Lori writes:
Hello TWiVers,
82 Fahrenheit, 28 Celsius , partly cloudy in Tomball Texas.
I have been an avid listener of TWiV (and Immune and the rest) since March 2020. In January I started listening to old episodes on my daily walks and love hearing the science unfold. Sometimes I am tempted to jump ahead to later episodes to see what happens but that would be like reading the last page of an Agatha Christie novel, where is the fun in that? Is the XMRV story really over? Where is that pan-influenza vaccine? What vaccine will cause the next pandemic? Did Vincent ever get around to watching Contagion? Oh, and by the way Alberta Canada is rat free (except for lab rats). Because of all the grain that is grown in the province, they are motivated to keep rats out. No pet rats allowed.
I just finished listening to TWiV episode 161 Concerto in B with Gabriel Victora, what a great episode. Loved learning about germinal centers which is so relevant today. Tomorrow’s episode is Virology 101, transcription, can’t wait.
It looks like I will soon be putting away my TWiV face mask which I wore with pride, it has served me well. Hoping the world is back to normal soon, thank you science.
Thanks you guys and gals for providing such great podcasts.
Best Regards,
Lori
Bob Krug writes:
I listened to part of your interesting cap-snatching program. I have a few comments.
There is no doubt that influenza cap-snatching occurs on nascent host pre-mRNAs as these RNAs are emerging from the host polII enzyme. Beautiful research from Cussack and Fodor established that it is required that the influenza polymerase bind to the LTR of polII to capture the capped 5’ ends of host pre-mRNAs.
Interestingly, Bartel’s lab (and other labs) has provided good evidence that a majority of the capped 5’ends of influenza mRNAs come from U1 and U2 snRNAs rather than pre-mRNAs. It is not clear how this result impacts the results of the Cell paper that you were discussing.
In contrast to the situation with influenza virus, it is not known how the bunyavirus cap-snatching mechanism accesses the capped 5’ ends of cellular mRNAs in the cytoplasm.
The new influenza antiviral that inhibits the cap-dependent endonuclease (Xofluza) has an added benefit that is not shared with the older antiviral Tamiflu. Specifically, because one dose of Xofluza eliminates essentially all the virus within 24 hours, the contagious period is shortened by 3-4 days. As a result, transmission within the household and in the public is strongly inhibited, leading to considerable reductions in the number of people infected and the number of deaths. For example, modeling predicts that administering Xofluza to only 30% of infected people within 48 hours of the onset of symptoms could spare about 20 million people from infection and reduce the number of deaths by 6,000. The problem is that many physicians stick to the older, less effective drug Tamiflu, which does not significantly inhibit virus spread.
All the best, Bob
Robert M. Krug, Ph.D.
Professor Emeritus
Department of Molecular Biosciences
University of Texas at Austin
Jon Yewdell writes:
TWiV Team,
Many thanks for discussing the UFO paper, a great reward for students and post-docs in the trenches doing the hard work of wrestling truths from nature.
UFO’s were discovered independently by three labs in Glasgow, Mt Sinai, and NIH, accounting for the cast of thousands.
Matt Angel, a recently departed post-doc in my lab, discovered UFOs via ribosome profiling (RiboSeq) of flu-infected cells. RiboSeq is an amazingly powerful technique developed by Ingolia and Weissman at UCSF that makes it possible to determine translation start and stop sites as well as pauses across all cellular translation products. The downside is that it is extremely demanding technically, expensive, and requires considerable bioinformatics expertise to make sense of hundreds of millions of short (28 nt protected fragment) reads. Matt did the RiboSeq in the paper, and as you surmised, my lab also did the antigen presentation work with SIINFEKL, a.k.a., the world’s most dangerous peptide (note irony).
We used SIINFEKL-tagging mainly to gather additional evidence for the translation of the upstream ORFs, not to establish the immunological relevance of UFO-encoded peptides. SIINFEKL is a sensitive tool for measuring translation due to the availability of transgenic T cells that recognize just a few class I (H-2Kb)-SIINFEKL complexes on the cell surface. While SIINFEKL is criticized as an unusually immunogenic/antigenic peptide, it is similar to bona fide immunodominant viral peptides in its abundance, affinity for class I, or ability to induce a diverse CD8+ T cell repertoire. As Vincent insightfully recognized, the UFO data do not directly demonstrate immunosurveillance of UFOs. There is no reason, however, why natural peptides encoded by these ORFs would not be surveilled, since to date, there isn’t a single viral protein that I know of that has the ability to avoid contributing peptides for immunosurveillance (joining death and taxes as inevitabilities).
Indeed, the ability of class I system to monitor translation of viral proteins is astounding. We reported that SIINFEKL on the influenza A virus negative strand is presented to T cells, as is SIINFEKL encoded by the M1 gene in the +1 reading frame after a stop codon using CUG to initiate translation (attached). Recently, we found an abundant highly immunodominant peptide (in mice expressing H-2Ld) derived from what is almost certainly a non-functional gene product, as it is encoded by a 14 residue ORF in the +1 frame of the NS1 gene. Weisan Chen had been searching for this peptide ever since he discovered it 20 years ago as a post-doc in my lab as an activity in a flu infected cell lysates. What’s super cool is that Matt found this ORF immediately from RiboSeq of flu-infected cells, providing proof of principle of RiboSeq for discovering peptides encoded by non-canonical ORFs.
Note that I don’t believe that the UFOs are particularly important for immunosurveillance. I think that this point was overemphasized in the very nice commentary that accompanied the UFO paper. There are plenty of peptides from conventional flu ORFs presented by human cells, which I will remind you have up to 6 different classical class I molecules, each presenting different flu peptides—at least dozens, and maybe even more than 100. Although they may not be generated as efficiently per molecule translated as peptides from uORFs encoding metabolically unstable proteins, translation of standard flu proteins is so robust (even low abundance viral proteins are synthesized at rates of high abundance cellular proteins) that DRiPs (defective ribosomal products) generated co-translationally are sufficiently abundant to result in highly robust peptide generation even from proteins that are exported from the cytosol (HA, NA, M2).
Happy to explain further on Zoom as needed.
Very best,
jon
Bonus information:
I have also attached a very recent paper where we correlate the translatome with the proteome and MHC I immunopeptidome (the repertoire of peptides recovered from class I as determined by mass spectrometry) in human B lymphoma cells. Remarkably, there are nearly eight times as many non-canonical translation products (in terms of variety) encoded by non-canonical proteins vs. standard annotated protein. Since most of the non-canonical proteins are very short, the total number of amino acids encoded in the non-canonical translatome is 11 million vs. 24 million in the standard translatome: still remarkable. Most of these proteins initiate with non-AUG codons. Many of these proteins are likely to be rapidly degraded and may have minimal biological activity, but this remains to be determined. There is so much to learn! Speaking of which, if you have an urge for self-flagellation, I have attached a pre-print of the antigen processing chapter in Paul’s Immunology written by Paul Roche (class II), Ike Eisenlohr (evasion), and me (class I).
Jay writes:
Good Morning Vinney,
Current temperature is 9 degrees C and it’s raining in Boise, Idaho. Keep the non SARS-Cov-2 podcasts coming. I really enjoyed the Cap snatching discussion. I did make it to the end and mostly understood it, although I got lost in some of the details. All of you did a great job in describing the paper.
The stick shift discussion brought back memories for me. I bought my first new car in 1993. Acura Integra. It was a sweet little car. This was the first year of the new body design. I bought a stick shift even though I did not know how to drive one! I was living in the Bay area at the time so had to learn how to drive up hills. It was an experience. I can remember many times trying to get up the hill to my house. I learned and the little car put up with me learning. I kept the car until 3 years ago. I sold it because my hips would hurt if I drove the car for more than 20 minutes. I put up with it for several years until I could not put up with it anymore. Now I drive a Toyota van. My hips do not hurt any more, and the van is more practical for me. Easy to fit mountain bikes, swords, spears and all the other stuff I tend to carry around. I still have fond memories of my Integra. Thanks again.
Jay
Boise, Idaho
David writes:
Hello Dr Racaniello, et. al.,
Listening to the ABC Science Show podcast this week, I thought their story about putting analysis of the coronavirus to music might be interesting to the team and other listeners.
The ABC segment can be found at: https://www.abc.net.au/radionational/programs/scienceshow/coronavirus-becomes-music/13363668 with summary of:
“Molecular biologist Mark Temple from Western Sydney University who is also a musician has created a “sonification” of the coronavirus genome. With fellow musicians he presented his music at Sydney’s City Recital Hall and spoke with Tegan Taylor. This is a taste of his creations, with more next week.”
Mark Temple’s site and more on the music can be found at:
Thought this would be a nice break for fans of the show.
Enjoy!
David