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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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CypA did not impair the nuclear export of influenza virus mRNA.The 293T/CypA+, 293T/CypA−, and CypA re-expression 293T/CypA− cell lines were infected with A/WSN/33 (MOI = 0.1). At 4 h post-infection, the CypA expression level was analyzed by western blotting using a polyclonal anti-CypA antibody in the three cell lines. β-actin was used as a control (A). Nuclear (N) and cytoplasmic (C) components were isolated from the various cell lines. One tenth of the components were used for Western blot by anti-α-tubulin and anti-lamin B1 (B). The rest of the components were used for the RNA extraction and real time PCR (C). M1 and NP mRNA levels were quantified by real-time RT-PCR using gene-specific primers. GAPDH was quantified as an internal control. Data are means ± SD of three separate experiments.
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pone-0031063-g004: CypA did not impair the nuclear export of influenza virus mRNA.The 293T/CypA+, 293T/CypA−, and CypA re-expression 293T/CypA− cell lines were infected with A/WSN/33 (MOI = 0.1). At 4 h post-infection, the CypA expression level was analyzed by western blotting using a polyclonal anti-CypA antibody in the three cell lines. β-actin was used as a control (A). Nuclear (N) and cytoplasmic (C) components were isolated from the various cell lines. One tenth of the components were used for Western blot by anti-α-tubulin and anti-lamin B1 (B). The rest of the components were used for the RNA extraction and real time PCR (C). M1 and NP mRNA levels were quantified by real-time RT-PCR using gene-specific primers. GAPDH was quantified as an internal control. Data are means ± SD of three separate experiments.

Mentions: As was shown, CypA did not affect the transcription and genome replication of influenza virus. To determine if CypA has an effect on viral mRNA nuclear export, we compared the nuclear and cytoplasmic abundance of M1 and NP mRNA upon influenza virus infection at 4 h post-infection in the 293T/CypA+ and 293T/CypA− cell lines. In addition, we also rescued the expression of CypA in the 293T/CypA− cell line and then analyzed the nuclear and cytoplasmic abundance of M1 and NP mRNA upon influenza virus infection. First, we determined the CypA protein level in the 293T/CypA+, 293T/CypA− and “CypA re-expression” 293T/CypA− cell lines (Figure 4A). Then nuclear and cytoplasmic fractions were isolated and analyzed by Western blotting for lamin B1 and α-tubulin (Figure 4B).The results showed that the nuclear/cytoplasmic fractions were isolated well. Further studies indicated that the ratio of nuclear/cytoplasm M1 mRNA was similar in all three lines (Figure 4C). In addition, we obtained similar results for the pattern of NP mRNA (Figure 4C). These results suggested that the presence of CypA in the cell had no effect on the nuclear export of viral mRNA.


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)

CypA did not impair the nuclear export of influenza virus mRNA.The 293T/CypA+, 293T/CypA−, and CypA re-expression 293T/CypA− cell lines were infected with A/WSN/33 (MOI = 0.1). At 4 h post-infection, the CypA expression level was analyzed by western blotting using a polyclonal anti-CypA antibody in the three cell lines. β-actin was used as a control (A). Nuclear (N) and cytoplasmic (C) components were isolated from the various cell lines. One tenth of the components were used for Western blot by anti-α-tubulin and anti-lamin B1 (B). The rest of the components were used for the RNA extraction and real time PCR (C). M1 and NP mRNA levels were quantified by real-time RT-PCR using gene-specific primers. GAPDH was quantified as an internal control. Data are means ± SD of three separate experiments.
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getmorefigures.php?uid=PMC3275614&req=5

pone-0031063-g004: CypA did not impair the nuclear export of influenza virus mRNA.The 293T/CypA+, 293T/CypA−, and CypA re-expression 293T/CypA− cell lines were infected with A/WSN/33 (MOI = 0.1). At 4 h post-infection, the CypA expression level was analyzed by western blotting using a polyclonal anti-CypA antibody in the three cell lines. β-actin was used as a control (A). Nuclear (N) and cytoplasmic (C) components were isolated from the various cell lines. One tenth of the components were used for Western blot by anti-α-tubulin and anti-lamin B1 (B). The rest of the components were used for the RNA extraction and real time PCR (C). M1 and NP mRNA levels were quantified by real-time RT-PCR using gene-specific primers. GAPDH was quantified as an internal control. Data are means ± SD of three separate experiments.
Mentions: As was shown, CypA did not affect the transcription and genome replication of influenza virus. To determine if CypA has an effect on viral mRNA nuclear export, we compared the nuclear and cytoplasmic abundance of M1 and NP mRNA upon influenza virus infection at 4 h post-infection in the 293T/CypA+ and 293T/CypA− cell lines. In addition, we also rescued the expression of CypA in the 293T/CypA− cell line and then analyzed the nuclear and cytoplasmic abundance of M1 and NP mRNA upon influenza virus infection. First, we determined the CypA protein level in the 293T/CypA+, 293T/CypA− and “CypA re-expression” 293T/CypA− cell lines (Figure 4A). Then nuclear and cytoplasmic fractions were isolated and analyzed by Western blotting for lamin B1 and α-tubulin (Figure 4B).The results showed that the nuclear/cytoplasmic fractions were isolated well. Further studies indicated that the ratio of nuclear/cytoplasm M1 mRNA was similar in all three lines (Figure 4C). In addition, we obtained similar results for the pattern of NP mRNA (Figure 4C). These results suggested that the presence of CypA in the cell had no effect on the nuclear export of viral mRNA.

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