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- Open Access
High level expression of the anti-retroviral protein APOBEC3G is induced by influenza A virus but does not confer antiviral activity
© Pauli et al; licensee BioMed Central Ltd. 2009
- Received: 31 October 2008
- Accepted: 16 April 2009
- Published: 16 April 2009
Human APOBEC3G is an antiretroviral protein that was described to act via deamination of retroviral cDNA. However, it was suggested that APOBEC proteins might act with antiviral activity by yet other mechanisms and may also possess RNA deamination activity. As a consequence there is an ongoing debate whether APOBEC proteins might also act with antiviral activity on other RNA viruses. Influenza A viruses are single-stranded RNA viruses, capable of inducing a variety of antiviral gene products. In searching for novel antiviral genes against these pathogens, we detected a strong induction of APOBEC3G but not APOBEC3F gene transcription in infected cells. This upregulation appeared to be induced by the accumulation of viral RNA species within the infected cell and occurred in an NF-κB dependent, but MAP kinase independent manner. It further turned out that APOBEC expression is part of a general IFNβ response to infection. However, although strongly induced, APOBEC3G does not negatively affect influenza A virus propagation.
- A549 Cell
- Deamination Activity
- Human H5N1 Influenza Virus
- Human APOBEC3G
In patients infected with HIV-1, the expression of human apolipoprotein (apo) B mRNA editing enzyme catalytic polypeptide 1-like protein 3G (APOBEC3G) was observed to be elevated , although this was not confirmed in cell culture experiments [2, 3]. Members of the APOBEC3 family are known to act with anti-retroviral activity against HIV [4, 5], but they also inhibit replication of hepatitis B virus (HBV) , and adeno-associated virus type 2 . The anti-retroviral activity of human APOBEC3 proteins is probably conferred by cytidine deamination of the newly synthesized first viral cDNA strand. This mechanism is counteracted by the HIV-1 protein virion infectivity factor (Vif) [8–12]. However, human APOBEC3 proteins may not only have anti-retroviral or anti-HBV activity. Two findings have triggered a broader interest in these proteins with regard to a potential antiviral action against RNA viruses. First, besides its DNA deamination activity, human APOBEC3 proteins were reported to also possess RNA deamination activity . Second, DNA deamination activity may not be the only antiviral action of these proteins [13–16] suggesting that APOBEC3s might possess functions that render them effective against other viruses, which do not have any DNA-intermediates during replication such as influenza A virus.
The IKK2/NF-κB module is another influenza virus-activated signalling cascade that is known to regulate a variety of genes. This includes IFNβ transcription, which is controlled by an enhanceosome, composed of the transcription factors IRF3/7, NF-κB, and AP-1 . To assess the involvement of IKK2 and NF-κB in virus-induced APOBEC3G expression, we used A549 cells that were retrovirally transduced with the vector pEGZ-IKK2KD. This transduction allows for the stable expression of the dominant negative mutant of IκB kinase 2 (IKK2), an approach that has been successfully used previously to efficiently blunt NF-κB activity [25, 26]. Upon infection of these mutant-expressing cells, APOBEC3G mRNA levels were reduced compared to control cells (Figure 2A) to a similar extent that was observed for the IFNβ gene (Figure 2B). The same pattern of APOBEC3G expression was also observed in infected cells pre-treated with the NF-κB inhibitor BAY 11–7085 (40 μM) (Figure 2C). Thus, NF-κB activity appeared to be crucial for viral APOBEC3G induction.
To independently analyse whether NF-κB might play a role in APOBEC3G induction, we stimulated cells with TNFα (20 ng/ml), a very strong activator of NF-κB . However, TNFα stimulation did not result in enhanced APOBEC3G gene transcription (data not shown), indicating that NF-κB activity alone is not sufficient to induce human APOBEC3G gene transcription.
Influenza virus infection results in type I IFN production (Figure 2B) and subsequent expression of IFN-responsive genes [28–30]. So far, it was not clear from the literature whether human APOBEC3 genes are induced by type I IFNs. While IFN-dependency was reported for the hepatoma cell lines HepG2 and Huh7 [18, 31] and for macrophages , human APOBEC3 proteins are not inducible in H9 cells by type I and type II IFN . To specifically address this issue for the lung epithelial cell line used in our study, A549 cells were incubated for different time periods with recombinant IFNβ (100 U/ml) (PBL), and the levels of human APOBEC3G and human APOBEC3F mRNAs were determined by qRT-PCR (Figure 2D).
IFNβ stimulation led to a nearly 20-fold induction of the human APOBEC3G mRNA (Figure 2D), which could also be observed at the protein level (Figure 2E); by contrast, the human APOBEC3F mRNA was not affected at all (Figure 2D). Strikingly, this pattern of human APOBEC3G versus human APOBEC3F expression exactly matched the results obtained upon virus infection (Figure 1B). This suggests that IFNβ, expressed upon virus infection in an NF-κB dependent manner, may be an indirect trigger of human APOBEC3G expression, leaving still open the question about the initial viral inducer.
The following reagent was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: anti-ApoC17 from Dr. Klaus Strebel. This work was supported by several grants from the Deutsche Forschungsgemeinschaft (DFG) (Lu477-11/2, SFB293 A17, Graduate School GRK1409), the Interdisciplinary Clinical Research Centre (IZKF) of the University of Münster, the FluResearchNet funded by the German Ministry of Education and Research (BMBF), and the EC funded STREP EUROFLU.
- Ulenga NK, Sarr AD, Thakore-Meloni S, Sankale JL, Eisen G, Kanki PJ: Relationship between human immunodeficiency type 1 infection and expression of human APOBEC3G and APOBEC3F. J Infect Dis. 2008, 198: 486-492. 10.1086/590212.View ArticlePubMedGoogle Scholar
- Stopak K, de Noronha C, Yonemoto W, Greene WC: HIV-1 Vif blocks the antiviral activity of APOBEC3G by impairing both its translation and intracellular stability. Mol Cell. 2003, 12: 591-601. 10.1016/S1097-2765(03)00353-8.View ArticlePubMedGoogle Scholar
- Mehle A, Strack B, Ancuta P, Zhang C, McPike M, Gabuzda D: Vif overcomes the innate antiviral activity of APOBEC3G by promoting its degradation in the ubiquitin-proteasome pathway. J Biol Chem. 2004, 279: 7792-7798. 10.1074/jbc.M313093200.View ArticlePubMedGoogle Scholar
- Bishop KN, Holmes RK, Sheehy AM, Davidson NO, Cho SJ, Malim MH: Cytidine deamination of retroviral DNA by diverse APOBEC proteins. Curr Biol. 2004, 14: 1392-1396. 10.1016/j.cub.2004.06.057.View ArticlePubMedGoogle Scholar
- Wiegand HL, Doehle BP, Bogerd HP, Cullen BR: A second human antiretroviral factor, APOBEC3F, is suppressed by the HIV-1 and HIV-2 Vif proteins. EMBO J. 2004, 23: 2451-2458. 10.1038/sj.emboj.7600246.PubMed CentralView ArticlePubMedGoogle Scholar
- Turelli P, Mangeat B, Jost S, Vianin S, Trono D: Inhibition of hepatitis B virus replication by APOBEC3G. Science. 2004, 303: 1829-10.1126/science.1092066.View ArticlePubMedGoogle Scholar
- Chen H, Lilley CE, Yu Q, Lee DV, Chou J, Narvaiza I, Landau NR, Weitzman MD: APOBEC3A is a potent inhibitor of adeno-associated virus and retrotransposons. Curr Biol. 2006, 16: 480-485. 10.1016/j.cub.2006.01.031.View ArticlePubMedGoogle Scholar
- Sheehy AM, Gaddis NC, Choi JD, Malim MH: Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature. 2002, 418: 646-650. 10.1038/nature00939.View ArticlePubMedGoogle Scholar
- Harris RS, Bishop KN, Sheehy AM, Craig HM, Petersen-Mahrt SK, Watt IN, Neuberger MS, Malim MH: DNA deamination mediates innate immunity to retroviral infection. Cell. 2003, 113: 803-809. 10.1016/S0092-8674(03)00423-9.View ArticlePubMedGoogle Scholar
- Mangeat B, Turelli P, Caron G, Friedli M, Perrin L, Trono D: Broad antiretroviral defence by human APOBEC3G through lethal editing of nascent reverse transcripts. Nature. 2003, 424: 99-103. 10.1038/nature01709.View ArticlePubMedGoogle Scholar
- Goila-Gaur R, Strebel K: HIV-1 Vif, APOBEC, and intrinsic immunity. Retrovirology. 2008, 5: 51-10.1186/1742-4690-5-51.PubMed CentralView ArticlePubMedGoogle Scholar
- Pillai SK, Wong JK, Barbour JD: Turning up the volume on mutational pressure: is more of a good thing always better? (A case study of HIV-1 Vif and APOBEC3). Retrovirology. 2008, 5: 26-10.1186/1742-4690-5-26.PubMed CentralView ArticlePubMedGoogle Scholar
- Bishop KN, Holmes RK, Sheehy AM, Malim MH: APOBEC-mediated editing of viral RNA. Science. 2004, 305: 645-10.1126/science.1100658.View ArticlePubMedGoogle Scholar
- Newman EN, Holmes RK, Craig HM, Klein KC, Lingappa JR, Malim MH, Sheehy AM: Antiviral function of APOBEC3G can be dissociated from cytidine deaminase activity. Curr Biol. 2005, 15: 166-170. 10.1016/j.cub.2004.12.068.View ArticlePubMedGoogle Scholar
- Shindo K, Takaori-Kondo A, Kobayashi M, Abudu A, Fukunaga K, Uchiyama T: The enzymatic activity of CEM15/Apobec-3G is essential for the regulation of the infectivity of HIV-1 virion but not a sole determinant of its antiviral activity. J Biol Chem. 2003, 278: 44412-44416. 10.1074/jbc.C300376200.View ArticlePubMedGoogle Scholar
- Cho SJ, Drechsler H, Burke RC, Arens MQ, Powderly W, Davidson NO: APOBEC3F and APOBEC3G mRNA levels do not correlate with human immunodeficiency virus type 1 plasma viremia or CD4+ T-cell count. J Virol. 2006, 80: 2069-2072. 10.1128/JVI.80.4.2069-2072.2006.PubMed CentralView ArticlePubMedGoogle Scholar
- Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods. 2001, 25: 402-408. 10.1006/meth.2001.1262.View ArticlePubMedGoogle Scholar
- Tanaka Y, Marusawa H, Seno H, Matsumoto Y, Ueda Y, Kodama Y, Endo Y, Yamauchi J, Matsumoto T, Takaori-Kondo A, Ikai I, Chiba T: Anti-viral protein APOBEC3G is induced by interferon-alpha stimulation in human hepatocytes. Biochem Biophys Res Commun. 2006, 341: 314-319. 10.1016/j.bbrc.2005.12.192.View ArticlePubMedGoogle Scholar
- Muckenfuss H, Hamdorf M, Held U, Perkovic M, Löwer J, Cichutek K, Flory E, Schumann GG, Münk C: APOBEC3 proteins inhibit human LINE-1 retrotransposition. J Biol Chem. 2006, 281: 22161-22172. 10.1074/jbc.M601716200.View ArticlePubMedGoogle Scholar
- Mariani R, Chen D, Schröfelbauer B, Navarro F, König R, Bollmann B, Münk C, Nymark-McMahon H, Landau NR: Species-specific exclusion of APOBEC3G from HIV-1 virions by Vif. Cell. 2003, 114: 21-31. 10.1016/S0092-8674(03)00515-4.View ArticlePubMedGoogle Scholar
- Ludwig S: Exploited defense: how influenza viruses take advantage of antiviral signaling responses. Future Virol. 2007, 2: 91-100. 10.2217/174607220.127.116.11.View ArticleGoogle Scholar
- Ludwig S, Pleschka S, Planz O, Wolff T: Ringing the alarm bells: signalling and apoptosis in influenza virus infected cells. Cell Microbiol. 2006, 8: 375-386. 10.1111/j.1462-5822.2005.00678.x.View ArticlePubMedGoogle Scholar
- Rose KM, Marin M, Kozak SL, Kabat D: Transcriptional regulation of APOBEC3G, a cytidine deaminase that hypermutates human immunodeficiency virus. J Biol Chem. 2004, 279: 41744-41749. 10.1074/jbc.M406760200.View ArticlePubMedGoogle Scholar
- Ludwig S, Hoffmeyer A, Moebeler M, Kilian K, Häfner H, Neufeld B, Han J, Rapp UR: The stress inducer arsenite activates mitogen-activated protein kinase extracellular signal-regulated kinases 1 and 2 via a MAPK kinase 6/p38-dependent pathway. J Biol Chem. 1998, 273: 1917-1922. 10.1074/jbc.273.4.1917.View ArticlePubMedGoogle Scholar
- Wurzer WJ, Ehrhardt C, Pleschka S, Berberich-Siebelt F, Wolff T, Walczak H, Planz O, Ludwig S: NF-kappaB-dependent induction of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas/FasL is crucial for efficient influenza virus propagation. J Biol Chem. 2004, 279: 30931-30937. 10.1074/jbc.M403258200.View ArticlePubMedGoogle Scholar
- Viemann D, Goebeler M, Schmid S, Klimmek K, Sorg C, Ludwig S, Roth J: Transcriptional profiling of IKK2/NF-kappa B- and p38 MAP kinase-dependent gene expression in TNF-alpha-stimulated primary human endothelial cells. Blood. 2004, 103: 3365-3373. 10.1182/blood-2003-09-3296.View ArticlePubMedGoogle Scholar
- Cao Z, Tanaka M, Regnier C, Rothe M, Yamit-hezi A, Woronicz JD, Fuentes ME, Durnin MH, Dalrymple SA, Goeddel DV: NF-kappa B activation by tumor necrosis factor and interleukin-1. Cold Spring Harb Symp Quant Biol. 1999, 64: 473-483. 10.1101/sqb.1999.64.473.View ArticlePubMedGoogle Scholar
- Geiss G, Jin G, Guo J, Bumgarner R, Katze MG, Sen GC: A comprehensive view of regulation of gene expression by double-stranded RNA-mediated cell signaling. J Biol Chem. 2001, 276: 30178-30182.View ArticlePubMedGoogle Scholar
- Geiss GK, An MC, Bumgarner RE, Hammersmark E, Cunningham D, Katze MG: Global impact of influenza virus on cellular pathways is mediated by both replication-dependent and -independent events. J Virol. 2001, 75: 4321-4331. 10.1128/JVI.75.9.4321-4331.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Geiss GK, Salvatore M, Tumpey TM, Carter VS, Wang X, Basler CF, Taubenberger JK, Bumgarner RE, Palese P, Katze MG, Garcia-Sastre A: Cellular transcriptional profiling in influenza A virus-infected lung epithelial cells: the role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza. Proc Natl Acad Sci USA. 2002, 99: 10736-10741. 10.1073/pnas.112338099.PubMed CentralView ArticlePubMedGoogle Scholar
- Bonvin M, Achermann F, Greeve I, Stroka D, Keogh A, Inderbitzin D, Candinas D, Sommer P, Wain-Hobson S, Vartanian JP, Greeve J: Interferon-inducible expression of APOBEC3 editing enzymes in human hepatocytes and inhibition of hepatitis B virus replication. Hepatology. 2006, 43: 1364-1374. 10.1002/hep.21187.View ArticlePubMedGoogle Scholar
- Peng G, Lei KJ, Jin W, Greenwell-Wild T, Wahl SM: Induction of APOBEC3 family proteins, a defensive maneuver underlying interferon-induced anti-HIV-1 activity. J Exp Med. 2006, 203: 41-46. 10.1084/jem.20051512.PubMed CentralView ArticlePubMedGoogle Scholar
- Hornung V, Ellegast J, Kim S, Brzozka K, Jung A, Kato H, Poeck H, Akira S, Conzelmann KK, Schlee M, Endres S, Hartmann G: 5'-Triphosphate RNA is the ligand for RIG-I. Science. 2006, 314: 994-997. 10.1126/science.1132505.View ArticlePubMedGoogle Scholar
- Pichlmair A, Schulz O, Tan CP, Naslund TI, Liljestrom P, Weber F, Reis e Sousa C: RIG-I-mediated antiviral responses to single-stranded RNA bearing 5'-phosphates. Science. 2006, 314: 997-1001. 10.1126/science.1132998.View ArticlePubMedGoogle Scholar
- Chen K, Huang J, Zhang C, Huang S, Nunnari G, Wang FX, Tong X, Gao L, Nikisher K, Zhang H: Alpha interferon potently enhances the anti-human immunodeficiency virus type 1 activity of APOBEC3G in resting primary CD4 T cells. J Virol. 2006, 80: 7645-7657. 10.1128/JVI.00206-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Kremer M, Suezer Y, Martinez-Fernandez Y, Munk C, Sutter G, Schnierle BS: Vaccinia virus replication is not affected by APOBEC3 family members. Virol J. 2006, 3: 86-10.1186/1743-422X-3-86.PubMed CentralView ArticlePubMedGoogle Scholar
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