TOM SCHALL didn鈥檛 exactly jump for joy after analysing the stretch of DNA
he鈥檇 plucked from his dish of cultured immune cells. Schall, then a graduate
student at Stanford University in California, had hoped the genetic snippet
would be the first building block of his thesis. But the DNA seemed to be so
scrambled by the cloning process that it was hard to tell whether it contained
any intact genes at all. Colleagues suggested he drop the project.
Instead, Schall persevered, combed through the dross and found one gene of
unknown function. He dubbed his discovery RANTES, after a character in
the 1986 Argentine movie Man Facing Southeast. The cinematic Rantes is
the inhabitant of an insane asylum who claims to be from outer space. During the
film, he reveals powers that makes his origins hard to doubt. Schall hoped that
his RANTES would prove to be just as fascinating. 鈥淢ake no mistake,鈥� he
muses, 鈥渘ames are very powerful things.鈥�
That was ten years ago. This past year, RANTES has finally earned
its dramatic moniker. With a speed that has left AIDS researchers breathless,
the RANTES protein made by Schall鈥檚 gene鈥攖ogether with the receptor to
which it binds and a few related receptors鈥攈ave rocketed to the centre of
HIV research. 鈥淚 have never seen as intense progress in AIDS research before,
and it鈥檚 still going at breakneck speed,鈥� says Bob Doms, a virologist at the
University of Pennsylvania in Philadelphia.
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RANTES is one of a group of 鈥渃ome hither鈥� proteins called chemokines. These
are released at sites of damage in the body and bind to protein receptors on the
surface of immune cells; they lure the immune cells to the injured or diseased
tissue. The reason behind the present frenzy of interest is that chemokines have
emerged as the key to three mysteries about HIV-1, the main virus that causes
AIDS. Firstly, it turns out that chemokines shield some cells from the insidious
advances of HIV-1. Secondly, chemokine receptors are used by HIV-1 to infiltrate
cells and, thirdly, natural mutations of one chemokine receptor already protect
some people from infection. Today, researchers are exploiting these insights to
search for novel drugs and vaccines.
Not surprisingly, chemokine researchers now find themselves treasured
commodities, although they鈥檇 like to keep an identity separate from the HIV
research juggernaut. 鈥淲e鈥檝e known we鈥檝e been in a hot field even prior to the
time we began to mesh with the AIDS investigators,鈥� says Schall, who is now a
researcher at the DNAX Research Institute in Palo Alto, California.
The chemokine field stretches back a hundred years to the work of Russian
biologist Elie Metchnikoff. Many of Metchnikoff鈥檚 contemporaries believed that
immune cells were passive travellers, carried along by the blood. If that were
so, he realised, immune cells should not be able to travel in animals without a
circulation, such as starfish larvae. Yet when Metchnikoff jabbed a rose thorn
into the skin of a larva, the wound became a swollen traffic jam of immune
cells. The immune cells found their own way to the injury. Other researchers
reasoned that the cells must be following a specific trail of chemicals, which
were later dubbed chemoattractant cytokines or chemokines for short.
By the 1970s, scientists had purified the first few chemokines from blood.
The number accelerated when scientists such as Schall started turning the tools
of molecular biology to bear on the problem. The list of chemokines ballooned to
a few dozen, and soon they began to uncover the 鈥渘oses鈥� that immune cells use to
sniff out these proteins鈥攖he chemokine receptors.
Because of the important role played by chemokines and their receptors in
controlling inflammation, researchers hope they will point the way to drugs for
inflammatory diseases such as arthritis and asthma. Then, last December, AIDS
researchers Robert Gallo and Paolo Lusso of the University of Maryland,
Baltimore, made the first link between chemokines and HIV-1. They were searching
for an elusive substance secreted by some immune cells that prevents HIV-1 from
infecting neighbouring cells. The researchers purified three factors that seemed
to be responsible. To everyone鈥檚 surprise, all three were chemokines: RANTES,
and its less theatrically named cousins, the macrophage inflammatory proteins
MIP1-a and MIP1-b.
While intriguing, the appearance of a few more test-tube inhibitors for the
HIV-1 retrovirus did not start a research stampede. 鈥淚f you pee into a test tube
you can purify something that inhibits HIV,鈥� notes Schall. But Schall and a
small community of chemokine researchers knew from unpublished work that the set
of chemokines identified by the Maryland team all latched onto immune cells via
the same receptor鈥擟CR-5. 鈥淚t occurred to all of us instantly that this
receptor was involved,鈥� says Craig Gerard of the Harvard Medical School, another
member of the chemokine elite. 鈥淪o we all hooked up with our own retrovirologist
and started working.鈥�
Even if HIV-1 needed contact with a chemokine receptor, it didn鈥檛 tell the
researchers why. Then in May, Ed Berger and colleagues at the National Institute
of Allergy and Infectious Diseases near Washington DC put forward an intriguing
possibility. Like Gallo and Lusso, Berger wasn鈥檛 interested in chemokines per
se, he was investigating another mystery鈥攖he identity of HIV-1鈥檚 partner
in crime, which helps it break into cells. It has been known for more than a
decade that HIV-1 steals into immune cells with the help of a protein on its
surface called gp120. This locks on to a receptor called CD4 on the surface of
the immune cells. But some cells with CD4 aren鈥檛 susceptible to HIV-1 infection,
so researchers knew that there must be at least one unidentified 鈥渃ofactor鈥� to
make cells susceptible to HIV-1.
Twelve years of attempts failed to identify any cofactor, but Berger and his
team succeeded by devising an ingenious assay that allowed them to witness gp120
in action. First, rather than use the tiny HIV-1 virus which can only be
followed with powerful microscopes, they built an artificial virus by
engineering mouse cells to express gp120 on their surface. Next, they added a
marker gene that turned these cells blue when they fused with a target cell
bearing a partner marker gene. Then the researchers added this second marker
gene to mouse cells which expressed CD4 but were nevertheless impervious to
HIV-1. To these surrogate immune cells, they also added a random collection of
human genes鈥攖o see if one of them produced a cofactor.
Window on infection
They reasoned that if any of the surrogate immune cells expressed the mystery
human cofactor, then the artificial virus would fuse with them and turn blue.
The assay worked. From their blue cells the researchers isolated a gene they
called fusin鈥攚hich turned out to code for a formerly unidentified
chemokine receptor (Science, vol 272, p 873). 鈥淚 knew we had a new
window on understanding how the virus infiltrates the cell,鈥� says Berger. 鈥淏ut I
had no idea it would be as explosive as it鈥檚 been.鈥� Indeed, news of the
discovery leaked out and two other laboratories had duplicated the results even
before Berger鈥檚 paper had been published.
But the race for HIV-1 cofactors wasn鈥檛 over. Once it has infected an
individual, the virus mutates, creating other strains. Berger found that the
fusin receptor only bound well to strains of HIV-1 that are abundant late on in
an infection, the types that appear when symptoms of AIDS develop. But these
viruses don鈥檛 seem able to transmit the disease from person to person. This
suggested that at least one other receptor must provide a handhold for the
strains that establish infection. Because Gallo and Lusso had implicated the
chemokines that bind to CCR-5, this receptor became the chief suspect.
Many labs now began to study whether CCR-5 served as a handhold for the virus
during cell fusion. That race ended in a five-way tie of publications鈥攆rom
groups including Schall, Doms, Berger and Gerard鈥攁ll appearing in June
(Cell, vol 85, p 1135 and p 1149; Nature, vol 381, p 661 and p
667; Science, vol 272, p 1955). When the dust settled, it emerged that
not only fusin and CCR-5 but two other chemokine receptors, called CCR3 and
CCR2b, also serve as cofactors. The rumour among AIDS researchers is that by the
end of December yet more examples will be published.
To protect someone from HIV-1, then, it would appear necessary to block every
receptor because the virus binds to so many. But in practice, it seems that one
receptor is more important than the others. This point emerged from research by
Ned Landau and Richard Koup at the Aaron Diamond AIDS Research Center in New
York who were studying another puzzling fact about the disease. There are people
around the world who have been exposed many times to the deadly virus, yet
appear not to get infected. While studying two such people, the researchers
found that they both had the same defect in the CCR-5 receptor gene (
Cell vol 86, p 367).
Studies have since shown that the immune cells of people with two copies of
this genetic mutation do not express the CCR-5 receptor at all, and that the
mutation is relatively common in Caucasian populations. The Aaron Diamond group
and other researchers estimate that about 1 per cent of Caucasians have two
copies of the mutation, while nearly 20 per cent have one copy. Yet in more than
2000 people infected with HIV-1 who have been studied so far, not one has proved
to have the double mutation, though according to chance researchers would have
expected about 20 people to have it. This suggests that individuals without a
CCR-5 receptor are highly resistant to HIV-1 infection鈥攅ven if they have
functional versions of the other chemokine receptors that HIV-1 exploits. It
also implies CCR-5 plays a specialised role in transmitting and establishing
infections in a new host.
So far, the mutation in CCR-5 has not been found in African or Asian
populations, though some people from these continents are known to resist HIV-1
infection despite repeated exposures to it. They may have a different mutation
in the gene for CCR-5, or a mutation in another gene that interferes with the
process of infection.
If knocking out CCR-5 is enough to halt most HIV-1 transmissions, then drugs
that block the receptor could be powerful therapeutics. Unfortunately,
chemokines such as RANTES are poor candidates because when they bind to CCR-5,
they activate immune cells in such a way as to enhance replication of any virus
that has already infected the cell. This could speed up, rather than slow down
the disease process.
More likely, drugs companies will focus on compounds such as the trimmed down
version of RANTES recently tested by Marco Baggiolini of the University of Bern
in Switzerland and his colleagues. This mini-RANTES can block CCR-5 but does not
rev the motor of immune cells and can reduce the rate of viral infection by more
than 90 per cent (Nature, vol 383, p 400). Unfortunately, it also binds
other chemokine receptors, and so is probably unsuitable.
AIDS and chemokines researchers say they are already working with companies
to search for drugs that will latch on to CCR-5. Researchers are encouraged by
the fact that some of the world鈥檚 best-selling drugs鈥攑ropanolol for angina
and cimetidine for gastric ulcers鈥攂lock receptors of the same general
design as CCR-5.
Ideally, to design the perfect drug, chemists would like to know exactly how
HIV-1 docks with CCR-5. Last Month, Gerard and colleagues, and independently a
group led by John Moore at Aaron Diamond, reported that HIV-1 binds to the
receptor in a two-step process. When viral gp120 binds to CD4, the protein duo
twists into a new shape which resembles a chemokine and locks tight on to CCR-5
(Nature, vol 384, p 179). 鈥淭his virus waits until it is already
tethered on the surface of the immune cells to expose this new surface and
attack CCR-5,鈥� says Gerard. The last-minute metamorphosis probably gives the
immune system no chance to attack the new face of the virus, which may explain
why patients can鈥檛 fight off the infection. But if researchers can develop a
vaccine that focuses an immune attack on this ersatz chemokine, that might be
enough to neutralise the virus.
Dire consequences
Most AIDS researchers are excited about where these new discoveries may lead,
but they add a few caveats to the list of hopes. They anticipate that it will be
only a matter of time before strains of HIV-1 are identified which can use
chemokines other than CCR-5 to infect cells, so a single drug will probably not
treat all patients. And while knocking out the RANTES receptor may stop HIV-1,
it could also have dire immunological consequences that have yet to be
discovered.
Schall hastens to add that the implications of this research go beyond AIDS.
For example, scientists know that one species of the malaria parasite,
Plasmodium vivax, depends on a chemokine receptor called the Duffy antigen
to invade cells鈥攁nd people with mutations in that protein are resistant to
malaria. Also, new significance is being attached to the CCR-5 mutation that is
so common in Caucasian populations. 鈥淲hen you have one in five people with a
mutation, that speaks of profound selection for that mutation,鈥� says Gerard.
鈥淧erhaps it helped defeat another viral scourge.鈥�
Because the mutation is found only in Caucasians, evolutionary geneticists
believe that the proposed ancient pathogen must have attacked only after
Caucasians split from their Asian and African cousins. And because the mutation
is so common, it must have hit them soon after that split.
All this suggests to Schall that RANTES, the other chemokines and their
receptors have always been on the front line of the battle against disease.
鈥淣ature鈥檚 telling us that using chemokines and their receptors to beat off viral
predators is nothing new,鈥� says Schall. 鈥淚t鈥檚 part of a cat and mouse game that
humans and pathogens have been playing for eons.鈥�
