A型流感病毒是一個具有外套膜並由8段負鏈單股RNA所組成的病毒，此RNA基因組會經由轉譯製造出11種病毒蛋白. 其中的Nuclear Export Protein (NEP)已知除了會藉由自己的出核訊息(Nuclear Export Signal, NES)和human chromosome region maintenance 1 protein (hCRM1)有交互作用，並參與在病毒核糖核蛋白(virus ribonucleoprotein, vRNP)的核外運輸外，也能負向調控病毒的轉錄及複製。然而，NEP在流感病毒生活史上或對細胞可能的影響還是有很多未知的地方。
在先前的研究中，我們發現NEP會跟許多細胞蛋白有交互作用，並可能會藉由宿主蛋白─主蛋白合成酶F1聚合體中α及β次單元體在病毒的繁衍中扮演一些角色. 由於近年來的報導中，病毒常被發現會藉由粒線體上的蛋白干擾粒線體，並調控細胞的免疫反應或將其作為能量的來源。我們推測NEP也許是一個多功能的病毒蛋白，並會藉由和α及β次單元體的結合搶奪粒線體ATP合成酶，調控細胞干擾素及ATP的生成，進而促進病毒的繁衍。我們首先利用免疫共沉澱法，在外加核糖核酸酶A的情況下，調查ATP合成酶F1聚合體中α及β次單元體與NEP之間是否為直接的結合。此實驗結果證實α及β次單元體與NEP的結合需要經由RNA的參與，而且GST pull down assay也證明相同的結果。另外，藉由免疫共沉澱法及螢光共軛焦顯微鏡，我們發現在流感病毒感染的細胞中，NEP會經由vRNP和ATP合成酶F1聚合體中α及β次單元體結合，並且和α及β次單元體共同存在粒線體中。在粒線體功能性的分析上，過量表達NEP會降低細胞內ATP及第一型干擾素的量；同時，過量表達ATP合成酶F1聚合體中α次單元體也會協助NEP抑制第一型干擾素(IFN-β)。令人驚訝地，雖然增加或降低α及β次單元體並不會造成細胞內病毒蛋白量的改變，但它們的表達量卻與IFN-β的激發層次呈現負向的調節關係，顯示ATP合成酶F1聚合體中α及β次單元體是拮抗IFN-β免疫反應所需。更有趣的是，降低α及β次單元體卻會使細胞外的病毒量下降，顯示與病毒在胞膜上的組裝或出芽有功能相關。
總結以上，我們認為NEP會經由vRNP和ATP合成酶F1聚合體中α及β次單元體結合，調控粒線體的功能，最後造成細胞外病毒量的變化，而可能影響到病毒繁衍。

Influenza A virus (IAV) is an enveloped virus with a segmented genome of 8 negative sense, single-stranded RNAs, which encode 11 viral proteins. The IAV Nuclear Exporting Protein (NEP) has been reported to involve in virus ribonucleoprotein (vRNP) nuclear export through Nuclear Export Signal (NES)-independent interaction with hCRM1, and can negatively regulate viral genome transcription/replication. However, the functional relevance of NEP in virus life cycle and possible cellular effects remain largely unknown. Our previous results suggested that NEP interacts with many cellular proteins and may have importance in viral egression by interacting with a cellular protein F1α/β ATP synthase. Recent studies indeed showed that certain viruses would subvert the cellular immune response or energy resource through mitochondrial protein to promote viral egression. In this study, we report that NEP is likely a multifunctional protein, which may hijack mitochondria ATP synthase by interacting with the F1α/β subunit to modulate cellular IFN response and cellular ATP level for IAV egression. First, by using in vitro GST pull-down assay and in vivo affinity purification in the presence or absence of RNaseA, we found that NEP interacts with ATP synthase in an RNA dependent manner and is associated with vRNP complex during IAV infection. We also found that NEP inhibited cellular ATP level and type I IFN response in a dose-dependent manner. In addition, overexpression of ATP synthase α subunit enhanced NEP to down-regulate type I IFN response. Surprisingly, after knockdown or over-expression of ATP synthase α or β subunit, intracellular viral protein production was unaffected, yet their expression levels are negatively correlated with the degree of IFN-β response after IAV infection. This result most likely indicated that ATP synthase α and β subunits are required for antagonizing IAV-induced IFN response. Moreover, knockdown of ATP synthase α or β subunit resulted in significant decrease of extracellular viral production, suggesting that ATP synthase α and β subunits have functional relevance with viral assembly or budding. These results collectively indicated that NEP had a role on extracellular viral production by targeting α and β subunits through vRNP in an RNA-dependent manner, and which may also correlate with the interruption of mitochondria function resulting in the disruption of energy production and innate host immune response that may be critical for viral egress.

Anand, S. K. and S. K. Tikoo (2013). "Viruses as modulators of mitochondrial functions." Adv Virol 2013: 738794.
Andrejeva, J., K. S. Childs, D. F. Young, T. S. Carlos, N. Stock, S. Goodbourn and R. E. Randall (2004). "The V proteins of paramyxoviruses bind the IFN-inducible RNA helicase, mda-5, and inhibit its activation of the IFN-β promoter." Proceedings of the National Academy of Sciences of the United States of America 101(49): 17264-17269.
Arnoult, D., F. Soares, I. Tattoli and S. E. Girardin (2011). "Mitochondria in innate immunity." EMBO Rep 12(9): 901-910.
Balaban, R. S. (1990). "Regulation of oxidative phosphorylation in the mammalian cell." American Journal of Physiology - Cell Physiology 258(3): C377-C389.
Basu, D., M. P. Walkiewicz, M. Frieman, R. S. Baric, D. T. Auble and D. A. Engel (2009). "Novel influenza virus NS1 antagonists block replication and restore innate immune function." J Virol 83(4): 1881-1891.
Beran, R. K., B. D. Lindenbach and A. M. Pyle (2009). "The NS4A protein of hepatitis C virus promotes RNA-coupled ATP hydrolysis by the NS3 helicase." J Virol 83(7): 3268-3275.
Broyles, S. S. (1991). "A role for ATP hydrolysis in vaccinia virus early gene transcription. Dissociation of the early transcription factor-promoter complex." Journal of Biological Chemistry 266(23): 15545-15548.
Bruns, A. M., D. Pollpeter, N. Hadizadeh, S. Myong, J. F. Marko and C. M. Horvath (2013). "ATP Hydrolysis Enhances RNA Recognition and Antiviral Signal Transduction by the Innate Immune Sensor, Laboratory of Genetics and Physiology 2 (LGP2)." Journal of Biological Chemistry 288(2): 938-946.
Bullido, R., P. Gomez-Puertas, M. J. Saiz and A. Portela (2001). "Influenza A virus NEP (NS2 protein) downregulates RNA synthesis of model template RNAs." J Virol 75(10): 4912-4917.
Chen, J., S. Huang and Z. Chen (2010). "Human cellular protein nucleoporin hNup98 interacts with influenza A virus NS2/nuclear export protein and overexpression of its GLFG repeat domain can inhibit virus propagation." J Gen Virol 91(Pt 10): 2474-2484.
Chen W Fau - Calvo, P. A., D. Calvo Pa Fau - Malide, J. Malide D Fau - Gibbs, U. Gibbs J Fau - Schubert, I. Schubert U Fau - Bacik, S. Bacik I Fau - Basta, R. Basta S Fau - O'Neill, J. O'Neill R Fau - Schickli, P. Schickli J Fau - Palese, P. Palese P Fau - Henklein, J. R. Henklein P Fau - Bennink, J. W. Bennink Jr Fau - Yewdell and J. W. Yewdell (2001). "A novel influenza A virus mitochondrial protein that induces cell death." Nature Medicine 7(1078-8956 (Print)).
Chua, M. A., S. Schmid, J. T. Perez, R. A. Langlois and B. R. Tenoever (2013). "Influenza A virus utilizes suboptimal splicing to coordinate the timing of infection." Cell Rep 3(1): 23-29.
Civril, F., M. Bennett, M. Moldt, T. Deimling, G. Witte, S. Schiesser, T. Carell and K. P. Hopfner (2011). "The RIG‐I ATPase domain structure reveals insights into ATP‐dependent antiviral signalling." EMBO reports 12(11): 1127-1134.
de Castro, I. F., L. Volonte and C. Risco (2013). "Virus factories: biogenesis and structural design." Cell Microbiol 15(1): 24-34.
Devenish, R. J., M. Prescott and A. J. W. Rodgers (2008). "The Structure and Function of Mitochondrial F1F0‐ATP Synthases." International Review of Cell and Molecular Biology 267: 1-58.
El-Bacha, T. and A. T. Da Poian (2013). "Virus-induced changes in mitochondrial bioenergetics as potential targets for therapy." Int J Biochem Cell Biol 45(1): 41-46.
Garrus, J. E., U. K. von Schwedler, O. W. Pornillos, S. G. Morham, K. H. Zavitz, H. E. Wang, D. A. Wettstein, K. M. Stray, M. Côté, R. L. Rich, D. G. Myszka and W. I. Sundquist (2001). "Tsg101 and the Vacuolar Protein Sorting Pathway Are Essential for HIV-1 Budding." Cell 107(1): 55-65.
Gorai, T., H. Goto, T. Noda, T. Watanabe, H. Kozuka-Hata, M. Oyama, R. Takano, G. Neumann, S. Watanabe and Y. Kawaoka (2012). "F1Fo-ATPase, F-type proton-translocating ATPase, at the plasma membrane is critical for efficient influenza virus budding." Proc Natl Acad Sci U S A 109(12): 4615-4620.
Huang, S., J. Chen, Q. Chen, H. Wang, Y. Yao, J. Chen and Z. Chen (2013). "A second CRM1-dependent nuclear export signal in the influenza A virus NS2 protein contributes to the nuclear export of viral ribonucleoproteins." J Virol 87(2): 767-778.
Hui, E. K. and D. P. Nayak (2001). "Role of ATP in influenza virus budding." Virology 290(2): 329-341.
Klumpp, K., M. J. Ford and R. W. Ruigrok (1998). "Variation in ATP requirement during influenza virus transcription." Journal of General Virology 79(5): 1033-1045.
Le Goffic, R., J. Pothlichet, D. Vitour, T. Fujita, E. Meurs, M. Chignard and M. Si-Tahar (2007). "Cutting Edge: Influenza A Virus Activates TLR3-Dependent Inflammatory and RIG-I-Dependent Antiviral Responses in Human Lung Epithelial Cells." The Journal of Immunology 178(6): 3368-3372.
Mancini, E. and R. Tuma (2012). Mechanism of RNA Packaging Motor. Viral Molecular Machines. M. G. Rossmann and V. B. Rao, Springer US. 726: 609-629.
McBride, H. M., M. Neuspiel and S. Wasiak (2006). "Mitochondria: more than just a powerhouse." Curr Biol 16(14): R551-560.
Medina, R. A. and A. Garcia-Sastre (2011). "Influenza A viruses: new research developments." Nat Rev Microbiol 9(8): 590-603.
Meylan, E., J. Curran, K. Hofmann, D. Moradpour, M. Binder, R. Bartenschlager and J. Tschopp (2005). "Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus." Nature 437(7062): 1167-1172.
Neumann, G., M. T. Hughes and Y. Kawaoka (2000). "Influenza A virus NS2 protein mediates vRNP nuclear export through NES-independent interaction with hCRM1." EMBO J 19(24): 6751-6758.
O'Neill, R. E., J. Talon and P. Palese (1998). "The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins." EMBO J 17(1): 288-296.
Ohta, A. and Y. Nishiyama (2011). "Mitochondria and viruses." Mitochondrion 11(1): 1-12.
Pérez, L. and L. Carrasco (1994). "Involvement of the vacuolar H+-ATPase in animal virus entry." Journal of General Virology 75(10): 2595-2606.
Palese, P. and M. Shaw (2001). "Orthomyxoviridae: the viruses and their replication. ." Fields Virology, 5th edition: 1647-1689.
Paterson, D. and E. Fodor (2012). "Emerging Roles for the Influenza A Virus Nuclear Export Protein (NEP)." PLoS Pathog 8(12): e1003019.
Peisley, A., B. Wu, H. Yao, T. Walz and S. Hur (2013). "RIG-I Forms Signaling-Competent Filaments in an ATP-Dependent, Ubiquitin-Independent Manner." Molecular cell 51(5): 573-583.
Puig, O., F. Caspary, G. Rigaut, B. Rutz, E. Bouveret, E. Bragado-Nilsson, M. Wilm and B. Seraphin (2001). "The tandem affinity purification (TAP) method: a general procedure of protein complex purification." Methods 24(3): 218-229.
Rambaut, A., O. G. Pybus, M. I. Nelson, C. Viboud, J. K. Taubenberger and E. C. Holmes (2008). "The genomic and epidemiological dynamics of human influenza A virus." Nature 453(7195): 615-619.
Reeves, M. B., A. A. Davies, B. P. McSharry, G. W. Wilkinson and J. H. Sinclair (2007). "Complex I Binding by a Virally Encoded RNA Regulates Mitochondria-Induced Cell Death." Science 316(5829): 1345-1348.
Richardson Jc Fau - Akkina, R. K. and R. K. Akkina (1991). "NS2 protein of influenza virus is found in purified virus and phosphorylated in infected cells." Arch Virol 116(0304-8608 (Print)).
Robb, N. C., D. Jackson, F. T. Vreede and E. Fodor (2010). "Splicing of influenza A virus NS1 mRNA is independent of the viral NS1 protein." J Gen Virol 91(Pt 9): 2331-2340.
Robb, N. C., M. Smith, F. T. Vreede and E. Fodor (2009). "NS2/NEP protein regulates transcription and replication of the influenza virus RNA genome." J Gen Virol 90(Pt 6): 1398-1407.
Rojo G Fau - Chamorro, M., M. L. Chamorro M Fau - Salas, E. Salas Ml Fau - Vinuela, J. M. Vinuela E Fau - Cuezva, J. Cuezva Jm Fau - Salas and J. Salas (1998). "Migration of mitochondria to viral assembly sites in African swine fever virus-infected cells." Journal of Virology 72(0022-538X (Print)).
Rossman, J. S. and R. A. Lamb (2011). "Influenza virus assembly and budding." Virology 411(2): 229-236.
Sato, M., H. Suemori, N. Hata, M. Asagiri, K. Ogasawara, K. Nakao, T. Nakaya, M. Katsuki, S. Noguchi, N. Tanaka and T. Taniguchi (2000). "Distinct and Essential Roles of Transcription Factors IRF-3 and IRF-7 in Response to Viruses for IFN-±/² Gene Induction." Immunity 13(4): 539-548.
Shimizu, T., N. Takizawa, K. Watanabe, K. Nagata and N. Kobayashi (2011). "Crucial role of the influenza virus NS2 (NEP) C-terminal domain in M1 binding and nuclear export of vRNP." FEBS Lett 585(1): 41-46.
Stauber N Fau - Martinez-Costas, J., G. Martinez-Costas J Fau - Sutton, K. Sutton G Fau - Monastyrskaya, P. Monastyrskaya K Fau - Roy and P. Roy (1997). "Bluetongue virus VP6 protein binds ATP and exhibits an RNA-dependent ATPase function and a helicase activity that catalyze the unwinding of double-stranded RNA substrates." Journal of Virology 71(0022-538X (Print)).
Stock D Fau - Leslie, A. G., J. E. Leslie Ag Fau - Walker and J. E. Walker (1999). "Molecular architecture of the rotary motor in ATP synthase." Science 286(0036-8075 (Print)).
Stuchell-Brereton, M. D., J. J. Skalicky, C. Kieffer, M. A. Karren, S. Ghaffarian and W. I. Sundquist (2007). "ESCRT-III recognition by VPS4 ATPases." Nature 449(7163): 740-744.
Sun, S., V. B. Rao and M. G. Rossmann (2010). "Genome packaging in viruses." Current Opinion in Structural Biology 20(1): 114-120.
Wang, C., X. Liu and B. Wei (2011). "Mitochondrion: an emerging platform critical for host antiviral signaling." Expert Opinion on Therapeutic Targets 15(5): 647-665.
Watanabe, R. and R. A. Lamb (2010). "Influenza virus budding does not require a functional AAA+ ATPase, VPS4." Virus Research 153(1): 58-63.
West Ap Fau - Shadel, G. S., S. Shadel Gs Fau - Ghosh and S. Ghosh (2011). "Mitochondria in innate immune responses." Nat Rev Immunol 6(1474-1741 (Electronic)).
Wise, H. M., A. Foeglein, J. Sun, R. M. Dalton, S. Patel, W. Howard, E. C. Anderson, W. S. Barclay and P. Digard (2009). "A Complicated Message: Identification of a Novel PB1-Related Protein Translated from Influenza A Virus Segment 2 mRNA." Journal of Virology 83(16): 8021-8031.
潘妍如 (2011). A型流感病毒NEP的蛋白質體學及NEP對粒線體ATP合成酶在ATP能量產生及病毒繁衍的可能交互作用. 碩士, 國立成功大學.