http://www.advocate.com/new_news.asp?ID=7231&sd=12/17/02

 Scientists photograph HIV’s path into human cells

Researchers at the University of Chicago, using fluorescent proteins
and time-lapse photography, have documented for the first time HIV’s
slow path in penetrating human immune cells, BBC News reports. The
pictures offer proof that the virus uses natural cellular machinery to
move from the cell membrane into the nucleus. HIV was shown to attach
to a cell protein called dyenin to travel through a series of tiny
microtubules from the cell membrane into the center of the cell. The
microtubules lead all the way to the cell’s nucleus where HIV inserts
its own genetic material, effectively turning the cell into a factory
that will make copies of the virus.

Time-lapse photographs were taken under the microscope at 15-second
intervals to reveal the virus’s steady–but sometimes
haphazard–progress toward the center of the cell. "They don’t make a
beeline for the nucleus," said Prof. David McDonald. "Their progress
is somewhat halting. They appear to jump from one microtubule to
another, moving in a jagged path, even sometimes moving backward, but
they eventually reach their destination." The pictures can be seen in
the Journal of Cell Biology.

The researchers hope their findings will help other scientists gain a
better understanding of how HIV invades immune system cells and offer
up new targets for treatments to slow HIV infection, including
compounds that aim to prevent HIV from linking with the dyenin
proteins or travel freely through cellular microtubules.

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http://news.bbc.co.uk/1/hi/health/2573667.stm

HIV spotted inside human cell

 Remarkable time-lapse pictures show, for the first time ever, HIV at
work in a human cell.

The pictures offer proof that the virus uses the cell’s own machinery
to be dragged inside the cell.

An estimated 45 million people worldwide are living with HIV,
according to the latest figures.

Scientists are learning more about how HIV causes damage once inside
the cell, but, until very recently, very little was understood about
how the virus gets in.

The study, by the University of Illinois at Chicago, reveals how HIV
"hitches a ride" aboard a cell protein called dynein as it makes it
way up tiny microtubules into the cell itself.

The tubules lead all the way to the cell’s nucleus, where the virus
can begin to start replicating by harnessing the cell’s own ability to
copy its genetic code.

Virus particles are only approximately 12 millionths of a centimetre
in diameter, yet the University of Illinois team managed to produce
pictures of them as they headed for their destination.

Jellyfish

They did this by attaching green fluorescent proteins from jellyfish
to them, then shining a blue light on them to make them glow.

Each HIV particle appears as a green dot on the background of the red
tubules, which have also been marked with another fluorescent protein.

 Time lapse photographs were taken under the microscope at 15 second
intervals to reveal the virus’ steady progress towards the centre of
the cell.

Professor David McDonald, one of the scientists leading the project,
said: "They don’t make a beeline for the nucleus.

"Their progress is somewhat halting. They appear to jump from one
microtubule to another, moving in a jagged path, even sometimes moving
backward, but they eventually reach their destination."

Professor Thomas Hope, another scientist involved in the project,
said: "We hope this basic research will one day lead to new targets
for drug therapy in the longstanding battle against Aids."

The pictures were published in the Journal of Cell Biology.

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http://www.sciencedaily.com/releases/2002/12/021212075622.htm

UIC Scientists Provide First Images Of HIV In Living Cells
In stunning color images using time-lapse microscopy, researchers at
the University of Illinois at Chicago have for the first time captured
the very earliest stages of HIV infection in living cells.

The researchers filmed individual HIV particles as they traveled to
the nucleus of a human cell and began taking over its genetic
machinery — the first step in the destruction of the body’s immune
system that leads to AIDS.

The movies not only offer tantalizing glimpses of HIV in action, but
provide visual proof that HIV enlists the assistance of its host to
wreak havoc on the body’s defenses.

The virus can be seen traveling along a part of the host cell’s own
skeletal framework of microtubules as it makes its way from the outer
membrane to the nucleus. The virus hitches a ride aboard a multi-unit
protein called dynein, commonly referred to as a molecular motor.

"Dynein is like a tractor trailer, the microtubules are the highway,
and the HIV particles are the cargo," said David McDonald, assistant
professor of microbiology and immunology at UIC.

McDonald and Thomas Hope, associate professor of microbiology and
immunology at UIC, are co-authors of the study, which appeared Nov. 11
in the Journal of Cell Biology. Science magazine named the paper an
"editor’s choice" in its Nov. 22 issue, and it will be featured in an
upcoming issue of Nature Cell Biology.

An editorial accompanying the paper said, "With the powerful
approaches developed by McDonald et al. and the incredible progress in
imaging single fluorescent molecules in living cells, … important
and fascinating questions of HIV cell biology can now be addressed."

Until recently, little was known about how HIV enters a cell. The
virus is made of an outer shell, or envelope, and a core, referred to
as a particle, which is composed of proteins and genetic material.
When the virus attacks an immune cell, it fuses with the cell’s
membrane and releases its particle core inside.

But what those particles do once they are inside — in particular, how
they arrive at the nucleus to hijack the cell’s genetic machinery and
begin reproducing their own DNA — had remained a mystery.

The tiny particles, only about 12 millionths of a centimeter in
diameter, have to cross a distance that is up to 500 times their size
to reach the nucleus. Moreover, the way is blocked by all kinds of
cellular structures, from energy-generating mitochondria to packets of
proteins. How do the particles get through this obstacle course?

The researchers were able to visualize individual HIV particles by
attaching green fluorescent protein to one of their components.
Derived from jellyfish, the protein has only recently been discovered
as a means of tagging individual molecules inside a living cell. When
blue light shines on the protein, it gives off a green glow.

The researchers also made the microtubules of the host cells glow a
deep red by incorporating another fluorescent protein into their
building blocks.

Pictures of living cells infected with HIV were taken under a
microscope at intervals as short as 15 seconds, creating a movie of
the viruses’ activities as they traversed the microtubular highway
toward their destination in the nucleus.

"They don’t make a beeline for the nucleus," McDonald said. "Their
progress is somewhat halting. They appear to jump from one microtubule
to another, moving in a jagged path, even sometimes moving backward.
But they eventually reach their destination."

The journey to the nucleus takes about two to four hours, he said.

At the periphery of the nuclei, the scientists saw the viruses form
complexes with genetic material of the host cells — appropriating the
tools that HIV needs to reproduce.

Dynein’s role was confirmed by injecting an off-the-shelf antibody
into the cells that prevents the molecular motors from working. When
the motors stop, the viral particles are found scattered throughout
the host cells, not congregated around the cells’ nuclei.

The paper represents four years of research, begun when Hope was a
researcher at the Salk Institute for Biological Sciences in La Jolla,
Calif.

"This work is confirmation of the dynamic new methods we are using to
study HIV," Hope said. "We hope this basic research will one day lead
to new targets for drug therapy in the longstanding battle against
AIDS."

Hope said he plans to extend the technique developed in this HIV
research to study Ebola, one of the deadliest viruses known and one
that could be used in a bioterrorist attack. Little is understood
about Ebola’s basic biology, including how it enters cells.

For more information about UIC, visit www.uic.edu