IF LARRY LOEB held a black belt in anything, it would be in molecular
biology, not judo. But like a martial arts expert, he’s discovered that the
secret of successful combat is not always strength. When Loeb goes into battle
in his lab at the University of Washington in Seattle, however, there are no
flying bodies and shattered glassware to worry about. His foe is a lot smaller,
more persistent and more deadly than any human adversary. It’s the AIDS
virus.
Ever since the AIDS epidemic burst onto the scene two decades ago, HIV has
proved difficult to control, largely because the virus mutates so rapidly that
it can often stay a step ahead of any drugs or vaccines doctors throw at it. But
if Loeb’s strategy works, the virus’s great strength may also prove to be its
downfall.
Just as a judo master uses the force of an opponent’s own blow to throw him,
Loeb thinks that doctors ought to stop fighting HIV’s mercurial behviour and
foster it instead. Boost the virus’s mutation rate just a little higher, he
suggests, and suddenly the advantage turns into a fatal flaw, leaving the viral
population so crippled by mutations that it fades away to nothing.
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Loeb and his colleagues certainly aren’t using the word “cure”. In fact,
they’ve yet to try their technique on anything bigger than a broth of cells. As
sceptics are quick to point out, many promising ideas perish on the long trek
from lab to doctor’s surgery. But if, by some combination of good instinct and
good luck, Loeb’s idea survives, it could herald a brand new approach to
controlling HIV.
All this molecular judo is possible because of a peculiarity in the way
populations of organisms react to a rising mutation rate. Almost three decades
ago, biophysicist Manfred Eigen of the Max Planck Institute for Biophysical
Chemistry in Göttingen showed mathematically that every replicating
system—such as a population of viruses infecting a host—has what he
termed an “error catastrophe threshold”, beyond which the population can no
longer reproduce successfully.
Think of all the HIV genomes in a person’s body as a collection of
manuscripts being diligently copied by generation after generation of monks who,
being only human, make errors now and then. As long as the monks make few
mistakes, each generation hands down enough error-free copies to transmit intact
the information held in the manuscripts.
Lost forever
But as the monks get sloppier in their copying, their successors have a
harder and harder time finding clean copies to transcribe. At some point, the
monks make so many errors that they can no longer produce even one clean
transcript, on average, from each clean copy they find, and the information in
the manuscripts is doomed to disappear. That’s the error catastrophe threshold.
“Once you cross it—and that’s a very sharp transition, like a true
physical phase transition—then you could say the information melts, or
evaporates, and you produce only erroneous information,” says Eigen.
As long as a virus population stays on the right side of the threshold,
mutations can be a good thing, because they provide populations with the raw
material they need to adapt to new conditions. “The higher the error rate, the
faster the system [or population] can adapt,” says Eigen. “So you want to work
close to the error threshold. And we found that the natural mutation rate in
viruses is very close to the error threshold.”
Loeb had heard these ideas many times at annual workshops organised by Eigen
since the mid-1980s, but he took the concept one step further. What if you could
somehow nudge up a virus’s mutation rate and push it over the edge? The
sharpness of the error threshold should mean that a fairly small change in
mutation rate should be rewarded by a much greater crash in viral numbers. And
he knew just the place to start his tests.
“If anything is going to be at the threshold of viability, it’s HIV,” says
Loeb. HIV normally suffers between 2 and 10 mutations each time it replicates,
and its compact genome and overlapping genes mean that they are very likely to
hit something important. Sure enough, when researchers sample the virus
particles in an infected person’s bloodstream, 90 to 95 per cent turn out to be
too defective to reproduce. The virus persists because the 5 to 10 per cent of
healthy viruses are fecund enough to keep the infection going. “It’s like a
herd. The population is able to survive even if very few members of the species
are capable of reproducing,” says James Mullins, a virologist also at the
University of Washington.
So Loeb and Mullins, together with John Essigmann, a chemist at the
Massachusetts Institute of Technology, decided to force that successful minority
to make more replication errors, in the hope of pushing the virus over the
threshold.
When HIV enters a host cell, its first step is to make a DNA copy of its RNA
genome (see Diagram).
During this process, individual DNA bases, of which
there are four kinds, (abbreviated as A, C, G and T) pair up with their matching
counterparts on the RNA strand and so form a new DNA strand. C always pairs up
with a G, G with C, T with A, and A with U, which is RNA’s equivalent of T. The
result is a DNA strand that exactly complements its RNA template.
Loeb’s team tampered with this orderly process by introducing a rogue element
to the cell—a non-standard type of DNA base called a base analogue. The
added base is structurally very similar to one of the normal bases and most of
the time it behaves just like it. But because its molecular structure differs
subtly, every now and then it pairs with the wrong partner. Each time that
happens, a new mutation is born. Some common AIDS drugs such as AZT are also
base analogues, but instead of causing mispairings, they simply shut down viral
replication altogether.
Of course, the goal is to introduce mutations into the virus’s genome but not
into the host cells, where they might harm the patient. Fortunately, there’s a
way to do that, at least most of the time. When the host cell copies its own
DNA, it uses an enzyme called DNA polymerase, which proof-reads as it goes, and
fastidiously clips out and replaces any analogues that find their way into the
DNA double helix. But when the virus copies its RNA into DNA, it uses its own
enzyme, reverse transcriptase, and these errors sometimes go unrepaired. So
Loeb’s team looked for base analogues that could evade repair during copying of
the virus’s genome but not the host’s.
An analogue called 5-hydroxydeoxycytidine (5-OH-dC) seemed to fit the bill.
Most of the time, this base acts like a normal C, but once in every 100 to 500
times, it mistakenly links up with an A instead of a G, roughly doubling the
usual mutation rate. The researchers have now screened a total of 14 base
analogues and have found two candidates that they hope will work even better
than 5-OH-dC.
Catastrophe strikes
With their new weapon in hand, the researchers set out to test Loeb’s idea.
They added 5-OH-dC to cultures of human T cells infected with HIV. Every few
days, they replenished the cultures by drawing off a standard quantity of
viruses and transferring them to fresh host cells. For the first 14 transfers,
nothing much happened—the virus continued to thrive. But then, on the 15th
transfer, catastrophe struck. Viral populations plummeted in all three cultures
containing the base analogue, and by the next transfer the virus had all but
vanished. Three comparable cultures without 5-OH-dC were still alive and well.
In a follow-up experiment, the virus effectively vanished after a series of
transfers in four of six analogue-treated cultures but in none of six
controls.
Promising as these initial results are, however, the researchers warn that
what they’ve dubbed “lethal mutagenesis” may not work as well in people as it
does in cell cultures. “A body is going to be a much more difficult target,”
says Mullins. The drug may turn out to have unacceptable side effects that
weren’t apparent in the cell cultures, for example. And indeed, the experiments
so far have used the drug at a concentration—1 millimole per
litre—far higher than drugs such as AZT can safely be given, notes Douglas
Richman, a medical virologist at the University of California at San Diego and
the San Diego Veterans Administration Medical Center. “As a practical clinician
I want to see more data and better activity to begin to think this is something
more than a laboratory curiosity,” he says.
Moreover, he notes, HIV is notorious for stashing away copies of its genome
in the chromosomes of host cells, where they can happily stay dormant for years
before reactivating. “This virus archives itself for years, so if you did
successfully clear the virus with this approach, the body could be reseeded,”
Richman says. And at least some of these reactivated viruses may come from early
generations, before many mutations had accumulated. Loeb agrees that lethal
mutagenesis is unlikely to cure AIDS completely because of this archive.
Instead, patients may be on drugs for the rest of their lives.
Still, Mullins and Loeb plan to take the next step later this year, testing
their approach in monkeys. If all goes well, they could begin tests in people
within two years. Their biotech company, Koronis Pharmaceuticals of Bellevue,
Washington, is already raising money to try the therapy commercially against HIV
and other viruses such as measles and hepatitis C.
Even so, Loeb doesn’t expect lethal mutagenesis to become the therapy of
choice for HIV, at least in the developed world, if only because the present
cocktail of drugs appears to work so well. But it could serve as a second line
of defence when the virus becomes resistant to the standard drugs or as an
add-on to the current mix. Indeed, Loeb’s experiments show that dosing his cell
cultures with 5-OH-dC increases the potency of AZT even before the virus
population reaches its error threshold.
But lethal mutagenesis may prove to be a much bigger boon for poorer
countries. The multi-drug cocktails now standard in Western countries cost
thousands of dollars a year, putting such treatment well beyond the means of
most of the world’s AIDS patients. “The problem is really overseas—Africa,
India and Thailand, and it’s growing in other places. We really do need
affordable therapies for the rest of the world,” says Essigmann. The base
analogues used today are much less expensive, so Essigmann suspects that the new
analogues such as 5-OH-dC will probably be relatively cheap, too.
Best of all, the virus may be less likely to evolve resistance to mutagenic
analogues than to today’s drugs. The virus population takes several generations
to accumulate enough new mutations to push it over the threshold, says Loeb. And
because the mutations quietly build up before the sudden catastrophic downfall,
the virus has little chance to adapt, by evolving a more selective enzyme, say.
New tricks
But even if the virus did find a way to resist, Loeb and Mullins have another
trick up their sleeves. They are now trying to tamper with the virus later in
the course of infection, when the DNA copy of its genome serves as a template
for many more RNA copies, which are then packaged into the viral protein coats
and packed off to infect other cells. To do this they would need mutagenic RNA
base analogues instead of DNA analogues.
The RNA copies are not made by the virus, but by one of the host cell’s own
enzymes, RNA polymerase—the same one responsible for making messenger RNA
as the first step in gene expression. That means the base analogues will end up
in the host’s mRNA as well as in the viral genome. But Loeb thinks that won’t be
a problem. A small proportion of aberrant mRNA ought to be like a few bad
photocopies in a stack—the original remains intact , so the bad copies
never multiply. But for the virus, those analogue-containing RNAs represent its
permanent genome, from which it hopes to make further copies, so they should
push it even closer to its error catastrophe threshold.
So far, no one has ever designed or used a mutagenic RNA base analogue, so no
one knows for sure how or whether they will work.”If we’re talking about really
prolonged, chronic therapy, I’d be sceptical that this would be non-toxic,” says
Richman. But if the RNA analogues do work, they offer a big advantage in the
ongoing battle against viral resistance. Because they are handled by the host’s
own enzymes, the virus has absolutely no control over them. “There’s no
conceivable way the virus could become resistant to the RNA analogues,” says
Loeb. Mullins agrees, though he takes a more cautious position: “We’re
optimistic, but we’re not so naive as to think it’s not possible.”
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Further reading:
Lethal mutagenesis of HIV with mutagenic nucleoside analogs
by Lawrence Loeb and others,
Proceedings of the National Academy of Sciences, vol 96, p 1492 (1999) -
Lethal mutagenesis of HIV by mutagenic ribonucleoside analogs
by Lawrence Loeb and James Mullins,
AIDS Research and Human Retroviruses, vol 16, p 1 (2000)