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Ubiquitin-specific protease 9X in host cells interacts with herpes simplex virus 1 ICP0.

Sato Y, Kato A, Arii J, Koyanagi N, Kozuka-Hata H, Oyama M, Kawaguchi Y - J. Vet. Med. Sci. (2015)

Bottom Line: The interaction between ICP0 and USP9X was confirmed by co-immunoprecipitation.Notably, USP9X depletion increased the ICP0 abundance and promoted viral replication.These results suggest that USP9X-dependent regulation of ICP0 expression is part of a complex feedback mechanism that facilitates optimal HSV-1 replication.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Virology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.

ABSTRACT
Herpes simplex virus 1 (HSV-1) expresses infected cell protein 0 (ICP0), a multi-functional protein with E3 ubiquitin ligase activity and a critical regulator of the viral life cycle. To obtain novel insights into the molecular mechanism by which ICP0 regulates HSV-1 replication, we analyzed HEp-2 cells infected with HSV-1 by tandem affinity purification and mass spectrometry-based proteomics. This screen identified 50 host-cell proteins that potentially interact with ICP0, including ubiquitin-specific protease 9X (USP9X). The interaction between ICP0 and USP9X was confirmed by co-immunoprecipitation. Notably, USP9X depletion increased the ICP0 abundance and promoted viral replication. These results suggest that USP9X-dependent regulation of ICP0 expression is part of a complex feedback mechanism that facilitates optimal HSV-1 replication.

No MeSH data available.


Related in: MedlinePlus

Co-immunoprecipitation of USP9X with ICP0 in infected cells. (A-B) HEp-2 cellsinfected for 8 hr with wild-type HSV-1 (F) at MOI 5 were harvested (WCE, whole-cellextract) and immunoprecipitated (IP) with anti-Flag, anti-ICP0 (A) or anti-USP9X (B)antibody. Immunoprecipitates were analyzed by immunoblotting (IB) with indicatedantibodies. (C) HeLa cells were infected for 8 hr with wild-type HSV-1 (F) or R7910(ΔICP0) at MOI 5, harvested and immunoprecipitated with anti-ICP0 antibody.Immunoprecipitates were analyzed by immunoblotting with indicated antibodies.
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fig_003: Co-immunoprecipitation of USP9X with ICP0 in infected cells. (A-B) HEp-2 cellsinfected for 8 hr with wild-type HSV-1 (F) at MOI 5 were harvested (WCE, whole-cellextract) and immunoprecipitated (IP) with anti-Flag, anti-ICP0 (A) or anti-USP9X (B)antibody. Immunoprecipitates were analyzed by immunoblotting (IB) with indicatedantibodies. (C) HeLa cells were infected for 8 hr with wild-type HSV-1 (F) or R7910(ΔICP0) at MOI 5, harvested and immunoprecipitated with anti-ICP0 antibody.Immunoprecipitates were analyzed by immunoblotting with indicated antibodies.

Mentions: To confirm the interaction between USP9X and ICP0, we performed co-immunoprecipitation inHEp-2 cells infected with wild-type HSV-1 (F) at MOI 5. Lysates were obtained 8 hrpostinfection, immunoprecipitated with anti-ICP0 or anti-Flag antibody, andimmunoprecipitates were analyzed by immunoblotting with anti-USP9X and anti-ICP0 antibodies.As shown in Fig. 3AFig. 3.


Ubiquitin-specific protease 9X in host cells interacts with herpes simplex virus 1 ICP0.

Sato Y, Kato A, Arii J, Koyanagi N, Kozuka-Hata H, Oyama M, Kawaguchi Y - J. Vet. Med. Sci. (2015)

Co-immunoprecipitation of USP9X with ICP0 in infected cells. (A-B) HEp-2 cellsinfected for 8 hr with wild-type HSV-1 (F) at MOI 5 were harvested (WCE, whole-cellextract) and immunoprecipitated (IP) with anti-Flag, anti-ICP0 (A) or anti-USP9X (B)antibody. Immunoprecipitates were analyzed by immunoblotting (IB) with indicatedantibodies. (C) HeLa cells were infected for 8 hr with wild-type HSV-1 (F) or R7910(ΔICP0) at MOI 5, harvested and immunoprecipitated with anti-ICP0 antibody.Immunoprecipitates were analyzed by immunoblotting with indicated antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4829507&req=5

fig_003: Co-immunoprecipitation of USP9X with ICP0 in infected cells. (A-B) HEp-2 cellsinfected for 8 hr with wild-type HSV-1 (F) at MOI 5 were harvested (WCE, whole-cellextract) and immunoprecipitated (IP) with anti-Flag, anti-ICP0 (A) or anti-USP9X (B)antibody. Immunoprecipitates were analyzed by immunoblotting (IB) with indicatedantibodies. (C) HeLa cells were infected for 8 hr with wild-type HSV-1 (F) or R7910(ΔICP0) at MOI 5, harvested and immunoprecipitated with anti-ICP0 antibody.Immunoprecipitates were analyzed by immunoblotting with indicated antibodies.
Mentions: To confirm the interaction between USP9X and ICP0, we performed co-immunoprecipitation inHEp-2 cells infected with wild-type HSV-1 (F) at MOI 5. Lysates were obtained 8 hrpostinfection, immunoprecipitated with anti-ICP0 or anti-Flag antibody, andimmunoprecipitates were analyzed by immunoblotting with anti-USP9X and anti-ICP0 antibodies.As shown in Fig. 3AFig. 3.

Bottom Line: The interaction between ICP0 and USP9X was confirmed by co-immunoprecipitation.Notably, USP9X depletion increased the ICP0 abundance and promoted viral replication.These results suggest that USP9X-dependent regulation of ICP0 expression is part of a complex feedback mechanism that facilitates optimal HSV-1 replication.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Virology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.

ABSTRACT
Herpes simplex virus 1 (HSV-1) expresses infected cell protein 0 (ICP0), a multi-functional protein with E3 ubiquitin ligase activity and a critical regulator of the viral life cycle. To obtain novel insights into the molecular mechanism by which ICP0 regulates HSV-1 replication, we analyzed HEp-2 cells infected with HSV-1 by tandem affinity purification and mass spectrometry-based proteomics. This screen identified 50 host-cell proteins that potentially interact with ICP0, including ubiquitin-specific protease 9X (USP9X). The interaction between ICP0 and USP9X was confirmed by co-immunoprecipitation. Notably, USP9X depletion increased the ICP0 abundance and promoted viral replication. These results suggest that USP9X-dependent regulation of ICP0 expression is part of a complex feedback mechanism that facilitates optimal HSV-1 replication.

No MeSH data available.


Related in: MedlinePlus