Fight the COVID-19 with Eterna

@jnicol this is a cool idea. There have been thoughts in the vaccine literature on making ‘attenuated virus’ vaccines based on changing the natural virus RNA sequences to be low expressing or easily degraded.

I think its something Eterna can explore in later stages of the pandemic. In particular, there is a ‘self-amplifying mRNA’ (SAM) technology for RNA vaccines that looks very promising for the COVID-19 vaccine but that involves using another virus (alphavirus) to deliver the mRNA for, say, the Spike protein. This idea is not being tested widely yet (to my knowledge), but may be later on in 2020, since some of the RNA vaccine companies hold the patents on it. Having attenuated or self-degrading mRNA could be important for that next-generation method and even have an impact in developing the safest vaccines for 2020-2021.

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I’m really excited to begin the lab!

Do nucleoproteins bind to specific RNA sequences?

I found an answer to the question if the nucleoproteins bind specific sequence wise in the RNA. The answer is no.  

WHO corona image

“We also find that the recruitment of nucleoprotein to nascent ribonucleoprotein complexes during replication of full-length viral genes is mediated through nucleoprotein–nucleoprotein homo-oligomerization in a ‘tail loop-first’ orientation and is independent of RNA binding.“

The role and assembly mechanism of nucleoprotein in influenza A virus ribonucleoprotein complexes

This paper also says that the nucleoproteins are important for translation. They removed them and it was possible to replicate up to 101 bases, but above that the nucleoproteins were needed.

“Using longer RNA templates we determined that the viral polymerase is also able to efficiently replicate and transcribe a 76-nucleotide-long template in the absence of NP. However, NP was found to be essential for the replication of templates of 101 nucleotides and longer”

Also I wonder: How do nucleoproteins know how to bind in a regularly spaced manner?

Ok, this was a negative strand virus, in need of a RNA polymerase. Not a positive strand RNA like corona. Do our corona virus contain a protein for a polymerase?

I have learned that positive strand viruses sometimes carry such a one, as this allows them to speed up viral production even more.

Here is a fine video explaining the different types of viruses. Like negative and positive strand ones. 

Link to video David Baltimore (Caltech): Introduction to Viruses from Ibiology

Ibiology has a bunch of fine lessons on viruses.

Nucleoproteins bind regularly to membrane proteins

Instead of nucleoproteins binding to specific RNA sequence, I found out that for corona, the membrane and the nucleoproteins specifically bind:

Just like in Ebola:

The same paper that answered my membrane versus nucleoprotein binding question, also answered another of my questions. 

Can nucleoproteins bind to each other?

Also I was wondering if nucleoproteins could bind to each other. As this could aid with structure and packaging too.

eli: Ok, and I did find confirmation that nucleoproteins can actually bind to each other and dimerize. Even more than that. 

rhiju  yup 

Now I suspect there are also sequence/structure motifs through the genome that engage the nucleoprotein but they haven’t been discovered.

Otherwise, how does the N protein know to encapsidate the coronavirus genome vs. all the other mRNAs in the cell?

such binding sites have been hard to decipher for any RNA virus. But here’s an example of how it was figured out recently for a classic model system the MS2 virus that infects E. coli:

The 3D RNA structure in this paper is incredible. We may want to take inspiration for both our understanding of CoV and also for how we want to ‘package’ our vaccines for stability.

PubMed Central (PMC)

In situ structures of the genome and genome-delivery apparatus in an ssRNA virus


eli: Oh, that is a really interesting question.

Can it be that the host mRNA are already having their own nucleoprotein chaperones?

@rhiju, I have searched, but have not yet found a good answer on this. I know the poly A tail in mRNA should be covered in some proteins to protect it. But if the mRNA is somehow covered, then these proteins are probably called something else.

I don’t recall which paper, but one of those I read, said that visualizing the nucleoproteins was rather hard, due to the capsid

Wow, you are right, they do not at all have that problem in the paper you linked. }

eli: However it was for the original SARS nucleoprotein. Which they weirdly call nucleocapsid.

rhiju: Ah yes, the name changes. The acronym N is pretty robust though. :smiley:

eli:  The 2D structure of the RNA MS2 genome, reminds me of the ribosome 2D structure 

Ms2 genome image

Nollers ribosome image

Although there are a lot more long stems in the MS2 one.

It seems to me from the images that the hairpin loops touches the capsid wall. But I’m not certain.

I think there is less structure variance in the M2 genome, than in the ribosome

There are more loops and stems of the same size.

Both the ribosome (2 half circles) and the MS2 fold into spheres.

The difference is where they have their proteins. Outside or everywhere.

The MS2 genome has a lot smaller multiloops.

They both seem to agree on small hairpin loops.

The MS2 2D overview kind of reminds me of cell glycosylation

N-linked glycosylation

The branching is kind of characteristic

Silly thought popped up as I went to bed. Not sure why one should do it, but if one wanted to, could one pack a ribo-T ribosome with a circular mRNA inside a virus capsule for sending it into a cell?

There isn’t that much size difference between a ribosome i bases and shape compared to the MS2 capsule.

rhiju: yea, I bet you could pack a ribo-T ribosome with an mRNA into a little capsule.

I think if you included a few more enzymes and tRNA that might even become a… minimal cell? Pretty cool basic science & engineering question — no one has achieved that before.


A piece of good news reported in the Washington Post today: The coronavirus isn’t mutating quickly, suggesting a vaccine would offer lasting protection.

From the article:  Scientists now are studying more than 1,000 different samples of the virus, Peter Thielen, a molecular geneticist at the Johns Hopkins University Applied Physics Laboratory who has been studying the virus, told The Washington Post.

There are only about four to 10 genetic differences between the strains that have infected people in the U.S. and the original virus that spread in Wuhan, he said.

“That’s a relatively small number of mutations for having passed through a large number of people,” Thielen said. “At this point the mutation rate of the virus would suggest that the vaccine developed for SARS-CoV-2 would be a single vaccine, rather than a new vaccine every year like the flu vaccine.”

It would be more like the measles or chickenpox vaccines, he said — something that would likely confer immunity for a long time.

“I would expect a vaccine for coronavirus would have a similar profile to those vaccines. It’s great news,” Thielen said.

Two other virologists, Stanley Perlman of the University of Iowa and Benjamin Neuman of Texas A&M University at Texarkana, both of whom were on the international committee that named the coronavirus, have told The Post that the virus appears relatively stable.

“The virus has not mutated to any significant extent,” Perlman said.

“Just one ‘pretty bad’ strain for everybody so far. If it’s still around in a year, by that point we might have some diversity,” Neuman said.

Neuman contrasted the coronavirus with influenza, which is notoriously slippery.

“Flu does have one trick up its sleeve that coronaviruses do not have — the flu virus genome is broken up into several segments, each of which codes for a gene. When two flu viruses are in the same cell, they can swap some segments, potentially creating a new combination instantly — this is how the H1N1 ‘swine’ flu originated,” Neuman said.

mobile – shooting for deployment in 1 month.
lab has begun!

I’m glad to hear. I’ve been waiting for mobile for a while

“In this study, one coronavirus isolated from Malayan pangolins showed 100%, 98.2%, 96.7% and 90.4% amino acid identity with 2019-nCoV in the E, M, N and S genes, respectively. In particular, the receptor-binding domain of the S protein of the Pangolin-CoV is virtually identical to that of 2019-nCoV, with one amino acid difference.”

Could pangolin antibodies be used in humans?

“It has been reported on March 14, 2020, that SARS-CoV-2 invasion is mediated not only by ACE2, but also by basigin. SARS-CoV-2 attaches itself to CD147 via spike protein (SP), although this research finding has not yet been peer reviewed.”–

SARS-CoV-2 invades host cells via a novel route: CD147-spike protein

So another target for an rna antiviral vaccine?

Basigin and neurothelin are related and I find neurothelin hits all over the E-protein C-terminus. Since E-protein is very strongly conserved and C-terminus is cytoplasmically oriented for antibody development this looks encouraging to me for E-protein consideration.

Here’s my silicon valley antiviral idea just for jollies.

An excerpt from an article explaining in plain language why the CDC test is running into problems and how the RT-PCR test works. Doesn’t give a lot of details on picking the best RNA segment but illustrates what can go wrong.

But if the test is so simple, why is the U.S. having trouble getting it to work?

The U.S. initially mandated the use of CDC-developed test kits for all coronavirus testing, but labs reportedly had trouble getting them to work. The CDC was criticized for not using test kits developed in Germany, which were successfully detecting coronavirus around the world and were backed by WHO. U.S. labs responded by developing their own tests, in some cases reporting quicker turnaround of results. This prompts the question: What are the differences between these tests and why do some work better than others?

The answer is relatively simple: each test chose a different “sentence” to copy from the viral RNA. Effectively, this changes the primers (first and last word of the sentence), the fluorescent probe (corresponding to some word in the middle), and the “positive control” (a tube of DNA containing the “sentence” that is used to make sure the test is working properly). In fact, the CDC’s test kits are really just this: a few tubes containing the three components above.

Choosing primers for any PCR experiment turns out to be tricky and sometimes unpredictable. Primers are just short pieces of DNA themselves, and some DNA has a tendency to fold in on itself, creating a “hairpin” structure which inhibits PCR. (This is a bit like the matching letters in a palindrome finding one another). These “palindrome” primers can produce a false negative — an infected patient whose sample appears to lack the virus. Alternatively, the primers can work just fine to make copies of coronavirus RNA, but might also be capable of copying some part of human DNA. Because patient samples (most often nasal swabs) contain both viral particles and human cells, these primers can produce a false positive — an uninfected individual testing positive for the virus. Other potential sources of RT-PCR failure are temperature issues, low primer or sample concentration, and contamination, among others.

  1. How a coronavirus test can fail. A “palindrome” primer can cause a false negative. Primers which can recognize human DNA can lead to a false positive.
    I generally avoid Medium as a source but the article was written by a graduate student:

Some statistics on the rate of false negatives:

Somehow this reminds me of our lab barcodes. If I recall correctly, Rhiju and co developed an algorithm to assign barcodes in an automated way.  

Also I guess in the above example, since the barcode is just next to the primer, and the primer is forming a helix too, there is coaxial stacking involved too. 

Nucleoproteins continued

I actually ended up finding a virus that was actually associated with histones. :slight_smile:

Simian Virus 40 drawn by David Goodsell

It is a DNA virus and as such it is not weird that it can bind with histones. But from what I gather, it doesn’t even bring along the gene for making histones, it just nicks the hosts histones. 

Most conserved sequences in coronavirus

I found a paper with crystal structure of the new corona virus nucleoprotein. They found conserved regions in it across 4 related coronaviruses:

From what I read, the part in coronavirus that has the most conserved sequence, is the nucleoprotein. 

“Viral N protein shows least variation in the gene sequence, therefore indicating it to be a genetically stable protein, which is a primary requirement for an efficient drug target candidate.” 

The SARS coronavirus nucleocapsid protein – Forms and functions

Can’t help wonder if I find similar images for E, M or S proteins, if there will also be conserved regions between SARS 1, 2 and MERS. I would like to kill them all in one go

eli  Rhiju, I have something I want to show you.

Octamer of Sars 1

The SARS coronavirus nucleocapsid protein – Forms and functions

It is kind of like histones made of 8 proteins, just that the histones would also be binding to each other too.

The N proteins connecting to each other could also explain the regular spacing between them. 

These N proteins have some tails sticking out. I won’t be surprised if they can be modified, just like histone tails.

The image said this is a 24 mer. Each of these sets are 8 nucleoproteins. Had they been drawn as spheres, they may have looked more like a histone.

There are size differences between the DNA and the viral histones.

“Interestingly, Chang et al. also observed the formation of an octamer in the asymmetric unit of the CTD crystal. Translationalstacking of the octamer forms a hollow twin helix structure with an outer diameter of 90 Å and an inner diameter of45 Å, with a pitch of 140 Å. The groove of the twin helix, which is lined with several positively charged residues, has a depth of 22.5 Å. The N-terminal 22 amino acid residues from a.a. 248–269 play an important role in protein–protein interaction in the octamer, accounting for the absence of the octamer in the crystal structure of CTD270–370.”

They are having the same spiral stairway, although opposite orientations. 

Quite a different way of wrapping though.

I read that RNA is stiffer than DNA. So it makes sense that DNA can be bent more.

Also this tying up of the RNA means it is much less prone to hydrolysis

There literally are no loops hanging out anywhere. Same goes for DNA when it is closely packed. 

By the way, I wonder why we can’t just cover our chosen mRNA in nucleoprotein from the virus and ship it as a vaccine. The RNA would be protected and the body might also learn to recognize the nucleoproteins as enemy as well.

So viral capsid, nucleoproteins and then a piece of mRNA (edited) 

rhiju  yes I agree that is a great idea. Vaccine folks have been working on synthetic ‘virus like particles’ for several years but hasn’t been deployed. The idea of using another actual not-too-toxic virus carrying the message from the bad virus is a very active area of exploration.

Look here:

(Link from DigitalEmbrace in the forum)

eli: I wonder if the corona viral shell could even be assembled without the RNA and nucleoproteins.

I kind of doubt. Because there seems to be a connection between all the parts.

Nucleoproteins are connected to M proteins. Envelope proteins are connected to M proteins. N proteins are connected to N proteins and RNA are connected to N proteins. So this is why another virus capsule is chosen for transport.

So the transporter needs to be one where all the parts are not interdependent.

Plus there would be way too much empty space inside the virus capsule. Ok, now I understand much better why a different virus is chosen as a transporter.

Packaging of RNA in histones or DNA in nucleoproteins?

eli Silly thought. Can RNA get packaged in histones. Have anyone ever tried?

Or DNA in nucleoproteins for that matter.

rhiju  histones will definitely stick to RNAs — but I think they aggregate. Assembling histones even with DNA in a test tube is actually pretty tricky. Next time I talk to my colleagues working on this, I’ll ask. There is a major effort to assemble in a test tube chromatin (DNA+histones) with RNA’s like ‘Xist’ or RNA-protein complexes like Polycomb-repressive complex, so your question is quite pertinent. I hadn’t previously considered the idea that RNA might even compete away the histones away from dsDNA. That is very intriguing.

eli Cool!

On the keeping of the structure of a vaccine RNA, I’m still pretty hot on the thought, to pack the RNA in viral nucleoproteins. I mean coronavirus does a pretty good job at keeping its RNA stable at room temperature. Imagine it was also well hydrated and cared for in a vaccine vial. And of course inside a small viral capsid too - for transport.

rhiju Good idea, keep it for later iterations of the vaccine challenges. 

eli One thing I’m still wondering very much about. When RNA like mRNA is inside the body, is it protected by any proteins? I mean like you wondered about why the nucleoproteins of coronavirus did not stick to host mRNA. Which I think is an excellent question.

rhiju  yes, in animals, there are proteins called hnRNP’s that might cover mRNAs

Also there may be chemical modifications

No one has really figured out how the proteins engage with the mRNAs.

It very well could be ‘beads on a string’!

eli  That is what I think would make sense. That would explain why the viral nucleoproteins don’t jump on just every available mRNA.

It would also make sense if those proteins binding to the single stranded RNA, would be something already available in the cell in huge amounts.

Just as the viral RNA polymerase removes the nucleoproteins as it tags along the viral mRNA (but still need the nucleoproteins for stabilizing the RNA), the ribosome would do similar.

Chemically modifying a virus out of existing

By the way, I found something really interesting today. I was earlier plotting on methylating the coronavirus to hell, or just as well demethylate it. Targeting its chemical modifications. 

Turns out someone has already been doing that, to another virus. With success. 

“Using high-throughput sequencing, Li scanned the virus’ genes and identified which one contains the greatest extent of m6A methylation before then knocking out these modifications to make a mutant virus. Looking at how these mutant viruses performed would shed light on the impact of m6A methylation.When human cells were exposed to the knock-out virus, they produced an antiviral protein known as type I interferon that pointed to the activation of the innate immune response, or the somewhat generalized response that the body has to a foreign pathogen. According to Li, “This opened up a big question. Why would a virus lacking this methylation produce a much higher innate immune response?”Upon reviewing the cellular signaling pathways involved, it became evident that m6A methylation makes the virus able to hide from the immune system by masking the RNA so that it didn’t stand out as being different than the host RNA. The immune system therefore doesn’t recognize the virus as being non-self RNA, and so it doesn’t summon the innate immune response—meaning that the virus is able to persist, undetected.”

Vaccine Targets Viral Epigenetic Modification, Could Prevent Respiratory Infection

Eli  On my question if nucleoproteins could bind to DNA histones or histones could bind to viral RNA, I found something extremely interesting:

“By itself, the SARS-CoV N protein is a non-specific nucleic acid-binding protein. It has been shown to bind to single-stranded RNA (ssRNA), single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) under in vitro conditions (Takeda et al., 2008, Tang et al., 2005).” 

The SARS coronavirus nucleocapsid protein – Forms and functions

rhiju das  Beautiful 

@Eli: Eterna lab barcodes are a little different than what DigitalEmbrace is talking about. There are in fact many publicly available software tools to optimize primers and choose the right ones.

As for Eterna lab barcodes, I don’t recall the Das lab using automatic barcode selection in any of the SHAPE experiments. I actually wrote such a tool back in 2014, and simulations showed that it could have improved the experimental data quality (by substantially reducing the interactions between designs and barcodes globally) but to my knowledge, it was never experimentally tested. Such tests would have been costly, double everything, cDNA libraries, experimental processing, etc, in order to compare player-selected barcodes vs automated selection. So it’s understandable that it wasn’t attempted…


I’ve been trying to come up with a COVID-19 project players could lead. Genome analysis/comparison is a possible candidate but I haven’t been able to identify a need/goal. Secondary structure is another area to consider. I believe only pieces of the SARS-CoV-2 molecular structure have been determined. I need to see if I can locate the molecular structure of SAR-CoV-1.

Why #masks4all is a good idea.…

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.

rhiju das  

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. :slight_smile:

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 :smiley:

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?

Ribosome footprinting

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?

rhiju das  

> 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

Sirt (deacetylase)

From the paper: Additionally, canine coronavirus (CCoV-II) infection has been shown to induce the expression of SIRT1, SIRT3 and SIRT4 [113], 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… 

I find SIRT1, 2 & 3 signatures as well as HDAC’s all over SARS-Cov2 E-protein.