Gene Regulation Of Antiviral APOBEC3G And APOBEC3F

Some intracellular molecules are discovered to function as the front line molecules of host defense against viral infection. Intracellar APOBEC proteins block viral replication by viral genome hypermutation of cytidine deaminase or unknown non-edited mechanism in the noncytolytic process. Among these deaminase, APOBEC3G(A3G) is the first one to be identified as an anti-HIV-1 factor. Recently, it is found to have broad antiviral activity against retrovirus, hepatitisB virus and endogenous retroelements. Another member of this family, APOBEC3F also possesses similar antiviral property. They may be incorporated into HTV-1 Δvif virions and leads to mutaion of deoxycytidine (dC) to deoxyuridine (dU) in newly synthesized minus strand DNA during reverse transcription in the next round of infection, resulting G-to-A hypermutation in viral genome and viral inactivation. Except this classical antiviral manner, they also can restrict virus infection by the deaminase-independent way. However, HIV-1 has evolved a protein named Vif(virus infectivity factor) to counteract A3G and A3F. It is well known that Vif targets A3G for ubiquitin-mediated proteasome degradation byforming a SCF-like E3 ubiquitin ligase containing Cullin 5 and Elongins B and C (Cul5-EloB-EloC-Rbx1). So it seems that the battle between A3G/A3F(host) and Vif(virus) may influence HIV pathogenesis. And induction of A3G/A3F expression in vivo is regarded as an effective antiviral strategy.Part â…  STAT1-Independent Cell Type-Specific Regulation of Antiviral APOBEC3G by IFN-αInterferons(IFNs) are widely used to treat disease of virus infection in clinic. The classical models assert that IFN-α induces transcription through a STAT1: STAT2 heterodimer in complex with the IFN regulatory factor (IRF)-9 cofactor, which binds to an IFN-stimulated response element (ISRE) in the promoter of IFN-α-responsive genes. IFN-γ pathway uses the STAT1 homodimer without a cofactor, which binds to the γ-activated sequence in gene promoters.In this study, the induction level of A3G expression by IFNs in various cells was examined by qRT-PCR. These cells included primary cells(hepatocytes, macrophages, and CD4⁺T cells) and cell lines(liver carcinoma cells,H9 cells and 293T cells).All of these cells, except T and 293T cells, up-regulated A3G mRNA in response to IFN-α. In liver cells A3G mRNA was also increased in treatment with IFN-γ. This showed that induction of A3G by IFN-α was cell-type dependent. In order to know whether there are quantitative differences in the levels of A3G after IFN-α treatment, we compared the A3G expression levels in control primary hepatocytes and cells treated with IFN-α from four donors (78, 79, 80, and 85). The results showed that some variation in A3G expression could be noted between the untreated cells, but a more marked variation could be observed after IFN treatment, indicating that the induction of A3G by IFN-α was donor dependent.The PKC-δ isoform was reported to be the kinase responsible for phosphorylating STAT1 at Ser⁷²⁷, a modification reportedly required for fulltranscriptional activation of ISG. We observed that preincubation of Hep3B cells with Rottlerin, reported to be a specific inhibitor of PKC-δ, for a hour before addition of IFN-α inhibited IFN-mediated induction of A3G. However, induction of other IFN-α-responsive genes such as PKR and ISG15 was not affected by Rottlerin. General inhibitors did not inhibit A3G induction by IFN-α in Hep3B cells, suggesting that none of the PKC isoforms participated in IFN-α signaling in liver cells. STAT1 was constitutively phosphorylated at Ser⁷²⁷ in Hep3B cells, but this phosphorylation was enhanced by IFN-α treatment. Rottlerin also did not reduce the enhanced phosphorylation of STAT1 at Ser⁷²⁷ by IFN-α. And we also found the Rottlerin did not affect the stability of A3GmRNA after IFN-α induction. Therefore, the specific inhibition of IFN-α-induced A3G mRNA expression by Rottlerin is apparently independent of PKC-δ and STAT1 activation.The results of siRNA experiments showed that STAT1 knockdown did not block IFN-α-mediated induction of A3G in Hep3B cells. However, this pathway was significantly inhibited by STAT2 siRNA. Phosphorylation of STAT1 at Tyr⁷⁰¹ by Jak is required for dimerization and transcription activation. STAT1 was activated by Jak in Hep3B cells, but it was not involved in IFN-α-mediated induction of A3G. Inhibition of the Jak potently blocked the induction of A3G by IFN-α, indicating that A3G required Jak for transcriptional activation. Therefore, induction of A3G by IFN-α was independent of STAT1, but dependent of STAT2 in Hep3B cells. IRF-9 expression was greatly increased in treatment with IFN-α, but induction of A3G and other ISG by IFN-α was reduced by IRF-9 siRNA. Hence IRF-9 was required for IFN-α induction of A3G and other ISG in Hep3B cells.We next investigated whether STAT1 was functional and required for IFN-γ pathway. We treated Hep3B cells with STAT1 and STAT2 siRNA. Then the induction of A3G and other ISG by IFN-γ was examined. As expected, knockdownof STAT1, but not STAT2 protein effectively blocked the induction of A3G by IFN-γ. This showed that STAT1 was required for the IFN-γ pathway of gene transcription in Hep3B cells.To determine whether our observation of the STAT1-independent IFN-α gene expression was unique to liver cells, we tested whether STAT1 was required in 293T cells for the IFN-α pathway of transcription. We firstly transfected 293T cells with control, STAT1 or STAT2 siRNA, then treated cells with either IFN-α and IFN-γ. Results showed that A3G was not inducible by IFN-α and IFN-γ in this cell line. But IFN-α induction of other ISG such as ISG15, MX1 and LMP2 was inhibited by both STAT1 and STAT2 siRNA. IFN-γ induction of IRF-1 as well as LMP2 was inhibited specifically by STAT1 siRNA and not STAT2 siRNA in 293T cells. Therefore, STAT1 was required for both the IFN-α and IFN-γ induction of ISG in 293T cells, consistent with the classical model of IFN-signaling complexes.Part â…¡ cell type-specific regulation of antiviral APOBEC3F by IFNsA3F and A3G are coordinately expressed in a wide range of human tissues and cells such as T cells, Macrophage cells and so on. Through analysis of the nucleotide sequence of A3G gene upstream region, it was found that the high sequence homology between A3G and A3F upstream region was existed. This indicated that gene expression of A3G and A3F might be regulated by similar transcription factor(s) or cis-regulator elment(s).In the present study the ability of IFNs to up-regulate A3F expression in various cells was also tested. Using a qRT-PCR method, it was observed that A3F was upregulated by IFN-α and IFN-γ in primary hepatocytes and multiple liver cell lines. In HepG2 cells, IFN-α induced A3F expression peaked between 4 to 12 hours after treatment. IFN-γ induced A3F expression peaked between 8 to 24 hours after treatment. Similar to HepG2 cells, both IFN-α and IFN-γ induced A3F expressionin Hep3B cells in a dose- and time-dependent fashion. The effect of IFN treatment on A3F expression in primary hepatocytes from several healthy donors was also determined. The results showed that A3F induction by IFN-α varied widely between approximately 2 to 9-fold among various donors. And IFN-γ was less effective than IFN-α in upregulating A3F in the primary hepatocytes although IRF-1 was efficiently induced by IFN-γ in these primary cells.The ability of IFN-α to induce A3F expression in primary CD4⁺ T lymphocytes was also evaluated. It was observed that A3F was not induced by IFN-α and IFN-γ in primary CD4⁺ T cells over a timecourse of 16 hours. However, other ISG, including PKR and IRF-1, were readily induced by IFN-α and IFN-γ respectively. These data indicated that IFN-mediated signaling pathway was functional in primary CD4⁺ T cells. Therefore, the transcriptional regulation of A3F by IFNs was cell-type dependent and might be distinct from those of PKR and IRF-1.Induction of A3F by IFN-α in macrophage cells was also discovered by qRT-PCR. IFN-α induced consistent A3F expression in macrophages from various donors. ERN-γ induced A3F expression in macrophages was more donor dependent. In one individual DFN-γ induced A3F expression in macrophages as efficiently as IFN-α while in others IFN-γ induced no appreciable A3F expression.Partâ…¢ Functional characterization of transcriptional regulatory region of APOBEC3GUsing NCBI tool, A3G can be found in a APOBEC3 cluster on chromosome 22(region: 37803082-37813694). The length of A3G gene is 10.61Kb. The translation start site is located in the first exon. And it includes eight exons and seven introns(Fig.1). All introns begin with GT and end with AG, consistent with the typical exon/intron boundary. It is also found several A3G mRNA sequencewith different transcription start sites are exited in Genbank.5'RACE were used to know whether other transcription start sites were existed in the cell lines and other tissues. Results showed that two transcription start sites(-308 and -40 site, the first base before ATG was set as -1) were existed in placenta cells, although it was considerable that the shorter one was generated from the interruption of reverse transcription. Two transcription start sites (-280 and -97) were also found in PBLC (peripheral blood lymphocyte cells) isolated from one donor. However, the transcription start sites in cell lines (HepG2, QSG7701) were not found partly(or mostly) due to the low expression level of A3G in these cell lines.When 5000bp regions(-5000 to -1) of APOBEC3 genes were aligned, it was found that the homology between A3G and A3F upstream region was 78.33% and other were all below 50%. 815bp(-815/-1) upstream region of A3G and A3F had even 97.41% identity. This indicated that A3G and A3F had the similar cis-regulatory elements for gene transcription, resulting co-expression of A3G and A3F in some tissues and cells.In order to delineate functional domains that control A3G gene transcription and respond to IFN-α stimulation, the 5'-flanking region of A3G was functionally characterized and its promoter activity was analyzed. The luciferase expression plasmids containing a series of fragments from A3G upstream region were constructed and transfected into some cell lines. The results showed two small regions(-397/-358 and -82/-1) were greatly responsible for A3G promoter activity. The transcription binding factors in these two identified regions were predicted by Transfac4.0.The regulatory transcription factor, Sp1 was found to bind to both small regions. The region(-397/-358) was containing specific Egr-1,RXR-alpha and Krox-20 binding sites. ICSBP binding motif was predicted to be in the region(-82/-1).In the experiment of IFN stimulation, it was found the A3G upstream region of -358/-223 was not responsible for IFN-α-mediated A3G up-regulation. We further found the regions of -5785/-2643, -3403/-1,-65/+1446 had no induced activity in HepG2 cells after IFN-α treatment. The luciferase activity of pGL2-1945(-1945/-1) also had no obvious change when Hep3B cells were treated with IFN-α. Therefore, IRSE of A3G in HepG2 and Hep3B cells still remained to be identified. However, the luciferase activity of pGL2 containing A3G upstream regions were increased about 2 fold in treatment with PMA. It was proposed that PMA might enhance the activity of A3G promoter activity.In summary, it was discovered that induction of A3G and A3F by IFNs was donor and cell-type dependent. In liver cells IFN-α up-regulated A3G expression through a novel STAT1-independent signaling pathway. From the study on A3G transcriptional regulatory region, it was known that A3G had two core promoter regions and several transcription start sites. They might play important roles in A3G expression and regulation.