1. Investigating the multiple proteins that contribute to apoptosis of infected and uninfected
CD4+ T cells during HIV infection
Juliana Nassali Kaggwa

2. Abstract
Over the years, a vaccine against HIV has been difficult to produce, but the virus has slowly
been easier to maintain through the better understanding of the different proteins that come into
play. Infected individuals show signs of a decline in immunity due to the virus killing both
infected and uninfected immune cells. During infection, the virus will attempt to kill off CD4+
and CD8+ T cells via apoptosis, but maintain macrophages, so as to keep them as viral reservoirs
(Busca et al. 2012). It protects them by utilizing their viral proteins, like Nef, to stimulate the
anti-apoptotic molecule Bcl-2 and inhibit caspase 3 and 8 activity within the immune cell. These
viral proteins, as well as pro-inflammatory cytokines such as IFNα, have either pro- or anti-
apoptotic effects during infection depending on concentration levels and disease progression
stage (Fraietta et al. 2012). It is this factor that contributes to conflicting ideas regarding the roles
these proteins play during HIV infection.
Introduction
Apoptosis, or cell programmed death, is a process that cells usually undergo in order to maintain
embryonic development and tissue homeostasis. This process can also contribute to the
pathogenesis of different diseases and infections from viruses such as human immunodeficiency
virus (HIV). HIV infection leads to the gradual decline in CD4+ and CD8+ T cells, and eventual
immune suppression over time leads to the development of acquired immune deficiency
syndrome (AIDS). Cells that have undergone apoptosis are determined by the use of flow
cytometry, a technique that counts cells with fluorescent labels and determines their size and
shape as they flow in a single file in a fluid medium past optical and electronic sensors.

3. The virus has also been found to utilize macrophages for the purpose of producing multiple
virions and killing off uninfected T cells. The question of how exactly HIV causes the death of
these T cells is crucial in trying to develop a vaccine against the virus.
It was initially believed that HIV infection only led to the direct apoptosis of infected CD4+ and
CD8+ cells, but the idea that bystander apoptosis plays a crucial role in the reduction in CD4+ T
cells in HIV -1 infected individuals is quite recent. This came about after previous studies
showed that the number of CD4 expressing T cells lost did not correlate with the number of
HIV-1 infected cells. A study looking at simian-HIV actually found that most CD4+ T cell
casualties were in fact uninfected bystanders (Matrajt et al, 2014).
A key protein in the bystander effect is the envelope (Env) glycoprotein. This viral protein has
been found to be expressed on the surface of both the virus and virus infected cells, and is
arranged as a hetero-trimer. Within each monomer, gp120 and gp41, are found, and these assist
in receptor-binding and act as a fusogenic transmembrane unit respectively.
During HIV infection, the virus utilizes multiple pathways within the host’s cell in order to
conduct apoptosis. This process is essentially grouped into either an intrinsic or extrinsic
pathway, though the pathways do overlap. The extrinsic pathway is composed of an external
membrane bound receptor that is activated by its specific ligand(s), which leads to a cascade of
steps that occur within the cell. The intrinsic pathway, also known as, the mitochondrial
pathway, is composed of the mitochondrion and the proteins that interact with it, such as, Bak,
Bax, Bcl-2 and Bcl-xL to name few. These proteins determine mitochondrial permeability and
work in unison to provide the desired effects of the cell (Garg et al. 2012).
Main Body

4. TNF super family
The tumor necrosis factor family are a group of around 19 ligand and 29 receptor proteins that
regulate complex signaling pathways which lead to inflammation, cellular differentiation, and
apoptosis. These proteins include TNFα, FasL, TNFβ, TNF- related apoptosis-inducing ligand
(TRAIL), TNFR1, Fas, TNFR2, TRAILR1 and many others, with the larger number of receptors
suggesting that these ligands interact with more than one receptor.
The first discovered TNF ligand, TNFα, is heavily involved in the increase in apoptosis observed
in HIV infected individuals. In the presence of inflammation, or viral infection, activated
macrophages and T lymphocytes produce TNFα, which goes on to trigger apoptosis. The ligand
binds to TNFR1 in CD4+ T cells (Fig 1), and this leads to the recruitment of an adapter protein,
TNFR- associated death domain (TRADD), which in turn interacts with a cytopathic death
domain region found on TNFR1. Additionally, TRADD also interacts with Fas- associated death
domain (FADD), which in turn activates caspase 8. This activation leads to the cleavage of BID
from its pro-domain and forming tBID. tBID then translocates to the mitochondria, binding to
the membranes, either associating with the pro-apoptotic proteins Bax protein on the outer
mitochondrial membrane, or Bak on the inner membrane (Busca et al. 2012). These interactions
lead to the formation of a pore that increases mitochondrial membrane permeability, allowing for
cytochrome c (Cyt c) to escape the organelle and end up in the cytosol. It is within the cytosol
that Cyt c interacts with caspase 9 and apoptosis protease activating factor-1 (Apaf-1) forming a
complex referred to as an apoptosome, which goes on to activate caspase 3. Caspase 3 then
initiates a cascade of events that cause chromatin condensation, cell shrinkage, blebbing etc.
(signs a cell is undergoing apoptosis). Fas and TRAIL, once bound to their respective receptors

5. undergo quite similar pathways, leading to the eventual activation of either caspase 3 or 9,
resulting in apoptosis (Fig 1).
Viral proteins
Within the HIV genome, five proteins actively influence apoptosis within the cells of a HIV
infected individual, which are Tat, Nef, Vpr, Vpu and gp160, and they all have either pro- or
anti-apoptotic effects depending on their concentration levels.
Tat
Trans-Activator of Transcription is a multifunction protein that has been found to increase the
expression of FasL and TRAIL in macrophages, thus playing a role in the increase in bystander
apoptosis, as the ligand on the surface of the infected macrophage interacts with the Fas receptor
on the uninfected CD4+ T cell surface. Tat has also been found to induce macrophages to
increase production and release of TNFα, and up regulation of the expression of caspase 8
(Mbita et al. 2014). The consequence of these actions leads to the increase in the chance of cells
undergoing apoptosis. It should be noted that, it’s only at high concentration levels where
apoptosis is increased. When concentration of Tat is reduced, T lymphocytes and macrophages
exhibit a decrease in sensitivity to signaling pathways initiated by FasL, TNFα, and TRAIL.
Vpr
Viral protein R has been found to be a potent apoptotic inducing agent, mainly by increasing
mitochondrial membrane permeability, thus, leading to a greater release of Cyt c and the
eventual apoptosis of the cell. High levels of the protein lead to apoptosis by activating Bax,

6. whereas, low levels lead to the transcription of an apoptotic inhibitor known as Survivin.
Survivin inhibits caspase activity and actually prevents signaling via Bax (Mbita et al. 2014).
In a study looking at how anti-apoptotic molecules Bcl-xL and Mcl-1 affect macrophages and
their predecessors, monocytes, during HIV-Vpr induced apoptosis, it was found that the
differentiated cells exhibited resistance unlike the monocyte. It was thought that Bcl-xL and Mcl-
1 played a role in their resistance to Vpr-induced apoptosis, but after transfecting the
macrophages with these proteins for 2hours, and then with Vpr for 24hours, it became clear that
only their survival in the absence of an apoptotic stimulus. But the introduction of inhibitor of
apoptosis proteins (IAPs) showed to provide macrophages with resistance, which could possibly
due to these IAPs binding to caspase 3, thus preventing its activation and leading to Vpr-induced
apoptosis (which is thought to be Fas-related) (Busca et al. 2012).
Nef
Negative factor is a protein found either within the membrane or cytosol. The protein has been
found to induce FasL expression in HIV infected T cells and macrophages, but protects them
against Fas-mediated apoptosis by stimulating Bcl-2 (Bad antagonist) and inhibiting caspase 3
and 8 activity (Muthumani et al. 2005). A downside to Nef’s protective nature, is that, it also
increases cells resistance to TNFα- induced apoptosis, leading to the pathogenesis of other
opportunistic infections, such as, Mycobacterium tuberculosis. Individually, both M. tuberculosis
and Nef increase TNFα production, but the presence of both leads to a decrease in TNFα
production. When it comes to uninfected T cells, Nef also increases FasL expression, but does
not protect these cells from Fas- mediated apoptosis. The protein has also been found to down

7. regulate Bcl-xL expression, thus allowing pro-apoptotic molecules like Bak and Bax to promote
apoptosis (Mbita et al. 2014).
Vpu
Viral protein U has been found to increase the sensitivity of T cells to Fas-mediated apoptosis
after a study deleted the Vpu gene within the HIV genome and transfected Jurkat T cells with the
virus. These cells were found to exhibit a decrease in sensitivity as compared to their uninfected
counterparts (Mbita et al 2014).
gp160 (gp120 and gp41)
The Env protein or gp160 is the predecessor to gp120 and gp41. Once it travels to the viral
membrane, it is cleaved into the two proteins. The glycoproteins are crucial members in the
overall Env glycoprotein of HIV, with gp120 acting as a bridge between the virus and the target
host cell; and the bridge being held down by gp41, which is important for the viral and host cell
membrane fusion (Garg and Joshi 2012). For fusion to occur, the gp120 of HIV must bind
specifically to the CD4 glycoprotein and an associated co-receptor (either X4 or R5) on the T
helper cell. The X4 co-receptor has been found to produce a more potent apoptotic effect than R5
(Cummins and Badley, 2010). CD4 binding leads to gp120 conformational changes that cause
the exposure of the co-receptor binding site and a heptad repeat region of gp41. Joshi and her
team, investigated the idea of gp41-mediated apoptosis determining the bystander effect. With
cells expressing the R5 co-receptor being exposed to different types of Env molecules
These interactions lead to 1 of 2 outcomes: either hemifusion or syncytia (Fig 2); both of which
lead to apoptosis.

8. Hemifusion and syncytia
Hemifusion is the interaction of the lipid bilayer of both the CD4+ T cell and HIV mediated by
gp41. It has been shown that it is actually hemifusion and not just gp120 binding that induces
apoptosis involving caspase 3. Unfortunately, no biochemical evidence is present to determine
what happens at the membrane contact site (Joshi et al. 2013). This information could shed light
on just how this hemifusion leads to apoptosis.
Syncytia formation is the fusion of the lipid bilayer and cytoplasmic contents and eventually
nuclear material of both an infected and uninfected cell, often characterized by ballooning, seen
with immunofluorescence. This phenomenon is the result of a host of molecular events that are
observed during the later stages of the disease progression. This formation triggers the events
that lead to the phosphorylation of the transcription factor p53 at serine 46 indirectly by p38
Mitogen Activated Protein Kinase (MAPK) and mammalian Target of Rapamycin (mTOR).
Usually, phosphorylated p53 undergoes cell cycle arrest, but it is the phosphorylation at serine 46
that ensures apoptosis occurs. This then leads to the increased transcription of Bax, thus,
increasing the activation off the mitochondrial pathway (Perfettini et al. 2005).
INFα
During viral infection, the cytokine interferon α (IFNα) is synthesized by plasmocytoid dendritic
cells (pDC) in an attempt to reduce viral replication. Due to conflicting ideas regarding INFα on
the immune system during viral infection, Fraietta and his colleagues attempted to determine the
effects of the cytokine on pro- and anti-apoptotic molecules. Interestingly, it was found that
IFNα not only significantly increased the expression of the pro-apoptotic protein Bak and Fas

9. receptor, but it also induced Fas-associated apoptosis in CD4+ and CD8+ T cells from both HIV
infected and healthy donor cells, but not TNFα and TRAIL- associated apoptosis. Just like in the
case of TNFα and the viral proteins, varying levels present at the different stages of infection
sow different effects, with persistently low concentration levels of IFN promoting apoptosis in T
cells, but high levels leading to the attempted control of viral replication. The effects of the
cytokine were also deemed to be stage specific, with acute infection exhibiting a protective effect
and chronic infection showing a pathogenic effect. This somewhat goes against the idea that HIV
infection increases IFNα expression, as one would expect an increase in IFNα as HIV became
chronic due to the increase in virions circulating in the body (Fraietta et al. 2013). This study, as
well as others, continue to prove that during HIV infection, Fas-mediated apoptosis tends to
dominate over TNF and TRAIL when looking at the extrinsic pathway (Fig. 1)
Discussion
HIV induced apoptosis is a process that is mediated and affected by multiple factors. Though
proposed ideas shed light on key proteins and pathways involved, it is clear that further studies
need to be conducted in multiple areas in order to come to a full understanding of how HIV
induces apoptosis, particularly bystander apoptosis, as it’s shown to be the largest contributing
factor to T cell loss (Matrajt et al. 2014). For example, the claim that the fusogenic activity
through gp41 “determines bystander apoptosis” could be up for debate, as other studies show
HIV proteins such as Tat and Nef also have this same effect (Muthumani et al. 2005).
The fact that viral proteins and other signal inducing proteins exhibit both pro- and anti-apoptotic
effects at varying levels, complicates the attempt in understanding the processes.

10. What is known is peripheral blood mononuclear cells (PBMCs), which consist of T, B and
natural killer cells, within HIV infected individuals show higher caspase 3 and 9 activity, lower
number of anti-apoptotic molecules (e.g. Bcl-2); and increased mitochondrial permeability (Garg
and Joshi 2012).
A majority of studies determined apoptosis within immune cells by looking at only Jurkat cells
(immortalized cancer cells), but only a few attempted to also look at primary cells in order to
compare and contrast, as was seen in a study conducted by Fraietta and his team (Fraietta et al.
2013). Results from only Jurkat lines do not show a true representation of what happens in vivo.
Despite being easier to use, Jurkat cells may also be contaminated with other viruses, as a study
conducted in 2008, highlighted the risk of this happening, especially when the correct testing
measures aren’t undertaken before using the cells for study (Takeuchi et al 2008).
Understandably, primary cells are much harder to work with, but including experiments that
utilize both cell types provides a clearer picture of what the results mean, as was also done by
Joshi and her team (Joshi et al, 2013).
Overall, the uncertainty in many aspects of determining the exact mechanisms that lead to
increased apoptosis of immune cells is a hindrance in the attempt to stop the effects of HIV
infection because many of the proposed pathways are interconnected (Fig 1) and using the
“inhibitor route” may potentially have adverse effects on other cellular processes.

11. References
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in human macrophage survival and cellular IAP1/2 (cIAP1/2) in resistance to HIV-Vpr-induced
apoptosis. The Journal of Biological Chemistry 287: 15118-15133
2) Cummins, N.W., and Badley, A.D. (2010) Mechanisms of HIV-associated lymphocyte
apoptosis: 2010. Cell Death and Disease 1, 1-9
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Kathuria, N., McGettigan, S.E., Lewis, M.G., Giavedoni, L.D., Jacobson, J.M., and Katsikis,
P.D. (2013) Type I interferon upregulates bak and contributes to T cell loss during human
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Figures
Figure 1. The cell signaling pathways undertaken through the binding of the TNF superfamily
ligands and their respective receptors, showing just how these paths overlap with each other in
order to cause apoptosis (Kumar et al. 2013)

13. Figure 2. The contributing factors that lead to either hemifusion or syncytia mediated apoptosis
(Garg et al, 2012)