James, there are lots of quite simple experiments that would be could be done, but I can't get anyone to think seriously about the problem - let alone do the experiments.
The attitude seems to be, "it can't be as simple as you suggest - someone would have noticed it". * Has anyone measured virulence vs temperature in cell culture? * This isn't what you've got in mind, but yes, it was noticed that the viruses that establish persistent infections of cell cultures (and they have to be less aggressive to do that) often *spontaneously* become temperature sensitive. And, conversely, temperature-sensitive viruses that are grown at low temp but in conditions where they can multiply fast, often spontaneously lose their initial temperature-sensitivity. Refs in my paper. Thse are all experiments that were done "by accident". In influenza a temperature-driven switch has been seen, switching between translation (high temp) and replication (low temp). See the link below for figures - described in words in my paper, below. Best wishes Patrick Figures showing the "switch": https://www.douglas.co.uk/f_ftp1/Seminar_by_Patrick_Shaw_Stewart_-_a_few_slides_for_Bill.pdf Paper: http://douglas.co.uk/f_ftp1/ShawStewart_final_1-s2.pdf Easy read part 1, about seasonality: https://oldwivesandvirologists.blog/ Easy read part 2 about Covid: https://oldwivesandvirologists.blog/Covid-19-and-the-trade-off-model-of-selection/ _________________________________ >From my paper: *The temperature sensitivity of viral transcription* Most laboratory respiratory viruses are propagated at 37 C, which may result in the rapid loss of ts characters, especially since viruses mutate very rapidly when they are introduced to new hosts. If, however, temperature sensitivity is a common feature of wild respiratory viruses, we might expect to see remnants of temperature sensitivity in the biochemistry of laboratory strains. It turns out that such remnants are quite common. For several decades virologists have found that maximum RNA transcription in influenza viruses occurs below normal body temperature. In 1977, Plotch and Krug [61] reported that the greatest activity of the RNA polymerase of WSN virus was at 30–32 C. This is similar to the optimum temperature of the polymerase of influenza C, which is 33 C [62,63]. Ulmanen et al. [64] found that the rate of transcription by detergent-treated WSN viruses (influenza A) was about 10 times greater at 33 C than at 39.5 C, and also that the binding of a cleaved primer cap (the ‘‘A13 fragment”) to the viral cores was ‘‘unexpectedly” much weaker at 39.5 C than at 33 C. Scholtissek and Rott [65] showed that the optimum for the polymerase of the Rostock strain of fowl plague virus was 36 C, five degrees below chickens’ normal body temperature (41 C). At least two reports show that temperature affects the balance between transcription and viral replication. Kashiwagi et al. looked at the effect of temperature on RNA production for five varied influenza A strains [66]. For all strains, vRNA unexpectedly decreased when the temperature was increased from 37 C to 42 C. The PA subunit of the viral polymerase caused this thermal sensitivity. In another interesting study, Dalton et al. showed that the production of mRNA by the PR8 influenza strain is favored at a higher temperature (41 C), with very little vRNA being produced at that temperature [67]. A plasmid- based recombinant system showed that as the incubation temperature increased from 31 C to 39 C the amount of replicative RNA products (c- and vRNA) decreased and a greater accumulation of mRNA was observed. The cRNA that is used as a template to make the vRNA formed a complex with the polymerase that was particularly heat-labile, showing rapid dissociation even at 37 C. The authors suggested that the ‘‘switch” that regulates the transition from transcription to replication is dependent on temperature, but made no comments about how shifts in the host’s body or respiratory tract temperature may influence this transition. On Sun, Mar 22, 2020 at 3:38 PM James Holton <jmhol...@lbl.gov> wrote: > Thank you Patrick, > > RNA structure is still structural biology, so I think relevant here. It > seems to me that RNA as a thermometer would be an easy hypothesis to test? > Has anyone measured virulence vs temperature in cell culture? > > The 3D structure of the genome is no doublt important. I wouldn't want to > try crystallizing the whole thing, but I wonder if this might be an > excellent target for cryoEM? A challenge for that "we can classify our way > out of anything" philosophy? And the result would most certainly be > interesting. > > -James Holton > MAD Scientist > > On 3/21/2020 8:41 AM, Patrick Shaw Stewart wrote: > > > James, this isn't conventional structural biology, but may be of interest, > and I haven't been able get any mainstream virologists to think about it. > > The protein sequences are obviously of interest, but so are the RNA > sequences at both ends of the Covid genome, which have conserved secondary > structure. A few years ago a paper came out suggesting that wild-type > influenza has multiple "RNA thermometers", which may play an important role > in the tropism of influenza. Similar mechanisms may exist in other > respiratory viruses, including Covid. > > My take on this, and the relevant papers, are below. > > Good luck to everyone and stay well, > > Patrick > > > > https://oldwivesandvirologists.blog/Covid-19-and-the-trade-off-model-of-selection/ > > > My paper in *Medical Hypotheses * > http://douglas.co.uk/f_ftp1/ShawStewart_final_1-s2.pdf > > Narberhaus, Franz, Torsten Waldminghaus, and Saheli Chowdhury. "RNA > thermometers." *FEMS microbiology reviews* 30.1 (2006): 3-16. > > Chursov, Andrey, et al. "Specific temperature-induced perturbations of > secondary mRNA structures are associated with the cold-adapted > temperature-sensitive phenotype of influenza A virus." *RNA biology* 9.10 > (2012): 1266-1274. > > Yang, Dong, and Julian L. Leibowitz. "The structure and functions of > coronavirus genomic 3′ and 5′ ends." *Virus research* 206 (2015): 120-133. > > > > > > On Fri, Mar 20, 2020 at 10:59 PM James Holton <jmhol...@lbl.gov> wrote: > >> You might think that as a structural biologist you won't be able to do >> much about COVID-19 anytime soon, but that is not true. Yes, real-world >> therapeutics and vaccines take time, but we have already seen just how >> fast we can get started. There are 21 PDBs already and some even have >> bound ligands. Good job Frank et al. BTW! And my personal thanks to >> all of you out there who are already hard at work on this. >> >> I believe this forum is an ideal place to share information and ideas on >> the structural biology of SARS-CoV-2 as we move forward. It's a big >> virus, but there are not that many proteins in it. If all of us >> independently do the same bioinformatics and literature searches and end >> up trying exactly the same thing in every lab all over the world, then >> that would be more than unfortunate. To that end, I am personally >> interested on ORF8 for reasons I will go into below. Has anyone tried >> to solve it yet? What happened? Didn't express? Bad diffraction? >> What? Do tell. >> >> Some of us, as you may have heard, are stuck at home, our beamlines and >> labs dark while we shelter-in-place. That doesn't mean our hands are >> tied. We are still allowed to think. The fraction of the human race >> that has a snowball's chance in Hades of figuring out this bug is very >> very small. Structure may be your main skill set, but you are still a >> biologist. Do you know how to run a PCR machine? Do you know how to >> pipette? You might think that anybody can do it, but that is really not >> the case. Ever trained a new student on sterile technique? How many >> days did that take? Now remember that your student was no dummy and >> already studying biology. Everyone reading this will make an excellent >> volenteer at the very least. I'm not saying this to belittle the >> average human, only to say that we scientists, moving in the circles we >> do, often forget that we have uncommon capabilities. >> >> For example, I also believe we can be useful in assay development. The >> void left by the dearth and delay of test results has been filled with >> fear, and that is a big problem. The tests, as defined, are >> straightforward, but also extremely regimented like any good laboratory >> protocol should be. The US CDC's instructions for academic labs are here: >> https://www.cdc.gov/coronavirus/2019-nCoV/lab/index.html >> My question is: how can this test be made faster, using more commonplace >> supplies, in high-throughput mode and still valid? Not just for >> clinical but for academic use? I think more than a few people on this >> list could be regarded as experts in making a complex biochemical task >> faster, more efficient, high-throughput and nonetheless valid. Yes, >> there are other people who do virus testing for a living, but right now >> they are all rather busy. Maybe if we put our minds to it we can help? >> >> As for why ORF8. I am basing my interest on the bioinformatics done in >> this article: https://dx.doi.org/10.1093/nsr/nwaa036. Search for >> "T8517C" and you will find what I'm talking about. The authors found >> two "types" of SARS-CoV-2. They call them "S" and "L" because the only >> conserved amino acid change involved is S84L in ORF8. The "S" type is >> believed to be the ancestor of "L". What is interesting is how tightly >> linked this mutation is to a silent mutation on the other end of the >> genome: the "L" type has a faster codon for Ser in ORF1. Such tight >> coupling (r^2=0.945) means there must be significant selective pressure >> preventing both of these mutations occurring in the same virus at the >> same time. That, I believe, is interesting. Espeically since they are >> so far apart I expect this selective pressure might work in trans: as in >> a super-infection. That is, the S and L genome types may interfere with >> each other. >> >> The authors fall short of claiming evidence of interference upon >> super-infection, and indeed they have already been criticised for >> calling "L" the "aggressive" type. But it is still interesting and >> points a finger at ORF8. >> >> ORF8 has only one homolog in the PDB: 5o32 with 25% identity over a >> stretch of 60 residues. This homologous region contains the S84L site >> (Val I544 in 5o32). I had a quick look and appears to be a >> cavity-filling mutation to me. Not very big, but maybe something could >> fit in there. To be sure we'd need a structure of ORF8. >> >> Good luck to you all, and stay healthy. >> >> -James Holton >> MAD Scientist >> >> ######################################################################## >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 >> > > > -- > patr...@douglas.co.uk Douglas Instruments Ltd. > Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK > Directors: Patrick Shaw Stewart, Peter Baldock, Stefan Kolek > > http://www.douglas.co.uk > Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 > Regd. England 2177994, VAT Reg. GB 480 7371 36 > > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > -- patr...@douglas.co.uk Douglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Patrick Shaw Stewart, Peter Baldock, Stefan Kolek http://www.douglas.co.uk Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
