Page 21 - O. Microbiology

Identification of a novel protein inhibitor acting on late-stage of HIV-1

replication
Kyung-Lee Yu, Ga-Na Kim, Hae-In Kim, Yu-Mi Jung and Ji-Chang You
National Research Laboratory for Molecular Virology, Department of Pathology, School of Medicine, The Catholic University of Korea
BACKGROUND AIM

Human immunodeficiency virus type 1 (HIV-1) infection induces innate immune responses
mediated mainly by IFN-I. IFN-Is are well-known cytokines eliciting broad anti-viral effects by
inducing the expression of anti-viral genes called interferon-stimulated genes (ISGs). Investigation the effect of SCOTIN on HIV-1
An anti-viral IFN-β inducible protein, SCOTIN, have recently reported to inhibit the Hepatitis C
Virus (HCV) replication by mediating the autophagic degradation of the HCV NS5A protein that replication.
plays a key role in HCV RNA replication. Therefore we investigated the effect of SCOTIN on HIV-
1 replication.
METHODS RESULTS

Figure 1. SCOTIN inhibits the HIV-1 replication
Proviral DNA
(A) (B)
HEK293T cells were transfected pNL4-3 GFP with control
(con) or SCOTIN-V5 and RFP expression plasmid (pDs-red ,
internal control). At 24 hours post-transfection, cell lysate
Experimetal Scheme
and viral soup were analyzed by western blot (A) and virus
titer of viral soup were measured by p24 ELISA (B)

Figure 2. SS, TM and PRD domain are important for anti-viral effect of SCOTIN
Viral soup of HEK293T
(A) (B)
Western Blotting Enzyme-Linked Immunosorbent assay (A) An illustration of the
(ELISA) truncated SCOTIN (B)
HEK293T cells were
transfected as described
in figure 1. At 24 hours
post-transfection, virus
titer of viral soup were
HEK 293T Cells HeLa cells measured by p24 ELISA.
Western Blotting Immnostaining
Figure 3. SCOTIN inhibit the Gag protein multimerization

HeLa cell transfected pNL4-
3GFP with control (mCherry)
Transmission electron microscopy (TEM) or SCOTIN-mCherry. Then,
immunostainig were
performed described as
Method section.

Figure 4. SCOTIN inhibits the HIV-1 budding via blocking the recruitment of Tsg101 to plasma membrane
(A) (C) (D)

(B)
(A) An illustration of HIV-1 Gag protein indicating ALIX and Tsg101 binding site. These interactions lead to the recruitment of members of the
host fission machinery, named the endosomal sorting complex required for transport (ESCRT) proteins, which promote membrane fission events
that separate virus particles from the cell (B) An amino acid alignment indicating similarity between SCOTIN-Tsg101, SCOTIN-ALIX (C) Tsg101
recruitment to plasma membrane is blocked by SCOTIN in HIV expression cells. (D) HIV-1 budding defect by SCOTIN overexpression.
CONCLUSION REFERENCES ACKNOWLEDGEMENTS

❑ SCOTIN overexpression reduced the virus replication (Figure 1) Kim N, Kim MJ, Sung PS, Bae YC, Shin EC,
Yoo JY. Interferon-inducible protein SCOTIN This work was supported by a research
❑ Almost all parts of SCOTIN, except CRD domain, are required for HIV- interferes with HCV replication through the
1 inhibition (Figure 2) autolysosomal degradation of NS5A. Nat foundation of Korea (NRF) Grant funded by the
Commun. 2016;7:10631. Korean Govermment (2017R1A5A1015366).
❑ In SCOTIN overexpression cells, decreased puncta formation of Gag
protein is observed, which indicates Gag mulimerization inhibition Draeby I, Woods YL, la Cour JM, Mollerup J,
Bourdon JC, Berchtold MW. The calcium
(Figure 3) binding protein ALG-2 binds and stabilizes Contact information
❑ It is well known that Gag protein recruits Tsg101 to sites of particle Scotin, a p53-inducible gene product localized
at the endoplasmic reticulum membrane. Arch
budding at the plasma membrane (PM) via a PTAP motif and previous Biochem Biophys. 2007;467(1):87-94.
data show that sequence similarity between SCOTIN and Tsg101
(Figure 4B). Contrary to our expectation that SCOTIN would be Martin-Serrano J, Zang T, Bieniasz PD. Role of supernova.yu@gmail.com
recruited at PM instead of Tsg101, SCOTIN block the movement of ESCRT-I in retroviral budding. J Virol.
Tsg101 to PM (Figure 4C) which resulted in HIV budding defect (Figure 2003;77(8):4794-4804.
4D) .