Friday, January 12, 2007

Drug Resistance

It's been a while since I've felt the need to post much new. Dissident debate is so stagnant that it's difficult to see anything original come to light.

A recent thread opening up on AME however reminded me about the problems there that they have with censoring any kind of intelligent poster (i.e. someone educated in virology). The questions arose - how to we know HIV is drug-resistant in some people with a test, and how can a virus acquire drug resistance anyway, when that's only possible in living organisms like bacteria?

The questions underline the biologic ignorance that is fundamental to misunderstanding much of HIV and AIDS. The saddest thing is that these people largely refuse to be educated.

Let's start simple - what is a virus. A virus is basically a collection of nucleic acid (be it RNA or DNA, it doesn't really matter) which is encased in a protein shell, and those proteins are encoded by the same nucleic acid they encapsulate. Viruses may or may not also possess an envelope. Again, it doesn't really matter. The only differences that the RNA/DNA or the enveloped/non-enveloped structure cause are subtleties in the replication cycle of the virus. The bottom line is that the protein is protecting nucleic acid that is able to produce more of the same protein.

So, since they're fundamentally only protein and nucleic acid, viruses obviously lack one of the key functions necessary to perform biologic activity. Namely, the ability to create energy.

Biology is really nothing more than chemistry with a purpose - you can boil down all biologic reactions to chemistry, and indeed all chemical reactions to atomic physics if you really want to go all out! The trouble is that the natural order of the universe is chaos - the second law of thermodynamics! Biologic organisms however are inherently ordered - fat molecules are lined up, proteins fold in certain ways, cells contain distinct organelles and bodies are made up of distinct organs. In order to create this order, energy must be put into the system.

Viruses lack this ability - and so they can only replicate if they can steal some from another living organism, a cellular organism that has it's own energy-producing machinery. As such, viruses are obligate parasites (meaning they HAVE to be parasitic). In some respects they might be thought of as sea monkeys - only "coming to life" when you add the magic ingredient.

Now obviously they are additions to this simple definition. Some viruses can produce additional DNA/RNA building blocks for example, others need to steal these also. Some will only infect dividing cells (which are mass-producing the biologic building blocks) and others will force non-dividing cells to start dividing. Some viruses replicate in the cell cytoplasm, others in the nucleus. They all follow similar themes however. Ultimately a virus must bind to a cell, get into the cell, unpackage it's genome, produce new proteins and new progeny genomes, package new viruses together, and get out of the cell. The patterns of replication that often define a virus species (certainly that's how viruses used to be defined, which is why HIV for example was originally called a type-C retrovirus) will depend on the structure of the virus and the additional proteins the virus encodes.

Moving on - how can a virus acquire drug resistance. Well, the real question should be how can they NOT acquire drug resistance! I think the trouble starts with people thinking that drug resistance is some kind of pseudo-intelligent decision made by a living organism to adjust to its environment. That's plainly rubbish. Bacteria and viruses can no more decide to become drug-resistant than you or I can decide to grow wings and fly. It's simply not a "decision" we can make. What CAN happen however is that certain genetic elements can be created by accident which might result in a different set of proteins or a modification of the existing set of proteins. The 'superbug' MRSA for example (Methicillin resistant Staphylococcus Aureas, a nasty multi-drug resistant bacteria) becomes resistant from the insertion of 'new' genetic elements into its genome. Now, I'm not saying that the genetic elements are literally "new", that SA suddenly creates a whole new set of genes to become MRSA, but in the context in which they cause a problem for us they are new. What most likely happened is that these genes envolved over millions of years in bacteria to protect them against toxins produced by fungi, viruses, or other bacteria. When we come in with our antibiotics (which are usually derived from natural compounds!) we cannot be surprised if mother-nature shows us what is up her sleeve.

Another example is the growing number of streptococcal infections that are drug resistant - in this situation the protein that is exploited by us to carry the toxin penicillin into the bacteria mutates subtly so it doesn't bind penicillin very well. Sure, they means that the bacteria has a slightly dodgy protein now - but faced with using a less-than-perfect protein or being dead, most bacterial colonies will end up containing an awful lot of less-than-perfect bacteria. It's plain and simple Darwinian Evolution.

So when we talk about the "spread" or "creation" bacterial drug-resistance, we are really only talking about the selection of pre-existing drug-resistance strains from a mixed population. It's a little like using a magnet to pull out iron filings from sand. If you kill off all the drug-susceptible bacteria then the drug-resistant ones will survive and grow to replace them, but it will appear to human eyes as if the population of bacteria somehow "acquired" or "developed" drug resistance. The bottom line - drug resistance was probably there all along, but we've just given the resistant bugs a chance to out-compete their cousins.

Here's a visual representation: x's are normal, o's are resistant.

xxxxxxxxxxxxxoxxxxxxxxxxxxxx = normal population

o = population after antibiotics

ooooooooooooooooooooooooooooooo = drug-resistant population ("acquired" drug resistance)

Viruses have one advantage and one disadvantage when it comes to drug-resistance, compared to bacteria. Firstly a single virus can create many hundreds or thousands of progeny virions. So their replication copy-number is huge. You have lots of room for error, because so long as you get a few working models the species stands a chance of survival. The disadvantage lies in that virus genomes are incredibly tightly packed - there isn't much room to add new, potentially advantageous genes. If a virus capsid is big enough to hold 10,000 bases of DNA, you can't expect it to work very well with an extra 5,000 bases of drug-resistant gene inserted! As a whole viruses are incredibly efficient with their nucleic acid - they have very little if any junk sequence for example (we on the other hand are 90% junk DNA) and may even use the same sequence to code for 2 or 3 different proteins! As such, viruses are typically forced to use simple point mutations, small deletions, or other minor changes in order to have any kind of genetic change. You're basically creating a vast batch of new viruses, and rolling the dice each time.

Aside from having large number of progeny, viruses are also inherently error-prone. RNA viruses in particular have no error-correction mechanisms in place. They rely on the base-pair matching between two strands of RNA, but if a mistake is made they have little to no recourse to correct it. Our own DNA replication on the other hand makes use of a veritable army of proteins to detect and repair DNA mismatches. Some DNA viruses may make use of this machinery as well of course, but HIV is an RNA virus.

The protein HIV uses to replicate itself (a cellular protein called RNA polymerase II) has an error rate of 1 in 10,000. HIV is around 10,000 bases long. So, on average, each HIV genome will contain one error - one deviation from the original template. HIV also uses its own protein reverse-transcriptase early in the life cycle, and that too has no error-correction mechanism. All in all this is a superb setup for creating a mixed population of viruses, of which some MIGHT, by chance, be drug-resistant. In addition, this error rate explains why many virus particles are non-functional -they are defunct genetically. It's important however to note that certain changes are better tolerated that others. A change to the cellular binding protein is very poorly tolerated (if the virus gets into the wrong cell type, or no cell at all, it's stuffed!) which is why antibodies to things like specific portions of the Env protein are consistently found in people with HIV. On the other hand subtle structural changes in the scaffold of the RT enzyme can be better tolerated to avoid binding to chemical X while still allowing chemical Y to bind (which is basically how mutations in RT exist to prevent drug binding while still allowing replication to go ahead). The alleged "inconsistencies" in the biology of HIV touted by the dissidents are very simply explained by the constraints of biology and Darwinian selection.

In the case of HIV, drug resistance is very well understood. For example, just like the penicillin-binding-proteins in strep, HIV protease and reverse-transcriptase can become modified so that it doesn't bind to the protease or RT inhibitors. Some of these mutations are very specific. The first mutation described, to AZT, was in a particular portion of RT. If you then exposed the virus (in tissue-culture) to a different RTI then this mutation reverted back to wild-type and a new mutation appeared. It seemed as if the virus could easily cope with one mutation or another, but not both. This was the basis behind using more than one drug at a time to combat HIV - and it worked. The virus took much longer on average to adapt to resisting two drugs that just one alone. Remember that this is all down to chance - and, on average, it will take longer to roll "all sixes" with 4 dice than with 2, right?

And that's assuming all things being equal - if successful, antiviral therapy can shut down replication and therefore should slow down the rise of resistant strains - rolling the dice every few minutes say, instead of every few seconds.

The beauty now with modern HIV care is that so many papers have been published linking specific mutations found in the HIV genome with drug-resistance. We can say with a large degree of accuracy, that if a virus contains mutation X it will be resistant to drug B. Some tests still rely on actually trying to grow a patient's virus in the presence of various drugs (after all, we can't yet know ALL the possible resistance mutations and combinations!) but genotype testing, as it's called, is extremely useful.

And this is an important point missed by the dissidents (no doubt because, as this AME thread shows, many of them simply don't understand the fundamentals). If HIV were a crock-of-shit, if the drugs really weren't attacking it, if the drugs weren't shutting down viral replication, how on earth do they explain the rise of specific mutations in response to exposure to a drug that are associated with improved viral replication in culture, AND as judged by viral load and CD4 count in real living people. The model is its own control - the scientists are not speculating that the virus is mutating around the drugs, they are seeing it with consistent, reproducible, predictable changes in the genetic code of the virus associated with real laboratory and clinical data. Genetic changes are inherently random - any kind of pattern is meaningful.

And this is the basis behind this new test the thread was kicked off by. If you look using sensitive techniques for drug-resistance genetic sequences, you will find even the rare viruses in the population that are, by luck, rolling sixes. You can therefore save the effort of killing off the non-resistant strains only to have the resistant ones pop up - you can select a different set of drugs that will attack all of them.

So, that is how a virus can acquire drug resistance, how you can test for it, and why it's meaningful in the case of HIV. It's also, incidentally, one part of the irrefutable science proving that HIV causes AIDS.


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