgmjournals.org/cgi/content/abstract/84/3/517
[/SIZE]
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</td></tr> </tbody></table> </td></tr></tbody></table> [SIZE=-1] ? 2003 Society for General Microbiology [/SIZE]
Reverse genetics studies on the filamentous morphology of influenza A virus


<nobr>Svetlana V. Bourmakina</nobr> and <nobr>Adolfo Garc?a-Sastre</nobr>
[SIZE=-1] Department of Microbiology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA [/SIZE]

[SIZE=-1]Correspondence
Adolfo Garc?a-Sastre
adolfo.garcia-sastre@mssm.edu<script type="text/javascript"><!-- var u = "adolfo.garcia-sastre", d = "mssm.edu"; document.getElementById("em0").innerHTML = '<a href="mailto:' + u + '@' + d + '">' + u + '@' + d + '<\/a>'//--></script>[/SIZE]
<!-- ABS --> We have investigated the genetic determinants responsible forthe filamentous morphology of influenza A viruses, a propertycharacteristic of primary virus isolates. A plasmid-based reversegenetics system was used to transfer the M segment of influenzaA/Udorn/72 (H3N2) virus into influenza A/WSN/33 (H1N1) virus.While WSN virions display spherical morphology, recombinantWSN-Mud virus acquired the ability of the parental Udorn strainto form filamentous virus particles. This was determined byimmunofluorescence studies in infected MDCK cells and by electronmicroscopy of purified virus particles. To determine the geneproduct within the M segment responsible for filamentous virusmorphology, we generated four recombinant viruses carrying differentsets of M1 and M2 genes from WSN or Udorn strains in a WSN background.These studies revealed that the M1 gene of Udorn, independentlyof the origin of the M2 gene, conferred filamentous buddingproperties and filamentous virus morphology to the recombinantviruses. We also constructed two WSN viruses encoding chimericM1 proteins containing the amino-terminal 1?162 aminoacids or the carboxy-terminal 163?252 amino acidsof the Udorn M1 protein. Neither of these two viruses acquiredfilamentous phenotypes, indicating that both amino- and carboxy-terminaldomains of the M1 protein contribute to filamentous virus morphology.We next rescued seven mutant WSN-M1ud viruses containing UdornM1 proteins carrying single amino acid substitutions correspondingto the seven amino acid differences with the M1 protein of WSNvirus. Characterization of these recombinant viruses revealedthat amino acid residues 95 and 204 are critical in determiningfilamentous virus particle formation.

 

[gsgs]

Re: Reverse genetics studies on the filamentous morphology of influenza A virus

thanks , I recently wondered about the pictures which showed
filamentous H5N1 but spherical H3N2 and had started a diary at fluwiki.


my summary:

free full article, 11 pages, at:
http://vir.sgmjournals.org/cgi/content/full/84/3/517
with some nice virus-pictures

J Gen Virol 84 (2003), 517-527; DOI 10.1099/vir.0.18803-0
Reverse genetics studies on the filamentous morphology of influenza A virus
Svetlana V. Bourmakina and Adolfo García-Sastre


A/WSN/33 (H1N1) is spherical with diameter ~100nm
A/Udorn/72 (H3N2) typically has 15% of virions larger than 400nm, while
they seem to be not much thinner than sherical virions, so the volume increases also.

The authors used reassorted and recombined versions to demonstrate
that the filamentous property is linked to the M1 gene and i.e.
to mutations R95K and E204D



"...The role that filamentous virus morphology may play in virus pathogenesis and disease is not known. Since long filamentous virions could theoretically infect neighbouring cells prior to their release, it has been suggested that filamentous virus morphology may facilitate cell-to-cell transmission of viruses in the respiratory mucosa (Roberts & Compans, 1998). On the other hand, small spherical virions would be expected to be more efficiently incorporated into aerosols and therefore may be easily transmitted from person to person. Future experiments are required to determine the biological role that filamentous viruses play in the influenza virus life-cycle and pathogenicity. The fact that clinical isolates of influenza A viruses invariably show filamentous phenotypes strongly suggests that this property is important for virus survival in nature. ..."


what is virus budding ?

 

[Anne]

Re: Reverse genetics studies on the filamentous morphology of influenza A virus

Our results show that both amino- and carboxy-terminal
regions of the M1 protein contain sequence determinants
responsible for filamentous morphology of virus particles.
There are seven amino acid differences between the M1
proteins of the Udorn and WSN strains used in our studies.
We found that the single amino acid mutations Arg-
95RLys and Glu-204RAsp caused a significant reduction
in the percentage of long virus particles. All other single
mutations did not significantly affect the filamentous
phenotype of WSN-M1ud virus as analysed by electron
microscopy of purified virions.

 

[JJackson]

Re: Reverse genetics studies on the filamentous morphology of influenza A virus

While the filamentous virons are larger they are still so small I do not see how that would cause a problem for aerosol transmission (even assuming this -- rather than droplet transmission -- is a major transmission method). However I can envisage this causing difficulties during endocytosis.

 

[LMonty]

Re: Reverse genetics studies on the filamentous morphology of influenza A virus

Educate me, JJackson (pretty please!)

how would a filamentous morphology affect endocytosis? Does filamentous morphology result in any differences in virulence? Assuming the 1918 origicial H1N1 was filamentous (was it?) did that morphology contribute to its lethality?

Thanks!

 

[gsgs]

Re: Reverse genetics studies on the filamentous morphology of influenza A virus

questions...
is the filamentous property implied by mutations
or can the same virus be spherical at one time
and filamentous at another time and how is this triggered ?
Do masks filter filamentous particles better ?
Is either sort needed to enter a human and infect cells ?

If WSN comes without filamentous virions, then why is it needed
at all ? What is it good for ?
What percent of filamentous virions do the 1918,1957,1968
pandemic strains have, how many has H5N1 ?

One other remarkable quote from that paper:

"The formation of extremely long cord-like structures with lengths
up to 500micrometer has been described upon infection of cells
with some influenza-C virus strains (Nishimura et al.,1990)"

that's half a millimeter long , so it could be seen
if it weren't so thin.

 

[Sonny]

host cell entry of filamentous influenza virus

host cell entry of filamentous influenza virus

http://www.springerlink.com/content/r9791181653n0172/

Characterization of the host cell entry of filamentous influenza virus
<table cellpadding="0" cellspacing="0"><tbody><tr> <td class="labelName">Journal</td><td class="labelValue">Archives of Virology</td> </tr><tr> <td class="labelName">Publisher</td><td class="labelValue">Springer Wien</td> </tr><tr> <td class="labelName">ISSN</td><td class="labelValue">0304-8608 (Print) 1432-8798 (Online)</td> </tr><tr> <td class="labelName">Subject</td><td class="labelValue">Medicine and Biomedical and Life Sciences</td> </tr><tr> <td class="labelName">Issue</td><td class="labelValue">Volume 150, Number 9 / September, 2005</td> </tr><tr> <td class="labelName">DOI</td><td class="labelValue">10.1007/s00705-005-0558-1</td> </tr><tr> <td class="labelName">Pages</td><td class="labelValue">1783-1796</td> </tr><tr> <td class="labelName">SpringerLink Date</td><td class="labelValue">Thursday, June 16, 2005</td></tr></tbody></table>



S. B. Sieczkarski¹ and G. R. Whittaker¹
<table><tbody><tr valign="top"><td>(1) </td><td>Department of Microbiology and Immunology, Cornell University, Ithaca, U.S.A.

</td></tr></tbody></table>Received: 2 April 2003 Accepted: 11 April 2005 Published online: 15 June 2005


Summary.
Most laboratory-adapted strains of influenza virus exist as spheres of approximately 100 nm in diameter, which are well established to enter cells by endocytosis in a pH-dependent manner. However, influenza virus isolated from the lungs of infected individuals is believed to exist as predominantly filamentous particles, up to several micrometers in length.

Here, we have attempted an initial characterization of the entry of purified influenza virus filaments into host cells ? in comparison to more commonly studied spherical forms of the virus. We demonstrate that the internalization of filamentous influenza virus particles is delayed, relative to spherical particles, and that this delay is a result of morphological rather than strain differences.

The filamentous influenza particles appear to retain their dependence on low-pH for entry, as demonstrated by a vacuolar-ATPase inhibitor, and viral trafficking to late endosomes, as demonstrated by the requirement for protein kinase C function. However, our data suggest that the endocytic uptake of the filamentous virus particles may be dynamin-independent, unlike spherical virions. Overall, these data provide a view of the entry of influenza virus in its filamentous morphology, demonstrating potential differences between the endocytosis of spherical virions in vitro and filamentous virions in vivo.

 

[JJackson]

Re: Reverse genetics studies on the filamentous morphology of influenza A virus

LMonty
My earlier comment was not based on anything I had read on the subject but was my own gut feeling based on what I know of the process. I had guessed that it would be more difficult to get the larger elongated viron to HA bond and cleave along its length so getting its polymerase complex and other RNA segments into the cell efficiently. As the paper implied that the viron shape was controlled by mutations in the M1 area of the M strand (which codes for the protein that makes the units that link together to create the structural scaffolding of the viron) this also makes sense as changes in the building block for the shell may well cause a change to the resultant shell. The M2 structure is not changed and it is groups of this protein, embedded in the M1 structure, which pump H+ ions into the viron reducing the Ph to a point where the bonds between the M1s loose cohesion so allowing the release of the contents. One other effect that might occur is a change in the ease with which these M1 to M1 bonds are able to dissolve i.e. if the M1 protein is bonding in a new way – evidenced by the change in viron shape – then it is also reasonable to expect that the strength of the bonds between them, and so the Ph at which these bonds break, may also change. As to virulence this seems to be more related to the NS and polymerase genes than the structural M1 protein so I was not expecting a major change.
All of the above is guess work on my part based on my incomplete – and quite possible incorrect -- understanding of the primary functions of the various proteins. The more I learn about this subject the more I come to realise that nearly all the genes have more than their obvious primary function and they all have subtle interactions.

 

[JJackson]

Re: Reverse genetics studies on the filamentous morphology of influenza A virus

What I should have added to the post above is that, again with no sound basis for my opinin, I think of the construction of the M1 structure of spherical virons as being much like that of the Carbon 60 molecule with elongated forms similar to C70 and the longer carbon fiber nanotubes. If you are unfamiliar with these you should find some good graphics if you search for Buckminster Fullerines.

 

[gsgs]

Re: Reverse genetics studies on the filamentous morphology of influenza A virus

the 1918 virus was somehow between the Udorn and WSN viruses,
V41A,K95R,I205V mutations from WSN, so that would be
M1udA41V,M1udR95K,M1udV205I in Fig.5 page 123.
Or an estimated 20% < 10nm , 70% 120-300nm , 10% >300nm.

It must be somehow advantageous for the virus to have a certain proportion
of lengthy ones.
It could improve infection of certain organs, while it's probably not so
good for transmission to other hosts, I speculate.