Cyclophilin A Restricts Influenza A Virus Replication through Degradation of the M1 Protein

et al. (2012) Cyclophilin A Restricts Influenza A Virus Replication through Degradation of the M1 Protein. PLoS ONE 7(2): e31063. doi:10.1371/journal.pone.0031063 Cyclophilin A Restricts Influenza A Virus Replication through Degradation of the M1 Protein Xiaoling Liu 0 Zhendong Zhao 0 Chongfeng Xu 0 Lei Sun 0 Jilong Chen 0 Lianfeng Zhang 0 Wenjun 0 Paul Digard, University of Cambridge, United Kingdom 0 1 Center for Molecular Virology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences , Beijing , China , 2 Graduate University of Chinese Academy of Sciences , Beijing, China, 3 China- Japan Joint Laboratory of Molecular Immunology and Molecular Microbiology, Institute of Microbiology, Chinese Academy of Sciences , Beijing , China , 4 Key Laboratory of Human Disease Comparative Medicine, Ministry of Health, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences and Comparative Medical Center, Peking Union Medical College , Beijing , China 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. - Funding: This work was supported by the National Basic Research Program of China (www.973.gov.cn, Grant No. 2011CB504705, 2012CB518900), the National Natural Science Foundation of China (www.nsfc.gov.cn, Grant No. 30972185, 81101253, 30901073), the Chinese Academy of Sciences Innovation projects (www. cas.cn, Grant No. KSCX2-YW-N-054, KSCX2-YW-R-158), the National Key Technologies Research and Development Program of China (www.most.gov.cn, Grant No. 2010BAD04B01), and the Beijing Municipal Natural Science Foundation (www.bjkw.gov.cn, Grant No. 6102018). Wenjun Liu is the principal investigator of the Innovative Research Group of the National Natural Science Foundation of China (www.nsfc.gov.cn, Grant No. 81021003). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Influenza virus is an enveloped negative-sense RNA virus that causes major public health problems worldwide. The matrix protein (M1) is the most abundant protein in the viral particle and forms the bridge between the viral envelope and the core. M1 protein is a multifunctional protein in the influenza virus life cycle including uncoating, transcription, the nuclear export of vRNP, assembly and budding. Several host cell factors have been determined possibly to be required for regulation of influenza virus replication through interacting with M1 at different stages of infection [1,2,3,4]. In the previous study, Cyclophilin A (CypA) was identified to interact with influenza virus M1 protein and impair the early stage of the viral replication [5]. In the present study, CypA might regulate the viral protein stability at the posttranslation level of influenza virus life cycle. Post-translational modification of proteins by ubiquitin is a key regulatory event in many cellular activities, such as signal transduction, transcription, nuclear transport, membrane protein trafficking, autophagy, and immune responses [6]. Previous studies suggest an important involvement of the ubiquitin proteasome system (UPS) in the influenza virus infection. For example, the ubiquitin-vacuolar protein sorting system is required during entry of influenza virus into cells [7]. Further studies indicate that inhibition of the UPS affects influenza virus infection at a postfusion step [8]. Influenza virus inhibits host interferon response through NS1 targeting the ubiquitin ligase TRIM25 [9]. Influenza A virus RNA replication was regulated through the ubiquitination and deubiquitination of NP protein [10]. However, the ubiquitination of influenza A virus M1 protein is still unknown. CypA is a member of the immunophilin superfamily that has peptidyl-prolyl cis-trans isomerase activity. Several lines of evidence implicate that CypA can aid protein folding due to its isomerase activity, and it is also active in cell signaling [11,12,13,14]. In addition, CypA is involved in the life cycles of several viruses, such as human immunodeficiency virus type 1 (HIV-1), influenza virus, vesicular stomatitis virus (VSV), vaccinia virus (VV), hepatitis C virus (HCV) and hepatitis B virus (HBV) [5,15,16,17,18,19,20,21]. Another member of the immunophilin superfamily, Pin1, has been reported to be involved in the UPS. Pin1 stabilizes the human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein and promotes malignant transformation [22]. Pin1 regulates NF-kB signaling through the UPS [23]. In the reports related to influenza virus, CypA was shown to be in the core of the influenza virion [24] and was up-regulated upon infection by avian H9N2 influenza virus in a human gastric carcinoma cell line (AGS) [25]. Furthermore, both human and chicken CypA specifically interacted with the M1 protein and suppressed the viral replication. In addition, the isomerase activity of CypA is not necessary for viral replication [5,26], but the precise functions and roles of CypA in the influenza virus life cycle have not yet been elucidated. Thus, it is of interest to further understand how CypA participates in viral replication. A cell line depleted of endogenous CypA would be a useful model to understand the precise functions of CypA in the influenza virus life cycle. Therefore, in the present study, a stable RNAi 293T cell line with maximally decreased CypA expression (293T/CypA2) was established as described in [27]. The replication of influenza A virus in the 293T/CypA2 and 293T (i.e., 293T/CypA+) cell lines was characterized to further determine the effects of CypA on virus replication. The present data indicated that CypA inhibited influenza virus replication through accelerating degradation of the M1 protein. CypA inhibited influenza A virus replication To better evaluate the functio (...truncated)