Page 19 - O. Microbiology

Construction and examination of a new type of Brec1
Recombinase derivatives for the Elimination of HIV Proviral
DNA from the Host Genome

National Research Laboratory for Molecular Virology, Department of Pathology, School of Medicine,
The Catholic University of Korea, Seoul.
Abstract Introduction

Even though combinatorial antiretroviral therapies (cART), which are used to treat Broad-range recombinase 1 (Brec1) was evolved by directed molecular evolution and, as a
HIV-1 infection, can effectively suppress and relieve symptoms of AIDS but does not Cre-type recombinase. Brec1 can remove the provirus from HIV-1–infected cells by
completely eliminate HIV-1 infection. In order to effectively cure HIV-1 infection, the specifically recognizing and recombining a highly conserved target site (loxBTR) located within
integrated proviral DNA must be removed from the virus infected host genome. We the LTRs of the majority of HIV-1 isolates. Brec1-mediated recombination of loxBTR sites
tried to develop a therapeutic protein drug using Broad-range recombinase 1 (Brec1) from HIV-1 provirus could delete or invert genomic sequences in the infected cell.
and a HIV nucleocapsid (NC). The Brec1 has previously been known to efficiently The HIV nucleocapsid (NC) is a small, highly basic, zinc binding protein that interacts with the
and safely remove integrated provirus from HIV-1 infected cells. However, it in its viral RNA to facilitate numerous processes in the viral life cycle. When NC is contained within
current form is difficult to being used as for a protein drug as it cannot get into cells the precursor Gag polyprotein, the NC domain is involved in genomic RNA packaging, Gag-
by itself. To overcome this problem, we fused the Brec1 with NC, which can be easily Gag multimerization and possibly initiation of particle production during virus assembly.
internalized into cells and capable of targeting integrated proviral DNA in HIV-1 Our previous work had established that NC can localize to the nucleus and we observed that
infected cells. We observed that the overexpression of NC-Brec1 lead to the excision NC can efficiently internalize into cells. In addition, NC has been known to interact with HIV-1
of co-transfected proviral DNA in cell lines. Virus production was also found to be proviral DNA. Therefore, we examined anti-viral effect against HIV-1 by fusing Brec1 with the
significantly reduced by HIV-1 p24 ELISA and western blot assay. Therefore, this HIV NC protein.
novel approach using a combination of Brec1 and NC might have a great potential to
develop a novel fundamental curative HIV-1 therapy.

Results

A

B
B

Figure 3. Recombination activity of NC-Brec1. (A) schematic diagram of NC-Brec1 fusion
protein and Brec1 expression construct pcDNA3.1-NC-Brec1, Brec1. (B) Luciferase activity of
293T cells co-transfected with reporter construct (pGL_LTR-F-luc-LTR), indicated plasmids
(pcDNA3.1, pcDNA3.1_Brec1, pcDNA3.1_NC-Brec1) and internal control plasmid (pDS-Red).
Figure 1. Internalization of NC into cells. (A) Confocal imaging of HeLa
cells transduced with FITC-NC. The cells were imaged at 3 hrs after
transduction. (B) Fluorescence imaging of HeLa cells incubated for 3 hrs with
FITC-NC at 37 ℃ or 4 ℃ and in presence of heparin.
1.2 Figure 4. Anti-viral efficiency against HIV-1 of NC-
Brec1. 293T cells were co-transfected with HIV-1
1.0
provirus (pNL4-3_EGFP), each plasmid (pcDNA3.1,
Relative HIV-1 p24 0.8 *** *** control plasmid (pDS-Red). ELISA analysis of virus
pcDNA3.1-Brec1, pcDNA3.1-NC-Brec1) and internal
supernatants from each transfected 293T cells by using
0.6
HIV-1 p24 ELISA kit.
0.4
0.2
0.0
Ctrl Brec1
Figure 2. Schematic representation of HIV-1 proviral DNA with LTRs NC-Brec1
and Brec1-mediated recombination.
Conclusion References

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