Genotype and mutation

[milker]

Hi all,

can someone explain what exactly is the genotype, that is, what is being checked, parts or the RNA strand or? And when we're talking about mutation, what is changing? Are the protein markers changing in any way or is it the RNA or both ?

Milker.

[Miss Philicia]

Well, I'm not the resident expert on such medical stuff, but mutations refers to I think "point mutations" which are the places/mechanisms that each different HIV medication attacks the virus.  Genotype and phenotype tests evalutate these point mutations to measure each individuals drug resistance.  Genotype measures codon changes that differ from the standard virus. Codon is a sequence of three nucleotides which form a unit of genetic code in a DNA or RNA molecule.  Phenotypic tests provide information about the relative amount of antiretroviral drug required to suppress replication of the patient virus, compared to the amount of drug needed to supress replication of a laboratory strain of virus

[milker]

 

Ok I have to do more research I didn't understand anything about what you said 

Milker (who will do more research and hopefully understand Philly)

[gerry]

Hi Milker,

Unfortunately, this is not really an easy thing to explain.  It would help to understand this process by first understanding what happens during the HIV life cycle.  Hopkins has an animated version of this: http://hopkins-aids.edu/hiv_lifecycle/hivcycle_txt.html

Genetic material (DNA and RNA are basically coded information that gets translated into amino acids which are the building blocks for proteins.  The usual sequence is DNA is transcribed to RNA and RNA is translated into amino acids.  The DNA and RNA sequence determines the amino acid sequence which in turn when linked together determines the type of protein produced.  Enzymes are basically proteins that perform specific chemical reactions.

With HIV (being a retrovirus, meaning its genetic material is RNA, rather than DNA), there is an extra step required to convert its RNA to DNA after it enters the host cell (for simplicity, lets assume its a T-cell, but we know there are other target cells).  To do that, it packs an enzyme (protein) called reverse transcriptase which was produced before the virus budded off the T-cell.  So once inside the cell, the reverse transcriptase does its work to convert the HIV RNA into DNA.  Once its converted to DNA, this can enter the cell and integrate into the T-cell, using another enzyme it packs called integrase.  Once incorporated into the T-cell DNA, it generally takes over the cell machinery to reproduce viral RNA.  This viral RNA then gets translated into proteins, including enzymes.  One of the enzymes is protease, which is in charge of breaking some newly manufactured viral proteins, including other enzymes (e.g., reverse transcriptase, integrase) which the new virus would need to restart the cycle when it binds to another uninfected T-cell.

The codons Philly mentioned are a series of 3 codes in the DNA and corresponding RNA that code for specific amino acids (the protein building blocks).  There are 4 different nucleotides (A, T, G, U) that are the building blocks for the genetic material, and any sequence of three generally (but not always) code for a specific amino acid. 

Point mutations causing resistance happen in the genetic material level.  However, because this genetic material is used to code for specific proteins, then the alteration in the genetic code gets translated into alterations in the protein (including enzyme) structure and function.  These alterations are the ones that enable the virus to resist the  actions of certain drugs. 

Currently, genotype testing is done for the reverse transcriptase gene and protease gene routinely.  It can also be done to investigate Fuzeon resistance but that is a test that is specifically ordered separately.

Genotype nomenclature starts with the letter designating the amino acid found in a "normal" or "wild type" virus, then the codon number or position where this happened, then the letter designating the amino acid found in the "mutant" virus.  Therefore, the M184V mutation that confers resistance to the NRTI lamivudine and emtricitabine means that the normal amino acid "methionine" in the reverse transcriptase gene at position (codon) 184 has been replaced with the amino acid "valine."

This is a long answer to your question.  But the shorter version is, both the genetic material (RNA) and the protein are altered, but that's mainly because the genetic material codes for the protein.

Hope that helps.

Gerry

[sdcabincrew74]

The quick and dirty ... genotype tests what kind of drugs will work with the virus that is in your body.  There are "mutations" or differences in the virus and everyone has some sort of "differences" or mutations.  The goal of the test is determine the virus you have and what meds will work against it.

In a perfect world, all meds will work on your virus and that is the case with most, my doc said, 80% of new infections.  About 15% according to him, in the San Diego area, are resistant to the class of drugs that contain sustiva.  The other 5% are resistant to more than one class of drugs. 

I am in the 15% group, no biggy, I take Reyetaz and Truvada instead of Sustiva and Truvada.

[milker]

Woah Gerry this is fascinating. Thanks for the explanation, I re-read philly's post and combined with yours I get it now (I had to read very very slowly). Do we know if external sources like nutrition, lifestyle, health, other illnesses have an effect on mutation, or is it just "dumb luck" that those alterations happen?

Thanks,

Milker.

[aztecan]

Hey Milker,

Well, sort of "dumb luck," with a dash of adherence issues thrown in for spice. HIV mutates so fast and makes so many copies resistance may be inevitable. Fact is, many of the copies HIV makes are untenable and don't survive simply because they are so poorly formed.

Sloppy adherence can accentuate this problem, because it allows strains of virus resitant to a particular med to replicate. The more resistant virus, the greater the chance of a viral breakthrough and drug resistance.

These days, people are being infected with already resistant strains, as SDCABIN alluded to. The non-nukes are particularly easy to develp resistance to for some reason.

I have never heard of nutrition or other outside factors helping to prevent resistance.

HUGS,

Mark

[JPinLA]

Hey Milker -

Everyone has provided a wonderful explanation of genotypes, phenotypes and point mutations relating to HIV drug resistance so hopefully you have a good understanding.  This type of thing (i.e. genetic mutations relating to drug effectiveness) is one of the areas of research i do in the biotech industry and is extremely prominent in drug development (not just antiretroviral but all drug development).  In any case, I'd like to add to Mark's post by saying that just by replication alone mutations will happen.  The replication process and the checks and balances that go along with it are imperfect and small mutations arise.  Thus, if the virus is allowed to replicate uncontrolled it will naturally mutate into different genotypic and thus phenotypic strains.   

I'm not too sure how fast this happens but it does and as Mark pointed out that inconsistent adherence to medications (once you begin) exacerbates the mutation process. 

I think of it kind of like natural diversity and evolution.  Diversity happened naturally through imperfect reproduction (replication) and evolution happens when characteristics are pushed or favored by the presence of a selection pressure.  In this case, medications are the selection pressures and without constant presence, the virus can find a way to survive if they are allowed.

It is entirely possible that there are other elements aside form drugs that may help or hinder the mutation process leading to drug resistance.  This could be diet (for instance the ECGC in Green Tea has been suggested to prevent HIV from binding to CD4 cells) or some other factor.

Anyway, that's my two cents.

JP

[gerry]

Hello Milker:

Mutations happen very commonly in HIV as the virus replicates because the reverse transcriptase enzyme that converts viral RNA to DNA in the host cell cannot “proofread” what it does; this gives rise to so-called “quasi-species” or variants of the original HIV strain.  This process gets magnified because HIV replicates very rapidly.  Some of the variants are viable and some are not.  The strain that predominates eventually depends on the fitness of the virus relative to the immune defenses of the host. 

In acute infection, the variability of the strain is limited; in chronic infection, this variability expands and this continues to do so as the infection progresses, even in the relatively long period in which symptoms are minimal or absent.  It is believed that the development of quasispeces has an adverse impact on the ability of the immune system to contain the virus.  This likewise makes it much more complicated to design a vaccine against HIV.

While the occurrence of random mutations are the general rule in HIV, these mutations in the absence of drug pressure do not lead to drug resistance.  The evolution of drug resistant mutations generally happens when viral replication continues to occur significantly in the presence of drug pressure from antiretrovirals.  There are several factors that lead to the development of drug resistant mutations (excluding transmitted resistance):
•   Inadequate number of drugs used to suppress the virus
•   Inadequate potency of the antiretroviral combination used to suppress the virus
•   Inadequate absorption of the drug(s)
•   Pharmacokinetic factors that may lead to inadequate drug levels achieved, or to potentially negative drug-drug interactions
•   Adherence problems

Any or all of these may play a role in development of drug resistant mutations and each one merits a separate discussion.  While adherence is emphasized and it is one aspect that is under the control of the individual (even though that in itself is influenced by factors that may be beyond the individual’s control, most notably, side effect issues), it is not the only factor that comes to play.

It’s tempting to go into these in more detail but that will make the post too long.  Hope this helps.

Gerry

[milker]

While the occurrence of random mutations are the general rule in HIV, these mutations in the absence of drug pressure do not lead to drug resistance.  The evolution of drug resistant mutations generally happens when viral replication continues to occur significantly in the presence of drug pressure from antiretrovirals. 

Thanks Gerry I didn't know that, but after reading documentation on how the virus replicates itself, I can understand that the places where the drugs attack are most likely to be affected by a protein change where as other changes in the shape are of no interest for the drugs anyway.

Milker.

[racingmind]

Milker,

    You have been asking a lot of "technical" questions lately....does this mean that the reality of the situation is finally sinking in? 

     A while back you were admittedly "happy-go-lucky" after diagnosis and concerned about it. Just wondering because the recent questions you are asking the forum are more of what one would expect from someone recently diagnosed. 

[milker]

I'm still a happy chappy  ! I'm asking all of those technical questions because i'm fascinated by this, and I want to UNDERSTAND how all this works, for three reasons:

1) I educate myself and I can educate others. If I don't understand the topic then I can't help others.
2) Being educated should help me understand my doctor better, and have him be confident that he can go technical. The more details I get the better it is.
3) If I have to take meds, I want to understand how this works, what i'm taking, why, and the side effects I should expect.

Knowledge is power, so i'll be asking more questions!

I don't think it's hilarious anymore, but I do not feel any of the symptoms of depression that I have seen from other newbies, I'm not scared, I don't think about death at all, which is good. My lifestyle hasn't changed at all, i still smoke and drink too much, that's for sure, and I'm working on it.

Milker.

[ndrew]

hi.

I find this all fascinating as well.  In fact, the more I am exposed to this stuff, the more it starts to make sense.  It's also kinda weird that all of this amazing biotechnology is happening in my body.  I am assuming some of this understanding is from recent research.

Drew

[gerry]

Milker:

I had wanted to respond to your other question in a related thread but I'm posting the response in this thread instead regarding phenotype and genotype:

So the HIV phenotypic transformation is harder to find because it could just be few amino-acids that changed place? I initially thought that phenotype was a faster exam?

Genotype tests are cheaper and easier to perform than phenotype tests.  As already mentioned, genotype tests measure the presence of specific point mutations which would usually (but not always) predict resistance to certain antiretroviral drugs.  Basically, the process (PCR) involves making many copies (“amplification”) of the HIV genome and then analyzing for the presence of certain mutations (genotypes) in the reverse transcriptase and protease genes. 

Phenotype tests, on the other hand, are more complicated, more expensive and take longer to perform.  It measures directly the ability of specific concentrations of individual antiretroviral drugs to suppress a person’s virus from multiplying and compares this to how a wild type virus would behave. 

Because of the high costs of traditional phenotype tests, one company developed what’s called the virtual phenotype.  It attempts to predict the phenotype from a genotype result by comparing it to a large database of historical genotype-phenotype results; thus, a virtual phenotype is not really a phenotype test (trivia!).

There are some genotypes that are quite easy to interpret in the sense that their presence always signifies resistance to certain antiretroviral drugs.  For instance, when analyzing the reverse transcriptase gene, an M184V genotype would indicate the presence of a mutant that is completely resistance to 3TC and FTC; a K103N genotype would indicate the presence of a mutant that is completely resistant to efavirenz, viramune and delavirdine. 

Not all resistance is complete, though.  For instance, the L74V genotype can cause 2-3 fold resistance to abacavir and 2-5 fold resistance to ddI.  K65R can cause intermediate resistance to tenofovir, abacavir and ddI.  In these situations, since resistance may not be complete, it would be useful to have a phenotype test as well if one wants to determine if any of these drugs still has any amount of suppressive activity left against the virus. 

The phenotype test is also useful in situations in which there is a complex combination of genotype mutations, especially in highly treatment-experienced individuals, the presence of which might cause cross-resistance to a number of antiretrovirals in the same class.  For instance, mutations in positions 41, 67, 70, 210, 215, and 219 of the reverse transcriptase gene (called TAMs) can occur sequentially in someone who remains in a failing regimen containing AZT or d4T.  The longer one remains in a failing regimen that contains AZT and d4T, the more TAMs accumulate.  This is important because not only do these cause resistance to AZT and d4T; when present in certain numbers and combinations, they also cause cross resistance to the other NRTIs abacavir, tenofovir and ddI, even if they have not been used.  So when TAMs are present (or any other complex sets of NRTI genotypes), it is useful to get a phenotype test as well to find out which NRTIs have been adversely affected through cross-resistance.

Phenotype testing is also useful when investigating PI resistance.   With most PIs, it actually takes several mutations (genotypes) to occur before complete resistance to the PI happens (they have the so-called “high genetic barrier to resistance”), especially with the more potent PIs and the advent of ritonavir boosting.  So it’s good to have phenotype tests in these situation, especially in treatment-experienced individuals because the genotype(s) when present may not necessarily predict the actual degree of PI resistance.

When performing baseline resistance tests prior to initiation of treatment, though, genotype testing is generally sufficient because it is mainly intended to rule-out the presence of pre-existing (transmitted) resistance.

Gerry

P.S. It's good to ask questions 

[milker]

Thanks Gerry for posting this information in this thread. I was under the initial impression that the phenotype was a shape test, where different parts of the virus that were going to be attacked should have an expected shape, and if not, the drug wouldn't work. So I was more thinking of a "visual test" where as the genotype test would check for specific glycoproteins. I'm slowly getting it

Thanks so much for taking time for explaining all this, Gerry, I truly appreciate it.

Milker.