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grokking pi mutations - the A156T story

May 31, 2010 - 6 comments

Lots of discussion about resistance lately.  Not sure whether the following will help or further confuse the issue, but anyway...

To understand resistance mutations you have to dig out a bit of dusty high school biology. Genetic information (think blueprints) is encoded in DNA/RNA, long molecules made up of 4 types of building blocks (the bases, G,A,T,C).  To make life happen something needs to read those blueprints and make machines that will actually get something done (digest lunch, take in some oxygen, sex!). An amazing ancient bit of machinery called the ribosome has been doing that since the days of the primordial ooze in all living cells in essentially the same way. A ribosome reads blueprints and cranks out proteins, molecular machines which are also long chains but made out of 20 types of  building blocks (the amino acids alanine. valine, threonine, etc.). Once made,  proteins float around in a cell, bumping into other molecules and doing what they know how to do  - that's life.

OK enough theory, enter an evil HCV virion from stage right. It only has one goal -  make more virus. To do this it enters a cell, unwraps its protective shell, and lets its 9600-long RNA molecule float around the infected cell where it soon runs into a ribosome. The ribosome doesn't know an HCV RNA from a human RNA, reads the blueprint, makes the corresponding machine and calls out 'next'. The resulting viral machine is actually half a dozen machines all stuck together. So before the virus can get anything else done it has to separate these pieces, sort of like unpacking a crate. To do this it relies on 'proteases' proteins that cut up other proteins, starting with some human ones, already floating in the cell, and, after the unpacking has started, using a specialized cutting tool of  its own to finish the job. The protein it uses to finish the unpacking/cutting is called NS3A - the HCV protease.

Now enter telaprevir/boceprevir from stage left (accompanied by  a greek chorus of eager investors chanting 'inhibit the protease, inhibit the protease'). Unlike the long DNA/RNA or protein chains, these are small molecules - but with a very specific shape. They were carefully designed so that if, as they are floating about in the cell, they bump into an NS3A molecule, they will stick to a specific part of the viral protein. Sure enough, the part they stick to is the very part of the molecule essential to get its job done -  think monkey wrench in the transmission. If the protease can't finish its unpacking job the rest of the viral machinery stays in the crate and the  viral life cycle (making copies of the viral RNA and of the protective wrapper proteins,  assembling the parts to make up fresh new virions and shipping them out of the infected cell) all comes to a screeching halt.

But... the evil virus is wily. It has discovered that a good way to survive is to try lots of different approaches - a variation on the theme of try and try again. So the 9600 bases in the HCV genome are not exactly the same in every virion, and as a result the NS3A proteases are not all the same either. For example, let's look at the A156T virions. These are virions whose RNA is such that their NS3A protein has the amino acid threonine (T) at position 156 instead of the alanine that most healthy virus carry at that spot. Up to now this has been a problem for the poor A156T virions and their descendants. The NS3A protein they produce is not as effective as that of healthy (wild type) virus, and consequently there's a lot fewer of them. The A156T tribe may be less fit, but their 15 minutes of fame has arrived and they are now taking to the spotlight with a vengeance. In cells with boce or tela floating around, A156T virus can still reproduce - but no wild-type HCV virus can. Did tela or boce cause the mutation? - no it was there all along but the drugs screened out everything else so the A156T (or similar mutations) are the only ones left standing.

At this point it may help to look at the image posted in my photographs list. The telaprevir molecule is shown in magenta. It's bound to the viral NS3A protease  most of which is shown in gray  in a summary shorthand ( with loops, arrows and helices) used to represent protein structure. The parts of  NS3A where mutation can interfere with tela/boce are in color (green, yellow, pink, etc.) Note that it's easy to see why position 156 is so crucial - it's right at the core of where tela sticks to the protease - make a change there and tela would just float off instead of binding. In fact the A156T mutation is one of the strongest in disabling tela effectiveness.

Questions for homework : what killed off the A156T virus in Jack who SVR'ed ? (hint think soc) What happened to the mix of A156T and wild-type virus in Jill who relapsed ? (hint remember A156T's fitness problem)

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Avatar universal
by mikesimon, Jun 01, 2010
Thanks so much willing.

148588 tn?1465782409
by desrt, Jun 01, 2010
Nice pic. I think I've got an extension cord on my truck that needs tela'. Much grokking needed till fullness is on my part.

Avatar universal
by cookie2554, Jul 06, 2010
Please see my comment attached to the photo. thanks,Cookie2554.

233616 tn?1312790796
by merryBe, Jul 09, 2010
for JAck, I would hazard a guess that maybe the riba is making the a156T less able to stick by causing more mutations by interfering with protein synthesis, or by interfering with transcription, one or the other, if not both.
but with no microbiology in my backgound, that's all it would be...a guess, based on Crotty's research into polio perhaps a good guess..

for Jill, I'd wonder if 156T and the wild child had a little midnight that's a scary thought!!

speaking of good old T....what do you think of the branch chained amino acids for liver supplemention then. It's a little confusing to me and I could use a primer in why they work or why certain other proteins should be avoided.
I wish someone with the background would explain it to we the wee people.  ; )

thanks for the journal entry...a good layman read really...every little bit like this helps.

Avatar universal
by spectda, Jul 16, 2010
Great explanation-Thank you for making it simple

233616 tn?1312790796
by merryBe, Nov 07, 2010
hey I forgot we started this conversation...shall we continue...

so I guess I guess right because look what I just found, old article but it answers the Jack part....
I'm still stumped as to Jill however...are you saying the 156 is all thats left or that it reverted to wild type.
It wouldn't surprise me if were the latter...but you tell me???


One of the few drugs available to treat hepatitis C has been shown to kill viruses by generating a flood of new mutations that overwhelm the virus - a mechanism known as error catastrophe, according to a new study from researchers at University of California, San Francisco. The newly discovered mechanism for the drug ribavirin should help pharmaceutical companies to create more effective versions of the drug to cure a larger proportion of hepatitis C patients.

That ribavirin destroys viruses by generating excess mutations comes as a big surprise because viruses that the drug attacks, those with RNA as their genetic material, usually profit from their ability to mutate, said Shane Crotty, BS, a graduate student in UCSF's department of microbiology and immunology.

RNA viruses like HIV and the influenza virus use a naturally high mutation rate to avoid and escape most treatments and vaccines, Crotty explained. "These viruses are incredibly clever. They use mutations to get around almost anything. But we now see that ribavirin adds so many extra mutations to the virus, that it is pushed into a kind of genetic meltdown," he said.

Crotty's main co-investigators on the study were Craig Cameron, PhD, assistant professor of biochemistry and molecular biology at Pennsylvania State University, and Raul Andino, PhD, UCSF associate professor of microbiology and immunology.

Previously, researchers had suggested that ribavirin kills viruses by blocking an enzyme necessary to prepare the genetic subunits, known as nucleotides, which the virus uses to copy its genome, Crotty said. But this idea was never very convincing, because more potent inhibitors of this same enzyme were not effective against the same viruses, he said.

The seed for the new experiments, which are published in the December issue of Nature Medicine, came from the demonstration by Penn State's Cameron that the ribavirin molecule can take the place of a nucleotide, meaning a virus would mistakenly insert ribavirin into newly formed copies of its RNA genome, Crotty said.

Crotty and his colleagues then found that this insertion of ribavirin into the virus' RNA created genetic mutations in a culture of the virus growing in cells in a petri dish. The more drug they added, the more mutations were generated, and as the number of mutations increased, fewer and fewer viruses survived, Crotty said. At the highest ribavirin concentrations tested, only one out of every ten million virus particles survived.

The idea of error catastrophe is not new, Crotty said, but this is the first time it has been demonstrated as a mechanism for a working drug.

Two pharmaceutical companies have already begun working with the study findings to develop a more effective form of ribavirin. Currently, ribavirin is prescribed together with the immune boosting drug interferon-alpha for patients who have hepatitis C, but it cures only about one-third of these cases.

The drug is also used to treat severe infections of newborns with the respiratory syncytial virus, which is the most common infection of the lower respiratory tract in children.

The companies will be searching for ribavirin-like molecules that generate mutations, but that also have different chemical features that could boost their effectiveness against hepatitis C, Crotty said. And because ribavirin's mechanism is unique among anti-viral drugs, companies will be searching for similar drugs that might work against other RNA viruses, Crotty said. "This paper shows that mutagenesis is a valuable anti-viral drug strategy. And it's reasonable to assume that there are other drugs like this out there, but until now we haven't known the right way to look for them," he said.

Other investigators on the study were David Maag and Jamie Arnold, both graduate students in the department of biochemistry and molecular biology at Pennsylvania State University; Weidong Zhong, PhD, Johnson Lau, MD, and Zhi Hong, PhD, all from Schering-Plough Research Institute.

This study was supported by grants from the National Cancer Institute, the National Institute of Allergy and Infectious Disease. Shane Crotty is a Howard Hughes Medical Institute doctoral fellow.

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