Use of Mammalian Two-Hybrid System to Detect Human p53-HIV TAT Interactions

Sydney Rapp, Biological Science; Dr. David Reisman, Biological Science

Research Question: Can we demonstrate an interaction between p53 and HIV TAT in vivo?
Project Goals and Objectives: The goal of the project is twofold; first, we want to generate the appropriate recombinant
DNA constructs required for the Mammalian Two-Hybrid System. Second, we want to use the Mammalian Two-Hybrid
System to determine whether p53 and HIV TAT form a complex in cells.

Project Impact: Although the current treatment for HIV is successful in reducing the circulation of HIV in patients,
complete eradication of the virus is not possible. By demonstrating an interaction between HIV-TAT and p53 in vivo, it is
possible that future experiments could find compounds that block this interaction and stop a complex from forming.
Preventing the p53-TAT complex in newly infected cells is hypothesized to release active p53 and drive cells to undergo
apoptosis or arrest via a p53-mediated pathway, thereby effectively preventing further replication and maturation of the
virus.

Background: HIV (human immunodeficiency virus) is the virus that can lead to acquired immunodeficiency syndrome or
AIDS if not treated (21). Unlike some other viruses, the human body cant get rid of HIV completely, even with treatment.
HIV specifically infects the CD4 cells (or T cells) of the immune system; these cells help the body fight infection and if
HIV destroys too many, the body cant fight the infections and the individual has AIDS (21). At this time there is no cure
for HIV however, the virus can be controlled using antiretroviral therapy. Although highly active antiretroviral therapies
(HAART) successfully reduce circulating HIV in patients to below the level of detection, complete eradication of the virus
is currently not possible due to the presence of the virus in cellular reservoirs, which permit constant low-level viremia (1-
8).
p53 is a protein that has long been recognized as a crucial tumor suppressor aiding cancer suppression via the
induction of apoptosis (cell cycle arrest) in response to many different cellular stress signals. Recently, p53 has been found
to have broader roles in physiology and pathology. The exact cell fate due to p53 activation is determined by cell type,
environmental conditions and the nature of the stress. The ancestral function of p53 is thought to be its ability to trigger
programmed cell death, or apoptosis, in response to DNA damage. Infection by many viruses leads to the induction of the
p53 gene, which promotes apoptosis of virus infected cells, thus limiting virus replication (9).
Numerous studies evaluating temporal gene expression during the infection of CD4+ T cells by HIV-1 have
reported that genes expressed early during infection, including p53, resemble those expressed in response to DNA damage
(16-19). In fact, p53 has been reported to be induced as early as eight hours post-HIV infection (16,18). One of the
characteristics of HIV infected cells is that the HIV-encoded TAT protein, in addition to numerous other activities, is
hypothesized to form a complex with the p53 tumor suppressor and block p53s cell-cycle arrest and apoptotic activity
(10-15). Preventing the p53-TAT complex in newly infected cells is hypothesized to release the active p53 protein and
drive cells to undergo apoptosis or arrest via a p53-mediated pathway, thereby effectively preventing further replication
and maturation of the virus.In a recent article about HIV Douglas D. Richman, a researcher at the Center for AIDS
Research, says that Research into a cure for HIV is in the early stages of a long and difficult path, and all innovative
options should be welcomed and investigated. My suggested experiment will utilize a Checkmate Mammalian Two-
Hybrid System to show that there is an interaction between p53 and HIV TAT. If this interaction can be shown, further
research by Dr. Reisman can be done to find a way to block this interaction, thus allowing p53 to function.

Project Design: The Checkmate Mammalian Two-Hybrid System is used to detect an interaction between two proteins in
vivo. For this experiment, those two proteins are p53 and HIV TAT. Specific domains, including a DNA-binding domain
and transcriptional activation domain, found in transcription factors form the basis of this system. The DNA-binding
domain binds to a certain DNA sequence and the association of a transcriptional activation domain and DNA-binding
domain may promote transcription. Association of these domains occurs when each is bound to a different protein and
these proteins interact. The interaction between these two proteins results in the transcription of the firefly luciferase
reporter gene. In this system a vector called pG5luc is used; this vector contains five GAL4 binding sites upstream of a
TAT box, which is upstream of the firefly luciferase gene. A pBIND vector is used because it contains the yeast GAL4
DNA-binding domain. A pACT vector contains the VP16 activation domain. The two genes encoding two potentially
interactive proteins, in this case p53 and HIV TAT, are cloned into the pBIND and pACT vectors to generate fusion
proteins that will also contain the DNA-binding domain of GAL4 and the activation domain of VP16, respectively. These
pGAL4 and pVP16 fusion constructs alongside the pG5luc vector are transfected into mammalian cells. An increase in the
transcription of the firefly luciferase gene indicates an interaction between p53 and HIV TAT. This process is overviewed
in the Figure 1, where X and Y represent the proteins p53 and HIV TAT.
 In order to use the Checkmate Mammalian Two-Hybrid
System, I must first create the necessary recombinant DNA constructs. To do this both p53
and HIV TAT must be inserted in the correct reading frames and the correct
orientation to generate fusion proteins into both the pBIND and pACT
vectors. I have initiated these procedures by PCR amplification of both p53 and
TAT using specific DNA primers that maintain the reading frames of the genes as well
as create the appropriate sequences at the ends of the products to allow directional cloning. This has
been accomplished and the results can be seen in (Fig. 2). These
products are now being ligated to the pBIND and pACT vectors Figure
that have been digested with BamHI and XbaI.
After ligation, the products are introduced into E. coli and the bacteria are selected
for resistance to ampicillin. Only E. coli carrying the introduced plasmid will grow
and those bacteria are analyzed to determine whether or not they now carry the
desired recombinant DNA construct. This analysis is accomplished by purifying the
DNA and performing a restriction enzyme digest and gel electrophoresis.
Once I have completed generating the correct expression vectors, these
DNAs will be purified and introduced into a series of human cell lines. These cells
include human U2OS (human osteosarcoma), T98G (human glioblastoma), and
HeLa (human cervical cancer). Two to three days after transfection, the cells are
lysed and the amount of firefly luciferase is quantified. This quantity is compared to
the control, which contains either p53 or HIV TAT. The transfection of control is expected to
result in a relatively low expression of the luciferase gene.

Project Timeline: The first task was to isolate and purify the p53 and HIV TAT sequences; this has already been
accomplished. The second task is to insert the p53 and HIV TAT sequences into the pBIND and pACT vectors (August-
October). The third task is to purify the resulting plasmids so that the DNA is free of protein, RNA and chemical
contamination (November). The fourth task is to transfect the vector constructs into mammalian cells (November-
January). The fifth task will be to identify whether or not an increase in transcription of the firefly luciferase reporter gene
has occurred due to the interaction of p53 and HIV TAT (January 2017). The sixth task will be to compare these results
with the transcription that results from the controls (February 2017). Finally, the results will be analyzed and put into a
written report.

Anticipated Results and Dissemination: I anticipate that the results of these experiments will indicate an interaction
between p53 and HIV-TAT. This interaction will lead us to the next step, which is to show that the interaction is inhibitory
(p53 function is blocked). After that, the goal is to find compounds that can prevent this interaction, thus extinguishing the
replication and maturation of the HIV virus.
The results of this project will be presented at USCs Discovery Day. In addition, the work I do may eventually
lead to more funding for Dr. Reismans research, which could facilitate the development of a better treatment for the HIV
virus.

Personal Statement: I began working in Dr. Reismans lab last semester and since then have gained valuable knowledge
on laboratory procedures as well as an understanding of the nature of Dr. Reismans research. By continuing my research I
will improve my laboratory techniques as well as my knowledge of the subject matter and become an even stronger
applicant for when I apply to dental school. Dental schools look for applicants with laboratory skills and many of them are
research based so an opportunity such as this would be an immeasurable experience. I would be honored to receive the
Magellan Grant for undergraduate research not only because it will allow me to dedicate more time to my project but
because it would further my comprehension of Dr. Reismans research as well as my appreciation for research as a whole.

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