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.
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.
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