new anti-HIV strategy: Progress report

marcos antezana marcos at uchicago.edu
Tue Jan 16 12:14:03 EST 2001


Dear Members of the Virology list

A month ago I posted in this list the outline of a new strategy to fight
HIV.  The strategy consisted in mutagenizing HIV genomes within virions
and in the cytoplasm as well as in inactivating the nuclear glycosylase
used by HIV to lower its C-to-U and G-to-A mutation rate during
reverse-transcription/DNA_double-stranding.  Meanwhile I have found out
that the glycosylase is already being targeted by some groups in ways
that however will most likely allow the virus to evolve resistance.

In the last weeks I have concentrated in exploring the potential of the
mutagenesis part of my proposal.  Below I present to you a reasoned plea
to start experiments to determine how much mutagen  would be needed to
inactivate HIV virions.  Furthermore I present a new view of why RNA
viruses mutate so quickly and why their reverse transcriptase is so
error prone.

But before that I would like to point out to you that last month it was
shown that one of the few drugs effective against viral hepatitis acts
by mutagenizing the virus (see below); and that simian AIDS can de
delayed indefinitely by keeping the viral load very low (below also).
Thus inactivating the virions through mutagenesis has the potential to
stop the progression to AIDS indefinitely.

Last, I would like to appeal to those of you that agree with the
rationale of my proposal below to write me:  I have contacted an HIV lab
that is ready to start experiments to measure HIV virion deactivation
through mutagenesis but the PI would like to know whether the community
of virologists at large would welcome such studies.

We`plan to measure the accumulation of mutations in HIV virions over
time and to determine how much mutagen one would need to inactivate
virions given plausible times of exposure to the mutagen.  We also plan
to study the time required for inactivation without any mutagen and how
making the medium acidic may enhance deactivation (C-to-U deamination
occurs faster in more acidic conditions).

Please write me and I will forward your comments.

Thanks and best to you all


   Dr. Marcos Antezana

   mantezan at midway.uchicago.edu
   marcos at uchicago.edu

////////////////////////////////////////  mutagenizing HIV virions
///////////////////

I am proposing studies to determine the dosage and exposure times
necessary to deactivate HIV virions with a polymerase-independent C-to U
deaminating mutagen, and then to determine if such dosages/exposures
would damage host cells and structures to any appreciable degree.

Given a certain mutagen dosage, damage to virion contents should be much
larger relative to that caused to host cells, because virions' have much
a higher surface to volume ratio.  Furthermore the nucleus has a battery
of extremely specific and active glycolsylases that remove Us from GU
mismatches so that nuclear DNA would be protected against the mutagen.

Deactivation through mutagenesis has the potential to become, in the
worst case, a last-resort form of chemotherapy for people who are not
responding anymore to available drugs and, in the best case, i.e. if it
can be accomplished with low toxicity, easily delivered compounds, *the*
solution to the problem of AIDS and a very cheap one at that, which is
especially important in order to fight the virus in developing
countries.

HIV deactivation through mutagenesis is very likely to be successful as
an anti-HIV strategy because i) there is much evidence that this can be
done in the laboratory and it was shown that one of the few drugs
effective against viral hepatitis acts by mutagenizing the virus; and
because ii)  there is overwhelming evidence that keeping low the virion
load can slow down the progression to human AIDS and recent evidence
shows that it can delay indefinitely the progression to simian AIDS.

Furthermore, when virions cannot infect or infect much less effectively,
viruses already within cells are condemned to kill their cells and
become extinguished even in sanctuaries.  To stick around, the virus
would have to become temperate.

Importantly, it is highly unlikely that HIV virions can evolve
resistance to polymerase-independent mutagenesis given their extremely
reduced enzymatic and structural homeostatic capabilities, and their
lacking evolutionary potential in this respect.  Resistance to
polymerase-independent mutagens would require evolving new
macroproperties (e.g. viral coatings, virion interior's biophysical
shifts), that would require more than simple point mutations in already
existing viral genes, the latter mutations at those at the basis of
viral resistance to current drugs.

HIV makes the big effort of recruiting the nuclear glycosylase to reduce
three to five times its C-to-U mutation rate during reverse
transcription and DNA double-stranding (L Mansky’s papers).  But the
time during which the glycosylase is used by HIV (around 1-2 hours) is
really short compared to the time during which new viral genomes can and
do mutate, i.e. the time between
viral_genome_transcription/virion_budding and new infection (one to a
few days if virion's average presence in the blood is an indication).
Thus the mutations affecting viral RNA genomes after RTion & integration
till they reach a new cell to infect, must be really many and those
caused by the RT may be insignificant (although the latter affect all
progeny genomes). This in fact explains the prevalence of G to A
transitions in viral mutagenesis studies (and also the discrepancy
between in vitro and in vivo RTion mutation production).  More
importantly, the huge losses of virions to deactivation through C-to-U
deamination could be the main reason why RNA viruses have to produce so
many virions, i.e. replicate so fast, which in turn requires them to
have compact genomes, i.e. delegate as much encoding of functions to the
nuclear genome as possible and encode only primitive proteins, e.g.
RTases:  Some not very highly mutated, decently infectious virions must
reach new cells to continue the viral cycle.

It is likely therefore that even a very slight increase in the
background mutation rate in the blood would do great damage to the viral
cycle of HIV by reducing drastically the number of infectious virions
that reach new cells.   The potential for an effect given a certain
mutagen dosage is much higher than when targeting viral nucleic acids
within the cell:  The exposure times are much longer and the virion's
very high surface to volume ratio should allow much more contact of the
mutagen with RNA genomes within virions than with cellular nucleic
acids.

There are three mutagens that have been proposed to me by local
geneticists:  nitrous acid, bisulfite, and hydroxylamine; they all
produce C-to-U deaminations.   Note that the quantities required are
likely to be very small since HIV as I understand is not heavily coated
unlike say phages.  Acidic conditions increase deamination rates so
acidifying the blood (while helping hemoglobin's oxygen delivery) could
be a "natural" way to mutagenize the virion by increasing spontaneous
deamination.  Moreover it has been shown that during transcription the
naked DNA minus strand is deaminated up to five times above the normal
rate, so one could target basic proteins that interact with the RNA
within virions also to let the nucleic acids’ own acidic nature increase
their own deamination (to this the virus could evolve resistance
however).

One should also test a non-C-to-U mutagen to check if  virions are
especially resistant to C-to-U mutations (they should be but possibly
only to physiological levels of deamination).  The nucleus has other
repair mechanisms so if the dosages required by non-C-to-U mutagen(s)
are much lower - hopefully to the point that most mutagen would be
neutralized in the cytoplasm - and have much lower general toxicity,
then one maybe should consider them rather than C-to-U ones.

Bisulfite is totally non-mutagenic on intact eukaryotic cells according
to local geneticists (possibly because the nuclear Uracyl glycosylase
protects them or because it is neutralized by the cytoplasm).  So its
real problem may be its general toxicity, whatever it means, but not
that it will give patients cancer or something.

My proposal is not a long shot since, as I already wrote above, much
literature has documented that i) viral infections can be extinguished
through mutagenesis (see below); and that ii) keeping low the virion
load delays the onset of AIDS.

Trying my proposal out would just cost killing a few cell cultures with
freshly-sampled, carefully mutagenized HIV virions until one can save
the cultures with a high enough mutagen dosage.  One should find out
however what is the likely time in vivo between virion budding and
infection.

Once inactivation dosages will be known, a toxicity lab could test how
damaging they are to host cells.

This studies should be paired with in-depth studies of the
physico-chemical conditions in the HIV virion's interior (I have not
found any literature about this and a few HIV-structure experts I have
contacted know of no works either).  The hope is that one can exploit a
physico-chemical peculiarity of virions to deliver and activate a
mutagen, an acid, or something else only inside them.

Finally, I have spent lots of time looking for reports about systematic
efforts to inactivate HIV virions with simple chemicals of low toxicity
but I could not find anything.  Maybe it's about time that money be
given to toxicologists and HIV culture experts so that they join forces
and start screening the millions of compounds, organic and not, that
have been described so far by mankind in order to find some that
inactivate the virions.  I bet that many among them will be effective
and some of them will have low toxicity  This may not be as elegant as
tampering with the molecular biology of the virus, but may prove more
effective and cheap and on top of everything if the effect would be
mediated by chemical rather than secondary-structural cues, viral
resistance may never arise.


///////////////////////////////  from Science december 2000
////////////////////

Enhanced: Preventing AIDS But Not HIV-1 Infection with a DNA Vaccine

Xuefei Shen and Robert F. Siliciano

Although there has been some success in treating human immunodeficiency
virus (HIV) patients with triple drug therapy
(highly active antiretroviral therapy or HAART), the best hope for
combating AIDS (the disease caused by HIV) could
be a combination of drug therapy and vaccination, according to Shen and
Siliciano in their Perspective. A new study in
rhesus monkeys treated with a DNA vaccine (Barouch et al.) demonstrates
that a powerful vaccine-induced CD8+
cytolytic T cell response reduces the amount of virus in the blood to
very low levels preventing the drastic decrease in
CD4+ T helper cells and subsequent immunodeficiency. As the Perspective
authors explain, vaccinating HIV patients
that are receiving HAART may enable HIV levels to be permanently brought
under control such that the drug treatment
can eventually be stopped.

/////////////////////////////   ////////////////////

>From a study published in the December issue of Nature Medicine;
Crotty's main co-investigators on the study were Craig Cameron, Ph.D.,
assistant professor of biochemistry and molecular biology at
Pennsylvania State University, and Raul Andino, Ph.D., UCSF associate
professor of microbiology and immunology.


A new study aimed at treating hepatitis C is suggesting that speeding up
a virus's ability to mutate could ultimately cause the virus to
overmutate and die off. University of California, San Francisco
researchers have discovered that one of the few drugs available to treat
hepatitis C, ribavirin, works by overwhelming the virus with a flood of
mutations. "That ribavirin destroys viruses by generating excess
mutations comes as a big surprise because viruses that the drug attacks,
those with RNA [ribonucleic acid] as their genetic material, usually
profit from their ability to mutate," said Shane Crotty, 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. "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," added Crotty. 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 a third of these cases. So what does the study mean for HIV? Could
the cure for HIV be as simple as forcing the virus to burn out by
overmutating? Crotty told the Bay Area Reporter that HIV presents a
unique challenge because although it's classified as an RNA virus, it
converts into its host's DNA. But Crotty said he is hopeful that other
scientists could use his study to develop new and unique strategies to
fight HIV. "I think now that we've shown that this strategy, this
mutationizing the virus, not only can work in a laboratory environment
but is actually the way the clinically used antiviral drug [ribavirin]
works, I do think that will excite drug companies to pursue that as an
avenue of anti-viral drug strategy, and hopefully even try that out
against HIV. I don't see why they shouldn't," said Crotty.











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