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Cyclophilin A restricts influenza A virus replication through degradation of the M1 protein.

Liu X, Zhao Z, Xu C, Sun L, Chen J, Zhang L, Liu W - PLoS ONE (2012)

Bottom Line: However, CypA decreased the viral protein level.Additional studies indicated that CypA enhanced the degradation of M1 through the ubiquitin/proteasome-dependent pathway.Our results suggest that CypA restricts influenza virus replication through accelerating degradation of the M1 protein.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Virology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.

ABSTRACT
Cyclophilin A (CypA) is a typical member of the cyclophilin family of peptidyl-prolyl isomerases and is involved in the replication of several viruses. Previous studies indicate that CypA interacts with influenza virus M1 protein and impairs the early stage of the viral replication. To further understand the molecular mechanism by which CypA impairs influenza virus replication, a 293T cell line depleted for endogenous CypA was established. The results indicated that CypA inhibited the initiation of virus replication. In addition, the infectivity of influenza virus increased in the absence of CypA. Further studies indicated that CypA had no effect on the stages of virus genome replication or transcription and also did not impair the nuclear export of the viral mRNA. However, CypA decreased the viral protein level. Additional studies indicated that CypA enhanced the degradation of M1 through the ubiquitin/proteasome-dependent pathway. Our results suggest that CypA restricts influenza virus replication through accelerating degradation of the M1 protein.

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In vivo ubiquitination assay suggested that CypA increased the ubiquitination of the M1 protein.293T/CypA− cells were transfected with expression constructs for Myc-tagged CypA, FLAG-tagged full-length M1, and His-tagged ubiquitin. Forty hours post-transfection, the proteasome inhibitor MG-132 (dissolved in DMSO) was added to the cells for 4 h at a final concentration of 10 µM. Cell extracts were then immunoprecipitated with Ni2+-NTA beads. The eluted proteins were analyzed by western blotting using an anti-FLAG monoclonal antibody.
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pone-0031063-g007: In vivo ubiquitination assay suggested that CypA increased the ubiquitination of the M1 protein.293T/CypA− cells were transfected with expression constructs for Myc-tagged CypA, FLAG-tagged full-length M1, and His-tagged ubiquitin. Forty hours post-transfection, the proteasome inhibitor MG-132 (dissolved in DMSO) was added to the cells for 4 h at a final concentration of 10 µM. Cell extracts were then immunoprecipitated with Ni2+-NTA beads. The eluted proteins were analyzed by western blotting using an anti-FLAG monoclonal antibody.

Mentions: To further determine if CypA affects the ubiquitination status of M1, an in vivo ubiquitination assay was performed in CypA−, M1- and His-ubiquitin-co-expressing cell lysates. Surprisingly, CypA decreased both the level of non-ubiquitinated M1 and the level of poly-ubiquitinated M1 species (Figure 7). When the proteasomal function was inhibited by MG-132, non- and poly-ubiquitinated M1 species accumulated to higher levels than in the control. Furthermore, CypA over-expression increased the non- and poly-ubiquitinated M1 species.


Cyclophilin A restricts influenza A virus replication through degradation of the M1 protein.

Liu X, Zhao Z, Xu C, Sun L, Chen J, Zhang L, Liu W - PLoS ONE (2012)

In vivo ubiquitination assay suggested that CypA increased the ubiquitination of the M1 protein.293T/CypA− cells were transfected with expression constructs for Myc-tagged CypA, FLAG-tagged full-length M1, and His-tagged ubiquitin. Forty hours post-transfection, the proteasome inhibitor MG-132 (dissolved in DMSO) was added to the cells for 4 h at a final concentration of 10 µM. Cell extracts were then immunoprecipitated with Ni2+-NTA beads. The eluted proteins were analyzed by western blotting using an anti-FLAG monoclonal antibody.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3275614&req=5

pone-0031063-g007: In vivo ubiquitination assay suggested that CypA increased the ubiquitination of the M1 protein.293T/CypA− cells were transfected with expression constructs for Myc-tagged CypA, FLAG-tagged full-length M1, and His-tagged ubiquitin. Forty hours post-transfection, the proteasome inhibitor MG-132 (dissolved in DMSO) was added to the cells for 4 h at a final concentration of 10 µM. Cell extracts were then immunoprecipitated with Ni2+-NTA beads. The eluted proteins were analyzed by western blotting using an anti-FLAG monoclonal antibody.
Mentions: To further determine if CypA affects the ubiquitination status of M1, an in vivo ubiquitination assay was performed in CypA−, M1- and His-ubiquitin-co-expressing cell lysates. Surprisingly, CypA decreased both the level of non-ubiquitinated M1 and the level of poly-ubiquitinated M1 species (Figure 7). When the proteasomal function was inhibited by MG-132, non- and poly-ubiquitinated M1 species accumulated to higher levels than in the control. Furthermore, CypA over-expression increased the non- and poly-ubiquitinated M1 species.

Bottom Line: However, CypA decreased the viral protein level.Additional studies indicated that CypA enhanced the degradation of M1 through the ubiquitin/proteasome-dependent pathway.Our results suggest that CypA restricts influenza virus replication through accelerating degradation of the M1 protein.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Virology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.

ABSTRACT
Cyclophilin A (CypA) is a typical member of the cyclophilin family of peptidyl-prolyl isomerases and is involved in the replication of several viruses. Previous studies indicate that CypA interacts with influenza virus M1 protein and impairs the early stage of the viral replication. To further understand the molecular mechanism by which CypA impairs influenza virus replication, a 293T cell line depleted for endogenous CypA was established. The results indicated that CypA inhibited the initiation of virus replication. In addition, the infectivity of influenza virus increased in the absence of CypA. Further studies indicated that CypA had no effect on the stages of virus genome replication or transcription and also did not impair the nuclear export of the viral mRNA. However, CypA decreased the viral protein level. Additional studies indicated that CypA enhanced the degradation of M1 through the ubiquitin/proteasome-dependent pathway. Our results suggest that CypA restricts influenza virus replication through accelerating degradation of the M1 protein.

Show MeSH
Related in: MedlinePlus