Page 47 - M. Immunology

Investigation of Anti-viral effect of SCOTIN and its derivatives

on Human Immunodeficiency Virus-1(HIV-1) production
Ga-Na Kim , Kyung-Lee Yu, and Ji-Chang You
National Research Laboratory for Molecular Virology, Department of Pathology, School of Medicine, The Catholic University of Korea, Seoul.

BACKGROUND AIM

Recently, many drug delivery system being development is important to Using a Drug Delivery System(DDS) is expected to increase
approach to delivering drugs on their effect site and achieving a significantly the effect of drugs. Cell Penetrating-Peptide(CPP) is
therapeutic outcome. Cell Penetrating-Peptide(CPP) is one of drug one of DDS that can be applied such purpose.
delivery system facilitate cellular uptake of various molecular equipment. Lately, Unpublished Advanced Cell penetrating-Peptide(ACP) that
SCOTIN expressed in the SHISA5 gene was first identified as a direct
target gene of the p53 transcription factor. SCOTIN has also been is studying in our laboratory has established a novel CPP, named
as ACP, which has a better cellular delivery activity than a well-
reported to interferes with HCV replication.
known CPP such as Tat of HIV-1. Also, it has been demonstrated
In the present study, we demonstrated anti-viral activity of SCOTIN thus far that SCOTIN expressed in the SHISA5 gene has anti-virus
against HIV-1 production and that could be enhanced using an effect against HCV. In this study, we have examined whether anti-
Advanced Cell penetrating-Peptide(ACP) that has established a novel viral activity of SCOTIN against HIV-1 production could be
CPP in our laboratory. enhanced and delivered by fusing our ACP.
METHODS

We have made various expression vectors of SCOTIN and ACP fusion SCOTIN. 293T cell was transfected with each of expression vectors. The
protein expression level of each recombinant vectors was verified by western blot assay. Next, they were co-transfected with NL4-3_GFP DNA
to 293T cells, and viral production level was measured by Western blot assay and enzyme-linked immunosorbent assay. Viral infectivity was
determined by GFP signaling. The same volume of virus containing each culture supernatants was used to infect MT4 cells.

RESULTS

Protein expression level of each recombinant vectors was compared
with SCOTIN. We observed the separation of ACP and SCOTIN by
signal sequence of SCOTIN when the combination of ACP is placed in
the upper sequence of SCOTIN. We examined whether ACP fusion
SCOTIN could reduce the virus level by co-transfection with a NL4.3-
GFP into 293T cell. We found that APC fusion SCOTIN significantly
reduced the viral level in the cell lysate and supernatants than
overexpression of SCOTIN alone. That is ACP fusion form of SCOTIN
reduced production of HIV-1 quite efficiently. Fig 1. Structure and Expression of the ACP-SCOTIN fusion Expression vector used in the study.
(A) Schematic diagram of ACP fusion SCOTIN expression vector constructs ( pcDNA3.1-SCOTIN,
pcDNA3.1_SCOTIN-ACP, pTriEx-1.1_ACP-SCOTIN). pTriEx-1.1 vector was used in when ACP placed
in the upper sequence of SCOTIN. 293T cell was transfected with each of expression vectors.
(B) That’s protein expression level was measured by western blot assay with SCOTIN alone.
Separation of ACP and SCOTIN by a putative signal sequence of SCOTIN in pTriEx-1.1_ APC-SCOTIN.

Fig 2. Investigation of Anti-viral effect of SCOTIN
and its derivatives on HIV-1 production. Fig 3. Analysis of Viral infectivity of SCOTIN
Each recombinant vectors were co-transfected with and its derivatives on HIV-1 production.
NL4-3_GFP DNA to 293T cells. (A) Viral production MT-4 cells were infected with the same volume of
level was verified by Western blot assay in cell lysate virus containing each culture supernatants
and (B) Data was normalized to internal control RFP. generated from co-transfected with NL4-3_GFP
(C) p24 of HIV-1 level was measured by enzyme- DNA to 293T cells. Resulting infected MT-4 cell,
linked immunosorbent assay in cell supernatants. GFP positive, were observed by (A) Fluorescent
microscope and (B) quantified by FACS analysis.
Taken together, This results suggested that ACP could enhance the effect of anti-HIV-1 by SCOTIN. Thus, this approach might
be useful approach for the development of a new anti-HIV-1 drug molecule.

CONCLUSION REFERENCES ACKNOWLEDGEMENTS

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from the National Research Foundation of Korean Ministry of Science
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The Catholic University of Korea, Seoul.