It’s
a disease that is well known all over the world: human immunodeficiency virus,
better known as HIV. It is often talked about in tandem with
acquired immunodeficiency syndrome, or AIDS, which develops in HIV patients
over time and is the end stage of the disease. HIV originated in chimpanzees as simian immunodeficiency
virus (SIV) and transferred over into humans in the 1800s. The first cases in the United States
were reported in 1981. Throughout
the 1980s, cases increased
dramatically, peaking in the early 1990s. However, a breakthrough in drug treatment for people living
with HIV and HIV prevention campaigns helped to bring the number of cases back
down. The drug therapy known as
antiretroviral therapy (ART) is still used today as the main way to help people
with HIV live normal lives, hopefully preventing/delaying the progression to
AIDS. They target different points
in the HIV virus replication cycle to try and slow down its progression through
the body.
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| Figure 1: HIV replication in a cell. It is able to dump its contents into the cell, reverse transcribe its RNA, integrate it into host DNA, and use the host to create new viral copies. |
HIV is a retrovirus, a form of RNA
virus that can be inserted into the host DNA, and then uses host cells to
replicate. The virus comes with
all sorts of proteins that let it do this. For example, reverse transcriptase allows the viral RNA to
be turned into viral DNA.
Integrase allows it to be inserted into the host DNA to then use the
host’s own protein making mechanisms to make new viruses and viral
proteins. Figure 1 shows an
outline of HIV replication. It is
possible to trace the viral RNA to viral DNA to host DNA and then back out to spread
to other cells.
HIV also specifically infects a
certain class of immune cells known as CD4+ T cells. CD4+ T cells are called this due to the presence of a cell
surface receptor called CD4. These
cells can differentiate into all different subtypes of CD4+ T cells called T
helper cells, or Th cells, whose name describes their function: they “help”
other immune cells mount responses to pathogens. CD4+ T cells and all of their progeny are crucial for
providing immunity to all sorts of infections, pathogens, and the like. HIV comes into play and infects the
CD4+ T cells. It creates a chain
reaction, infecting, spreading, and slowly killing all the CD4+ T cells in the
body. In simple terms: HIV is slowly
knocking out an entire branch of the immune system. Any further immune function that would need a CD4+ cell to work
won’t be able to work once the CD4+ T cells are gone. Figure 2 lays this out in a graph showing CD4+ T cells in
blue, viral RNA copies in read, and time in weeks on the x-axis. It is possible to see that as the RNA
copies go up, the CD4+ T cells go way down. Many of the ART drugs target these proteins that prevent the
HIV from infecting cells as easily or spreading once it has infected a cell. However, it is still unknown how HIV
actually kills CD4+ T cells.
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| Figure 2: Timeline of HIV infection. As viral RNA increases, CD4+ cells decrease. A latent period exists where the person may not know they are infected until their cell count reaches a certain point. |
Cooper et al demonstrate one
potential way HIV could kill CD4+ T cells. They first infected cells with HIV and stained for a
particular viral protein called p24.
They noticed that the CD4+ T cells that were killed didn’t express this
viral protein, while cells that weren’t killed did. Next, they looked at whether these cells that were lacking
expression of this viral protein had been infected with the virus before they
died. More T cells were infected
with HIV that also encoded for green fluorescent protein (GFP), which
fluoresces green. GFP is often
used as an indicator for protein production. The gene for GFP is placed within
the HIV genome, so if HIV proteins are being produced, GFP will also be
produced. If the cells are dead,
no GFP will be detectable. This is
a commonly used method to visualize and also quantify protein production. They analyzed the cells for GFP
expression and cell viability, as well as viral cDNA. Non-viable GFP- cells were found to have copies of viral
cDNA. When viable GFP+ cells were
watched over time, the researchers saw that many of these cells eventually died
(therefore losing their GFP expression) but retained viral cDNA. These data together suggest that the
cells that were killed died after successful HIV gene expression.
The
authors next wanted to examine what part of the HIV replication process was
causing cell death. One of the
earlier processes is when integrase inserts HIV DNA into host DNA. Cells that were treated with raltegravir,
an integrase inhibitor use in ART, had much higher viability than cells
untreated with the drug. Then,
cells were infected with HIV containing a mutation that eliminates its ability
to integrate into the host DNA. The
viral DNA that wasn’t integrated also fused into little circles. These cells didn’t die, and taken
together with the previous data, strongly suggested that the integration step
in HIV replication was what was causing cell death. They also eliminated the
cell’s ability to create these little DNA circles, and the cells still didn’t
die, supporting the idea of the actual integration step causing cell death.
HIV
viral gene expression was then knocked out while the ability to integrate was
left intact. The cells then
started to die again, showing that integration alone is enough to cause cell
death. Immune cells were then
taken from subjects already infected with HIV. The cells were stimulated for viral replication, and death
of CD4+ p24- cells was once again observed. However, when raltegravir was introduced, cell viability
went up, showing that integration dependent cell death also occurs in natural
infection in people and not just lab induced infection in cells.
Lastly,
the authors looked at what proteins in the cell were responsible for activating
cell death. They noticed the
activation of a protein called DNA-PK (which is part of a cell death pathway) by
HIV with the integration ability, but not by HIV with its integration ability
eliminated. This suggests some
sort of link between integration and the protein DNA-PK. When this protein was inhibited, cell
death was blocked, similar to what happened when HIV integrase was
blocked.
Overall,
the researchers were able to demonstrate that HIV integration is a main cause
of cell death in CD4+ T cells and this is done somehow with DNA-PK. How this occurs still has to be looked
at, but they were able to lay a foundation and propose a mechanism for how HIV
actually causes cell death. This
research was important, however, because it’s the first strong evidence
implicating integration and DNA-PK in HIV dependent cell death. HIV’s ability to evade the immune
system and go through rapid evolution to avoid the drugs companies have
developed against it is a problem.
Having a wide variety of drugs available to combat these rapid changes is
useful to help treat patients. If
scientists can better understand the mechanism of how HIV causes cell death,
they can create drugs that inhibit that mechanism, therefore prolonging and
improving the quality of life of people with HIV.
Other information about HIV:
http://www.cdc.gov/hiv/basics/whatishiv.html
Images
http://www.thenakedscientists.com/HTML/uploads/tx_naksciimages/Hiv-timecourse.png
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