The road to effective HIV vaccines is littered with many
hurdles and blockades that cause vaccines to be ineffective. One of the main
reasons why HIV leads to many ineffective drug vaccines is because of its
ability to mutate rapidly. The rapid mutation of HIV allows the virus to avoid
potential vaccines such as antibody vaccines that could neutralize the virus or
help facilitate the death of the virus. As a result of the rapid mutation, many
current scientists are looking into new ways to fight off the virus. A couple
of the new methods in stopping the virus include targeting specific proteins
that make up HIV that allow it to mutate and infect immune cells. Scientists
are also looking into methods that enhance the host antiviral proteins found in
the immune cells. In a new study from Kumamoto University, researchers have
discovered a new method in reducing the infectivity of HIV-1 (the usual HIV
strain found in humans). What the researchers discovered could lead to a
potentially new development of vaccines in fighting HIV. The scientists
discovered the use of a recent protein called GANP (germinal center-associated
nuclear protein) that enhances the facilitation of the host antiretroviral
protein called A3G (its function causes hypermutations in newly synthesized
viral DNA) into the viral capsid in the absence of the HIV accessory protein
Vif.
The function of HIV’s accessory protein Vif is to promote
the synthesis of proviral DNA by inhibiting a host antiviral protein. The host
antiviral protein that Vif inhibits is APOBEC3G where Vif degrades the protein
by facilitating the APOBEC3G to a proteasome. In the absence of HIV, APOBEC3G
normally functions as an antiviral cytidine deaminase enzyme that converts a
cytidine to a uracil (C to U) in viral RNA.
In trying to get a visual understanding of the importance of
APOBEC3G, here is a video that will demonstrate the general function of
APOBEC3G on HIV:
In an effort to take advantage of APOBEC3G’s ability against
viral RNA, the researchers tried to experiment and explore the combination of
A3G (a protein of the family of APOBEC proteins that has the same ability) with
GANP since GANP has shown in a previous study that it enhances the use of one
of the other APOBEC proteins called AID. GANP is a protein that is upregulated
when CD4+ T cells are activated so the researchers want to see how
GANP interacts with A3G in the HIV virion during an HIV infection.
The researchers wanted to figure out the
effect of the GANP within the virion core of HIV-1. In order for them to test
out GANP’s effect in HIV-1 virions, the researchers produced a control HIV-1
virion versus a HIV-1 virion that was deficient in the accessory protein Vif,
which they named ΔVif HIV-1 virion. As a result of testing the GANP
protein in the two virions, they detected less of the GANP in the control
compared to the Vif deficient HIV-1 virions. The difference in the levels of
GANP between the control and Vif deficient virions meant that the Vif not only
inhibits the action of A3G, but Vif also inhibits the action of GANP. Likewise,
the absence of Vif increases the level of activity of the GANP. Afterwards, the
researchers examined whether GANP affects A3G in the HIV-virion. To examine the
affects, the researchers made another experiment where they separated the HIV-virion
of both the control and Vif deficient cores from the rest of the cell
components by pelleting the cores to see whether GANP and A3G were both located
in the cores or in the supernatant liquid above the pellet. As a result, the
GANP was located in the pellet where the cores are located while the A3G was
located in both the pellet and the supernatant. Next, the researchers checked
the location of A3G when GANP is absent. In the absence of GANP, A3G was mostly
found in the supernatant liquid with about 10% A3G found in the pellet. From
the data that the researchers gathered, they found out that GANP significantly
increased the amount of A3G within the pellet while reducing the amount of A3G
in the liquid outside of the core so GANP ultimately helps the activity of A3G facilitating
inside the virion core of HIV.
In using the ΔVif HIV-1 virion, the researchers also found
out the interaction of RNA with GANP and A3G. They had known from a previous
study that the binding of A3G to 7SL RNA is a plausible mechanism for its efficient
packaging into virions (2). So they tested GANP’s binding of A3G to RNA in the
absence and presence of GANP, and found GANP to increase the levels of A3G
bound to RNA where GANP binds A3G to specific regions of RNA and thus mediating
packaging of A3G into virions.
As a result of finding out that the GANP mediates A3G into
the virions, the researchers wanted to see the anti-HIV-1 activity of A3G. The
researchers compared the effect of A3G with and without GANP in infectious
HIV-1 on target cells. With the addition of GANP, the amount of hypermutations CàU deaminases increased
significantly to about 89% after sequence analysis (1). As a result of the
mutations, the proviral DNA for the virions contained an increase of GàA because of the base
pair for uracil (U) in the RNA complements for the production of adenine (A) DNA
nucleotides. Because of the mutations, the proviral DNA coded for truncated or misfolded
proviral proteins.
To finally confirm the potential antiviral effect of A3G
with the addition of GANP, the researchers compared the effect of reduced
expression of GANP versus overexpression of GANP in fully infectious HIV-1
virions on target cells. In reduced GANP, the target cells experienced
increased infections while the overexpression of GANP significantly reduced the
level of infection from HIV-1 virions.
With this new discovery, the researchers are one step ahead
in the War on HIV/AIDs. They believe GANP’s potential role in mediating
APOBEC3G can lead to a new development of vaccines for HIV. The next step in
furthering this research is to experiment in
vivo on model organisms such as mice since the experiment tested in vitro on cell cultures. Testing in vivo will allow the researchers to
see whether the effect of GANP is successful or not in CD4+ T cells
of live mammals as well as seeing any side effects as a result of overexpression
of GANP.
Primary Source:
1.
Maeda,
K., S.A. Almofty, S.K. Singh, M.M.A. Eid, M. Shimoda, T. Ikeda, A. Koito, P.
Pham, M.F. Goodman, and N. Sakaguchi.
2013.
GANP Interacts with APOBEC3G and Facilitates Its Encapsidation into the Virions
to Reduce HIV-1 Infectivity. J. Immunol.
193:6030-6039.
Secondary Sources:
2.
Zhang, W., J. Du, K. Yu, T.
Wong, X. Yong, and X. Yu. 2010. Association of Potent Human
Antiviral Cytidine Deaminases with 7SL RNA and RNP in HIV-1 Virions. J. Virol. 84(24):12903
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