Thursday, December 19, 2013

New Possible HIV Treatment reduces HIV-1 Infectivity with the use of GANP to mediate APOBEC3G Packaging in HIV-1 Virions

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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