Comments on: Release of influenza viral RNAs into cells

By: Steve

Steve — Sun, 17 Jan 2010 12:40:14 +0000

Hey Karla,

All inflenza virions have 8 RNA strands within them. The RNA within the swine flu is from human, pig and avian flu virions. At the beginning of this thread you can see the intact flu virion at the left. Its hard to see the 8 RNA strands until the virion releases them in the target healthy cell's cytoplasm in the next to last illustration. In the last illustration at the right side, the RNA strands “are then transported into the cell nucleus where viral RNA replication occurs.”

By: Karla Ward

Karla Ward — Sun, 17 Jan 2010 07:42:29 +0000

Hi Am I right in thinking that the (swine flu) Novel H1 N1 RNA is made up of aproximatly 8 or so influenza viruses and not just swine influenza?? if so can you tell me which ones I am a student nurse and trying to do a presentation on the swine flu virus and although I can remember reading somewhere about Swine influenza virus containing 8 or more influenza viruses I cant remember where or which ??
thanks Karla Ward

By: Chris

Chris — Sat, 12 Dec 2009 19:35:47 +0000

Oops sorry, I see that question is addressed below.

By: Chris Murray

Chris Murray — Sat, 12 Dec 2009 19:32:36 +0000

How do the imported RNAs transported to the nucleus?

By: Steve

Steve — Thu, 06 Aug 2009 19:40:45 +0000

Thanks so much herr professor. I've been trying to get the information you've shared for quite some time specificly for influenza. First- I have seen 500 for cocksackieviruses (Vernon Knight- Viral and Mycoplasmal infections of the Respiratory tract 1973 page 155) but nothing in the same book for influenza. I'd love to get a reference from you so I can read more about this.
I am interested in multiplication in epithelial cells as that is what flu loves to munch in our respiratory tract.

Next-I had no idea that only 10% of budded virions were viable. Is that typical for most virions? You called them particles. What is the difference?

You rule,

Steve

By: profvrr

profvrr — Thu, 06 Aug 2009 19:23:01 +0000

The yield of influenza virus would depend upon the cell type, but
typically about 1000 particles, of which ~100 are infectious.

By: Steve

Steve — Thu, 06 Aug 2009 11:32:23 +0000

Great Teaching. Here's my question: how many influenza virions does a typical hijacked cell eventually bud out?
Do each of those (or what %) then hijack new epithelial cells?

Thanks,

Steve

By: profvrr

profvrr — Mon, 20 Jul 2009 17:52:17 +0000

M1 protein is multifunctional, at least in terms of influenza virus
replication. It has a structural role in the virion, and also a role
in entry. Exactly how it functions won't be known until its structure
is determined. There are similar proteins in other enveloped RNA
viruses which have structural roles. None have yet been shown to
function during entry as does influenza M1.

By: Reassortment of the influenza virus genome

Reassortment of the influenza virus genome — Wed, 01 Jul 2009 12:59:01 +0000

[...] an influenza virus infects a cell, the individual RNA segments enter the nucleus. There they are copied many times to form RNA genomes for new infectious virions. The new RNA [...]

By: pp

pp — Tue, 23 Jun 2009 19:30:54 +0000

I have a slightly offtopic question regarding M1. I have studied influenza A viruses and have a basic understanding of what role plays each protein, and how they work.

However it seems M1 is everywhere and nowhere all at once - intervening in many steps of the viral infection as well as affecting the virion structure, but still lacking a very definite role. It almost appears as if it was some sort of all-purpose molecular glue. So I'm left wondering how this protein actually functions? Is it a sort of molecule that is specific to influenza, or rather something to be found wildly in nature?

Thanks a lot for exposing and sharing your knowledge, it's extremely interesting!
Cyril

By: JJackson

JJackson — Mon, 01 Jun 2009 15:32:43 +0000

Sorry to go back to it but just to be clear are there two lots of ion pumping going on? (PS yes I know this is not a body building web site).

On first reading I took there to be an decrease in ph being created between the inside of the endosome relative to the cytoplasm

“When the endosomal pH reaches 5.0, the viral HA protein undergoes a conformational rearrangement. This change exposes a fusion peptide on the HA – a short, hydrophobic sequence that inserts into the endosomal membrane and causes it to fuse with the viral envelope. When this occurs, the viral RNAs are released into the cytoplasm.”

Which lead to the fusion peptide ‘bursting’ the the endosome. Does this release the vRNA or just release the intact viron? If the pump is in the endosomal wall why is it there, is the reduction of ph to 5.0 normal for an endosome during transport to the nucleus?

If there is only one pump in this story – M2 – then it is pumping H+ into the virus’ interior which causes the HA to re-jig its tertiary structure to expose the fusion peptide which breaks the endosome and frees the polymerase complex from the M1.

PS where does HA0 cleavage by the host protease fit in to all this.

PPS Thanks for the time you have taken to run explain all this.

By: profvrr

profvrr — Mon, 11 May 2009 17:31:43 +0000

The M1-shell doesn't have any symmetry (e.g. icosahedral, found in
many capsids) nor can a T number be defined.

By: Ascription is an Anathema to any Enthusiasm › Searching the Alternate Routes

Mon, 11 May 2009 13:17:32 +0000

[...] of an anti-viral. The stupid viruses can mindlessly find a route around his clever invention. Adamantine was approved for use in 1966, by the 2005-2006 US flu widespread flu strains had routed around the [...]

By: Debate in science… « The Virelegy

Debate in science… « The Virelegy — Fri, 08 May 2009 17:45:11 +0000

[...] Debate in science… From the Racaniello blog, post May 6th: http://www.virology.ws/2009/05/06/release-of-influenza-viral-rnas-into-cells/ [...]

By: Mauricio Carrillo-Tripp

Mauricio Carrillo-Tripp — Fri, 08 May 2009 14:29:13 +0000

I am also curious about what you said not being technically a capsid. Copies of M1 form a shell encapsulating the genetic material inside the lipid envelope. Is the structure of this shell similar to those capsids of other non-enveloped spherical viruses in terms of symmetric arrangements? or is it more of a 'loose' structure (weaker inter subunit interactions)? Can you define a T number for example?
Thanks again for all your answers!

By: Mauricio Carrillo-Tripp

Mauricio Carrillo-Tripp — Fri, 08 May 2009 13:35:53 +0000

thanks for clearing that up, and the link, very helpful. I'm looking forward to read the following posts on the subject!

By: Influenza viral RNA synthesis

Influenza viral RNA synthesis — Fri, 08 May 2009 12:15:14 +0000

[...] the (-) strand influenza viral RNAs enter the nucleus, they serve as templates for the synthesis of mRNAs. These molecules are then transported back to [...]

By: profvrr

profvrr — Fri, 08 May 2009 01:25:42 +0000

You are correct, the M2 forms a proton pump in the viral membrane. It does not traverse the underlying shell composed of the M1 protein. Yet the protons end up in the virion interior, where they dissociate the RNA-protein complex from M1, allowing dissociation of the shell formed by M1 (technically it is not a capsid). It is believed that the protons enter the virion before membrane fusion occurs, to prime the viral RNA for release into the cytoplasm. Yes, the structure of M2 and amantadine are known - see http://bit.ly/O0cJE.

By: profvrr

profvrr — Thu, 07 May 2009 18:54:22 +0000

The NP NLS has only been tested in mammalian systems. It is a 'nonclassical' NLS because it does not resemble classical import sequences.

Amantadine has neurological side effects due to its dopaminergic and adrenergic activity, and to its activity as an anticholinergic.

Yes, there are new NA inhibitors in development – see http://www.virology.ws/2009/01/28/new-influenza... and the reference therein.

As for drugs that target the NP NLS…no, none have focused on that step. It's a difficult assay for drug screening and others (NA, polymerase) are much easier to adopt to a high throughput format.

By: phogdog

phogdog — Thu, 07 May 2009 17:40:35 +0000

Good article and great Q's and A's too.

By: Mauricio Carrillo Tripp

Mauricio Carrillo Tripp — Thu, 07 May 2009 16:08:29 +0000

This whole discussion seems very interesting to me since is one of my research areas, although I am not an experimentalist and I'm kind of new in the field. I have a few comments and questions I'd like to add.
As far as I know, there is a difference between an ion channel and an ion pump. The former acts according to a gradient, while the later can go against this gradient, therefore it needs to get energy from somewhere (eg ATP).
“As the endosomal vesicles that contain the virus particles move towards the cell nucleus, their pH drops. This change in pH is accomplished by a cellular channel that pumps protons (H+) into the vesicle.” I imagine this is a proton pump, not a channel, the same that takes protons out of the cell, using ATP found in the cell interior, only now it pushes them into the endosomal vesicle.
When the endosomal membrane fuses with the viral lipid envelope, the capsid, formed by many copies of M1, should be exposed now. ” To get around this problem, the influenza virion has a few copies in its membrane of a protein called M2. This viral protein forms a channel in the membrane that actively pumps protons from the endosome into the interior of the virion.” From this description it seems that M2 is actually a proton pump, using ATP from the cell to work? Do we know if this happens before, after or while the fusion is taking place? From this illustration (http://www.virology.ws/wp-content/uploads/2009/...), it appears as if M2 traverses both the viral lipid envelope AND the capsid, but from the illustration above this is not clear. So, where do the protons pumped by M2 end up, is it inside the capsid? Could this explain why the capsid disassembles? Do we know the 3D structure of M2 and the Amantadine?
Thanks!

By: Mao Asada » ESCRT

Mao Asada » ESCRT — Thu, 07 May 2009 08:23:20 +0000

[...] Release of influenza viral RNAs into cells [...]

By: Corey Philipp

Corey Philipp — Wed, 06 May 2009 23:59:43 +0000

If the NP protein has an NLS then is the NLS specific to eukaryotes? Or is it more specific, like mammals? Not all NLS signals are created equally...

With amantadine, would the side effect be the interaction of the porins or with importin?

Are there any new M2 or NA inhibitors in the development?

Last question...Has any work been done on drugs that target the NP protein to either cleave the NLS or change the confirmation such that importin doesn't recognize the NLS?

By: profvrr

profvrr — Wed, 06 May 2009 23:38:19 +0000

Viral RNAs are transported into the nucleus through a pore-complex, shown in the illustration. This so-called nuclear pore complex is used by the cell to actively more large assemblies in and out of the nucleus. Proteins have to have very specific amino acid sequences to be transported through these pores; such sequences are called nuclear localization signals (NLS). Influenza viral RNAs are bound to a viral protein, NP, which has an NLS and therefore hijacks this import system.

Side effects of drugs occur because the drugs inhibit a cellular target. For example, in the case of amantadine, the drug may block a cellular pore, or might even inhibit a completely different kind of protein. The NA inhibitors might in the same way inhibit other proteins - they look like sialic acids, which could potentially interfere with many cellular processes. It's usually not something in the antiviral that is added.

Scientists figure out complex processes by using a model system, and then breaking down the process into small steps. Then they propose a hypothesis and do experiments to test it. Let's take nuclear import of influenza RNA. People wondered how the RNA could get into the nucleus because it doesn't have an NLS. So they proposed that one of the viral proteins bound to the RNA had an NLS. This NLS was found on the NP protein. Then the sequence was changed which prevented the NP from entering the nucleus. These experiments were done in cells in culture.

When I teach virology, I always teach not only the models, but how the models were obtained. I can't do that here because of time/space limitations. Anyone can teach facts. Not everyone can teach how the facts were obtained. If you learn how these processes are sorted out, then you will learn how science is done.

By: Trine

Trine — Wed, 06 May 2009 22:02:24 +0000

OK, a few questions: How do the viral RNAs make it into the nucleus?
And, learning about how amantadine and oseltamivir work make me wonder about side effects…why do side effects come about? Is it from something happening when the drugs are doing their job, so to speak, like actually clogging the ion channel, or is it something that happens on the way to that channel? or is it something else in the drugs – maybe some added, like a preservative?
And, lastly, how do we know all this? How do scientists figure these really complex processes out?
Okay, sorry about the endless questions…thanks!