IFI44L expression is regulated by IRF‐1 and HIV‐1

Interferon (IFN)‐inducible 44 like (IFI44L) is an IFN‐stimulated gene, the expression of which is induced by IFN and human immunodeficiency virus (HIV)‐1 infection. However, the mechanism has not yet been determined. In this study, we cloned the promoter of the IFI44L gene and found that interferon regulatory factor‐1 could bind directly to the IFN‐stimulated response element in the IFI44L promoter to activate IFI44L. Furthermore, we demonstrated that HIV‐1 can activate the IFI44L promoter to influence the expression of IFI44L.

Interferon (IFN)-inducible 44 like (IFI44L) is an IFN-stimulated gene (ISG), which is located on the same chromosome as the known antiviral ISG IFI44. Expression of IFI44L is induced by IFN and HIV-1 infection. However, the mechanism by which IFN-I induces IFI44L production has not yet been determined. In this study, we analyzed transcriptional regulation of IFI44L via cloning of the IFI44L promoter. We found that IFI44L has two IFN-stimulated response elements (ISRE), which are necessary for the basal level of IFI44L transcription. IFN-I and IFN-II can activate the IFI44L promoter through one of the two ISREs. IFN regulatory factor (IRF)-1 can activate transcription of IFI44L by binding to one of the ISREs. Additionally, co-transfection of the IFI44L promoter with an HIV-1 infectious clone or HIV-1 infection activated IFI44L promoter transcription, but did not upregulate IFI44L expression via ISREs. These findings will help to understand the interaction between IFI44L and HIV-1, and aid in elucidation of the role of IFI44L in the antiviral innate immune response.
Interferon (IFN)-inducible 44 like (IFI44L) is a part of the IFI44 family and is located on the same human chromosome as the previously identified antiviral interferon-stimulated gene (ISG) IFI44, which is involved in numerous signaling pathways of the innate immune response. At the same time, IFI44L is also an ISG and can be induced by many different viruses such as influenza and the HIV-1 [1,2]. HIV-1 proteins can affect the expression of IFI44L through different regulatory pathways in different cell types. For example, HIV-1 gp120 can downregulate the expression of IFI44L by interaction with a4b7 [3]. Also, the transcription level of IFI44L is significantly upregulated in HIV-1 gp120treated vaginal epithelial cells [4]. Additionally, HIV-1 Vpr can upregulate the expression of IFI44L [5]. However, the mechanism by which HIV-1 induces IFI44L up-regulation has not been determined. In addition to HIV-1, when rhesus monkeys are infected by the simian immunodeficiency virus, IFI44L is significantly upregulated up to 38-fold on the 10th day of infection, and the up-regulation of IFI44L is accompanied by up-regulation of ISGs [6].
There are three types of IFNs: IFN-I, IFN-II, and IFN-Ⅲ, which participate in a variety of biological activities, including antiviral response, antitumor, inflammatory response, and immunomodulatory activity [7][8][9]. IFNs regulate transcription of ISGs through the Janus kinase/signal transducer and activator of transcription (STAT) pathway [10]. Using the IFN-I signaling pathway as an example, after IFN binds to its receptor, the receptor-associated tyrosine kinases Tyk2 and Jak1 are activated, and then, STAT1 and STAT2 are phosphorylated to form a heterodimer complex and translocated to the nucleus. The complex is assembled with interferon regulatory factor (IRF)-9 and binds to the IFN-stimulated regulatory element (ISRE) on the ISG promoter to induce ISGs transcription [11][12][13][14][15].
Interferon regulatory factors participate in the IFNmediated signaling pathway. Nine members of the IRFs family have been identified in human cells. For example, IRF-1 can bind the promoter region of IFNb and activate transcription. IRF-2 can also bind to the IFN-b promoter, but it inhibits IFN-b expression [16]. IRF3 plays a major role in the transcription of the gene encoding IFN-1. IRF-5 plays a role in the immune response against viral infections. IRF-7 can bind to MyD88 and TRIF and increases the production of IFN-I induced by the stimulation of some TLRs [9,[17][18][19].
The expression of IFI44L in HIV-1 progressor blood samples are significantly higher than that in nonprogressors, and IFI44L levels are upregulated by IFN-a [2]. However, the mechanism by which IFN-I induces IFI44L production is not yet determined. In this study, we cloned the promoter of IFI44L gene to better understand its transcriptional regulation. We found IRF-1 could bind directly to the ISRE in the IFI44L promoter to activate IFI44L. Furthermore, we demonstrated that HIV-1 can activate the IFI44L promoter to influence the expression of IFI44L.

Primer
Sequence

Luciferase reporter assay
HeLa cells were transfected with luciferase plasmids and bgalactosidase expression plasmids. Luciferase activity was determined with luciferase report system (Promega) and normalized to b-galactosidase activity.

Promoter activity analysis of IFI44L
To clarify the mechanism how IFI44L gene is induced by IFNs, we first studied the transcriptional regulation mechanism of IFI44L. We used the translation start site (ATG) as +1 and determined that the first exon of IFI44L is located at À16 to À183 bp through comparing the IFI44L genomic sequence in UCSU, Ensembl, and the IFI44L mRNA sequence in NCBI. Then, we cloned the 1190 bp DNA upstream of the transcription initiation site and performed sequence analysis. The results showed that it contained two ISRE components (Fig. 1A). To verify whether the cis-acting elements are involved in the transcription, a series of deletions of the promoter were constructed (Fig. 1B). We transfected these reporter plasmids into HeLa cells and measured the luciferase activity 48 h post-transfection. As shown in Fig. 1C, the basal transcriptional activity of truncated IFI44L promoter from +1 to À274 (with the ISRE-2 deleted) decreased moderately, while the basal transcriptional activity of the IFI44L promoter was further reduced when the truncation spanned position À237 to À1190 (with the ISRE-1 deleted). In addition, we also studied the basal transcriptional activity of pGL-390 in Jurkat cells to ensure the reliability of the above results, and similar results were obtained (Fig. 1D). In order to further confirm whether the two ISREs are involved in regulating the basic transcriptional activity of the IFI44L promoter, we constructed a series of mutations on pGL-274 and pGL-237-1190 (Fig. 1E). As shown in Fig. 1F,G, the basal transcriptional activity decreased by 72% (ISRE-1 mutation) and 14% (ISRE-2 mutation). Furthermore, when the two elements were mutated, pGL-143 completely lost the promoter activity compared to the empty vector. Therefore, we concluded that the two ISREs are essential elements in the basal transcription of IFI44L.
The ISRE-1 element is essential for IFNs to activate IFI44L promoter To verify whether the IFI44L promoter responds to IFNs, HeLa cells were transfected with pGL-274 or pGL-237-1190; 36h post-transfection, cells were treated with IFN-a or IFN-c for 12 h, respectively. As shown in Fig. 2A, both IFN-a and IFN-c could activate pGL-274 (with ISRE-1). Besides, we also transfected pGL-274 into Jurkat cells and treated with IFNs, and got similar results as 2A (Fig. 2B). Furthermore, IFN-a was more potent than IFN-c at inducing promoter response. However, IFN-a and IFN-c could not activate pGL-237-1190 (with ISRE-2). To further confirm whether IFNs activate pGL-274 via the ISRE-1, we constructed ISRE-1 mutant plasmids (pGL274mISRE-1). We found that the IFI44L promoter did not respond to IFNs when the ISRE-1 was mutated (Fig. 2C,D). These results demonstrated that the ISRE-1 is essential for IFNs to activate the IFI44L promoter.

IRF-1 binds to the ISRE to increase IFI44L expression
The IRF family can participate in the transcriptional regulation of the target genes. In order to identify whether the IRF family is involved in regulating IFI44L, we co-transfected Myc -IRF-1, 3.1-IRF-2, Myc-IRF-3, Myc-IRF-5, Myc-IRF-7 encoding plasmids with pGL-390 into HeLa cells and measured the transcription activity of pGL-390. As shown in Fig. 3A,B, overexpression of IRF-1 significantly activated pGL-390 in HeLa cells, the trend measured by western blot was consistent with luciferase results, but protein levels were not as sensitive as transcriptional levels. In order to further confirm that IRF-1 can upregulate the IFI44L promoter, we co-transfected the IFI44L promoter and dose-gradient Myc-IRF-1 into 293T cells, and found that IFI44L promoter could be upregulated by IRF-1 in a dose-dependent manner (Fig. 3C). We then further explored whether IRF-1 could activate the IFI44L promoter via the ISRE element. We co-transfected Myc-IRF-1 and pGL-274mISRE-1 or pGL-237-1190mISRE-2 into HeLa cells. As shown in Fig. 3D  with IFN-a and followed by PCR. The results showed that endogenous IRF-1 could interact with the ISRE of IFI44L (Fig. 3F). The results above indicate that IRF-1 can bind to the ISRE of IFI44L promoter to regulate IFI44L expression.

Discussion
Interferons can activate the expression of ISGs through the JAK-STAT pathway, which inhibit viruses at different stages of replication. HIV-1 is able to regulate a number of ISGs to ensure its own infection [20,21]. As an ISG, IFI44L can be induced by IFNs and HIV-1. In this study, we explored the mechanism of how IFI44L is induced by IFNs and HIV-1.
First, we cloned and analyzed the features of the IFI44L promoter and we found that the IFI44L promoter has two ISREs, but no TATA and GC boxes. Two ISRE components are located in the complete repeat sequence [22,23]. We identified that the basal transcriptional activity of IFI44L is significantly decreased when the ISREs are mutated, indicating that these two ISRE elements are essential for the transcriptional regulation of IFI44L, especially ISRE-1. This is similar to some other ISGs, which are controlled by ISRE. After IFN-I stimulates cells, a heterodimer complex is formed by phosphorylation of STAT1 and STAT2, which is assembled with IRF-9 and induces ISGs production by binding to ISRE [11][12][13][14][15]. Furthermore, we determined that IFN-I can activate IFI44L promoter and that it is through ISRE-1 rather than ISRE-2. IFN-Ⅱ can also induce up-regulation of IFI44L promoter activity, but the upregulate level is lower than that of IFN-I. We suspect that this upregulation of IFN-II is indirect because we cannot be sure that there is a gamma-interferon activation site element on the IFI44L promoter. IFN regulatory factors are a class of transcriptional regulatory proteins that regulate the expression of IFNs and ISGs. Therefore, we also tested whether IRFs could activate IFI44L. We observed that IRF-1 can significantly activate the transcription of IFI44L. This is because IRF-1 can recognize ISRE and activate the transcription of IFN-I and ISGs [24,25], which indicates that IFI44L may participate in the host's innate immunity through these IRFs.
As mentioned earlier, HIV-1 proteins can affect the expression of IFI44L through different regulatory pathways in different cell types. Furthermore, we demonstrated that HIV-1 can affect the expression of IFI44L by upregulating the activity of the IFI44L promoter in 293T cells. Besides, Lu et al. [26] reports that high-throughput screening experiments at the cellular level have detected a significant increase in HIV-1 replication levels after IFI44L has been knocked out. We speculated that IFI44L plays an immunoregulatory role for HIV-1 in host cells, one possible reason is that HIV-1 can ensure latent infection by up-regulating the expression of IFI44L. However, after mutation of ISRE, HIV-1 can still upregulate the IFI44L promoter activity, therefore, HIV-1 did not pass the ISRE to affect the IFI44L promoter activity. The mechanism by which HIV upregulates the promoter activity of IFI44L needs further study.
In summary, this study found that IFN-I and IFN-II can upregulate the transcription of the IFI44L promoter, and HIV-1 can activate the IFI44L promoter to influence the expression of IFI44L. We will continue to explore the specific mechanism by which HIV-1 upregulates IFI44L initiation activity. This will help to understand the more physiological functions of IFI44L and the interaction between IFI44L and HIV-1, and determine the status of IFI44L in antiviral innate immune response.