Wednesday, December 4, 2013

A New Mechanism of Cell Death by HIV

            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.

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.
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.

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