Nucleoproteins modifiable like histones
eli I have been thinking about why nucleoproteins don’t just bind on to host mRNA. I have been thinking about what is different between mRNA’s and corona genomic RNA. Normal mRNA has a 5’ cap and a poly A tail. Genomic viral RNA I assume is different. It is one long RNA. No cap or tail. How do genomic RNA keep its integrity from the ends?
So perhaps for nucleotides to tag on to the RNA, the RNA has to be rather long. Also it needs to not have large caps on either ends for nucleoproteins to get on.
Which made me think of the different hypothesized models for how nucleoproteins did grab onto the RNA.
In section 7.1 this paper lists 4 possible ways, nucleoproteins can pack with the RNA.
Model 2 is about growth. Sliding or hopping on of N proteins.
Model 3 is packaging. I love how they describe it: “Thus, the N protein binds to RNA in a fashion resembling that of an octopus clinching onto its prey (RNA) using all its tentacles”
Model 4: “Thermodynamic basis: Electrostatic interaction drives the formation of N-RNA complex but the multitude of weak protein–protein interactions contributes towards the self-assembly of the helical RNP”
The SARS coronavirus nucleocapsid protein - Forms and functions
Now if sliding is the manner, then mRNA would be protected from nucleoproteins from both ends.
Or perhaps the first set of nucleoproteins would have to slide on to the mRNA, for further recruitment of n proteins.
Oh and by the way, the reason I bring this up is that I have been playing around with the GFP mRNA in the lab. I simply think the mRNA is mainly associated with itself most of the time. I tried to untangle it from the 5’ end, one strand at the time. To see if that would leave a large 3’ tail dangling lonely. It didn’t. So I basically think the 3’ tail is set up to have a large potential to either fold with itself or fold with nearby regions or make a neck with the 5’ end if matching sequences are around. That way most of the mRNA is kept together and less prone to cutting. I’m aware I’m using just a simulated version of Vienna2 to show me.
Thanks for insights. Please do note that’s Coronavirus RNA genomes do have caps and polyA tails!
eli Oh, interesting.
So the coronavirus is just like one massive long mRNA.
So if it weren’t for that it needed to be confined to a small place, like its virus capsule, it may not have needed all the N proteins.
eli: Rhiju, I have been thinking about something. I may have an idea why nucleoproteins binds so regularly to the viral RNA, when it is packaged.First something from a video that helped me better visualize what the genome of a cell looks like when it is in use.
DNA Doesn’t Look Like What You Think!
I think something similar goes on with the viral RNA when it is in use. Just think about the 2D view of the MS2 bacteriophage. It suspiciously looks like something to me that will become a sphere when folded out and not packed in nucleoproteins.
So I think a good deal of the nucleoproteins fall off the viral RNA or is spread out, just as it does for the DNA in a cell when it is not replicating.
I have also kept thinking about how and why of the regularity of the nucleoproteins are packed around the viral RNA. In particular what you said: It very well could be ‘beads on a string’!
What about making that magnetic beads?
The RNA/DNA is highly negative. The histones and nucleoproteins are positively charged.
Electric forces and magnetics alone could explain the regularity of the binding.
While I checked if the histones were positively charged, as I suspected, I began wondering if the chemical modifications of the histones could change the charge of the histones, as this could help explain the winding and unwinding. The answer I found was yes. It is called acetylation and deacetylation of histones.
“Deacetylation is simply the reverse reaction where an acetyl group is removed from a molecule. Acetylated histones, octameric proteins that organize chromatin into nucleosomes basic structural unit of the chromosomes and ultimately higher order structures, represent a type of epigenetic marker within chromatin. Acetylation removes the positive charge on the histones, thereby decreasing the interaction of the N termini of histones with the negatively charged phosphate groups of DNA. As a consequence, the condensed chromatin is transformed into a more relaxed structure that is associated with greater levels of gene transcription.”
Now I wonder if some of the chemical modifications that can happen to nucleoproteins, can do something similar with relaxing or condensing the viral RNA. I recall reading that nucleoproteins can get phosphorylated.
And if these chemical modifications affect the charge of the nucleoprotein too.
And thus how condensed or relaxed the coronavirus genome is.
It kind of all makes sense now. The RNA viral genome has to be neutral in change when it has to get packaged and the RNA has to get really close to itself. The nucleoproteins work as charge neutralizers. So the corona nucleoproteins being rather large compared to the RNA also makes sense as that effectively shields the RNA from itself.
Now it also makes sense why there are some conserved regions in the nucleoproteins. I bet some of the charged regions are among.
Another thing I am wondering about is if the order of the genes, also has a function in relation to when a gene is made. I took note of that in one of the papers I read, it says that the genes (Those of membrane, spike, envelope and nucleoproteins - mentioned in no particular order here) of coronavirus always came in the same order. The nucleoproteins last. So is this something that has to do with whether they are also needed last? Is there anything written about the order of which proteins get used in a virus?
Eli Another question. Are there charge differences between RNA and DNA? Because it seems to me from the images that I have seen that DNA can get packaged closer together than RNA.
And I now have examples on acetylation of nucleoproteins in viruses.
It has been recently reported that the nucleoprotein (NP) of influenza virus is acetylated in infected cells, and this modification contributes to the RNA polymerization activity of the virus. As the influenza virus, the Ebolavirus contains single-stranded negative-sense RNA as its viral genome, which interacts with NP and other viral proteins. In this study, we performed a series of biochemical experiments and revealed that the recombinant Ebolavirus NP and the viral matrix protein VP40, which binds with NP, were acetylated by eukaryotic histone acetyltransferases, such as P300/CREB-binding protein (P300/CBP) and P300/CBP-associated factor (PCAF), in vitro.
Acetylation of lysine residues in the recombinant nucleoprotein and VP40 matrix protein of Zaire Ebolavirus by eukaryotic histone acetyltransferases
Also I have an example of a host enzyme, the deacetylase HDAC6, that by deacetylating the influenza A virus, can inactivate the virus. I don’t think the paper is clear on the mechanism. It mentions several candidates for the deacetylation. Among them the NP. But I believe it is the Nucleoprotein that is at the receiving end.
In other words, I believe in RNA histones.
HDAC6 Restricts Influenza A Virus by Deacetylation of the RNA Polymerase PA Subunit
Right now I feel like deacetylating coronavirus
By the way I read about ribosome profiling. It is my feeling that nucleoproteins will just get moved out of the way by a massive ribosome and it as such can not be used to find where the nucleoproteins bind. Are there any ways to digest RNA in a way that would leave the nucleoproteins on, where they are?
So when the nucleoproteins are to pack the RNA for replication, the nucleoproteins ought to get deacetylated.
Now I wonder if charge plays a role in some of the conserved regions of the corona viral genome in general.
Are there any patterns to what amino acids are conserved?
> By the way I read about ribosome profiling. It is my feeling that nucleoproteins will just get moved out of the way by a massive ribosome and it as such can not be used to find where the nucleoproteins bind. Are there any ways to digest RNA in a way that would leave the nucleoproteins on, where they are?
Yes there are strategies that crosslink proteins to the RNA, digest the RNA that is not protected by the protein, and then figure out which RNA segments are left. I don’t know if this has been applied to coronavirus genomes yet
Eli Cool. Perhaps it has already been applied to another RNA virus genome that would demonstrate the concept.
I have learned that these proteins in the host are involved in Acetylation (HAT) - Histone Acetyl Transferase and Deacetylation (HDAC) - Histone deacetylation.
DNA and chromatin regulation | Biomolecules | MCAT | Khan Academy
I am wondering if the virus brings its own or it uses the host’s proteins. I have found some cases that involve the latter.
Also that SIRT’s can be involved.
Histone Deacetylases in Herpesvirus Replication and Virus-Stimulated Host Defense
From the paper: Additionally, canine coronavirus (CCoV-II) infection has been shown to induce the expression of SIRT1, SIRT3 and SIRT4 , suggesting that the regulation of individual sirtuins is virus specific.
Eli I got lucky, someone has been doing that experiment you mentioned to influenza A.
Nucleoprotein footprinting. It is a most interesting read.
Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication
“NP binds short fragments of RNA (~12 nucleotides) non-uniformly and without apparent sequence specificity. Moreover, NP binding is reduced at specific locations within the viral genome, including regions previously identified as required for viral genome segment packaging. Synonymous mutations designed to alter the predicted RNA structures in these low-NP-binding regions impact genome packaging and result in virus attenuation, whereas control mutations or mutagenesis of NP-bound regions have no effect. Finally, we demonstrate that the sequence conservation of low-NP-binding regions is required in multiple genome segments for propagation of diverse mammalian and avian IAV in host cells.”
This makes me think of your latest paper with conserved regions of coronavirus that had specific structures.
I would be interested in if these regions of the coronavirus also were low in nucleoproteins.
“In this study, we set out to determine how IAV NP interacts with vRNA during infection in cells. We show that the NP of IAV binds the vRNA non-uniformly and that regions of low-NP binding are enriched for predicted RNA secondary structures. Synonymous mutations designed to destabilize the predicted RNA structure attenuate IAV replication, whereas synonymous mutations that maintain the predicted RNA structure or mutations in NP-bound RNA regions have no effect on virus replication in vitro or in vivo. Viral attenuation is associated with an increase in defective virus production, suggesting that low-NP-binding regions and the predicted RNA structures are required for viral genome packaging.”
rhiju das i am hoping that someone in the world is doing those experiments now…