key: cord-0702354-hi3i1mwl
authors: Taylor, Jared P.; Cash, Melanie N.; Santostefano, Katherine E.; Nakanishi, Mahito; Terada, Naohiro; Wallet, Mark A.
title: CRISPR/Cas9 knockout of USP18 enhances type I IFN responsiveness and restricts HIV-1 infection in macrophages
date: 2018-02-13
journal: Journal of Leukocyte Biology
DOI: 10.1002/jlb.3mia0917-352r
sha: 4fb5e7fb5d3ec9366bfa98bb436d3b34dc4755fb
doc_id: 702354
cord_uid: hi3i1mwl

The IFN-stimulated gene ubiquitin specific proteinase 18 (USP18) encodes a protein that negatively regulates T1 IFN signaling via stearic inhibition of JAK1 recruitment to the IFNα receptor 2 subunit (IFNAR2). Here we demonstrate that USP18 expression is induced by HIV-1 in a T1 IFN dependent manner. Experimental depletion of USP18 by CRISPR/Cas9 gene editing results in a significant restriction of HIV-1 replication in an induced pluripotent stem cell (iPSC)-derived macrophage model. In the absence of USP18, macrophages have increased responsiveness to stimulation with T1 IFNs with prolonged phosphorylation of STAT1 and STAT2 and increased expression of interferon-stimulated genes that are key for antiviral responses. Interestingly, HIV-1 requires some signaling through the T1 IFN receptor to replicate efficiently because a neutralizing antibody that inhibits T1 IFN activity reduces HIV-1 replication rate in monocyte-derived macrophages. USP18 induction by HIV-1 tunes the IFN response to optimal levels allowing for efficient transcription from the HIV-1 LTR promoter while minimizing the T1 IFN-induced antiviral response that would otherwise restrict viral replication and spread. Finally, iPSC and CRISPR/Cas9 gene targeting offer a powerful tool to study host factors that regulate innate immune responses.

It is well established that type I IFNs (T1 IFNs) can restrict acute HIV-1 infection in vitro. [1] [2] [3] [4] [5] In clinical trials treating human patients with recombinant IFN-2a, T1 IFNs can suppress viral replication in the absence of antiretroviral therapy (ART) in some patients. 6, 7 However, this approach failed in long-term treatment when study subjects became refractory to IFN treatment and viral loads returned to previous levels. T1 IFNs are important in establishing early control of HIV-1 in an in vivo SIV rhesus macaque model, 8 Signaling through IFNAR results in the phosphorylation and activation of STAT proteins including STAT1 and STAT2. The consequence of STAT1/2 phosphorylation is induced expression of hundreds of ISGs. [15] [16] [17] The protein products of ISGs then target host and viral machinery as a means to restrict viral replication. However, a small subset of the ISGs expressed are negative feedback mechanisms that turn off IFN signaling so that resolution of the immune response can occur.

One of these important negative regulators is ubiquitin-specific proteinase 18 (USP18).

USP18 is an IFN-inducible deISGylating enzyme that specifically removes the ubiquitin-like posttranslational modification, ISG15, from target proteins. [18] [19] [20] During an IFN response, many newly synthesized proteins are ISGylated, 21 which can have a variety of effects depending on the target. Some targets, such as IFN regulatory factor 3 (IRF3), are protected from ubiquitin-mediated proteasomal degradation. 22 It has also been shown that viral proteins, including HIV-1 Gag, can be ISGylated. 23 Durfee and colleagues 21 propose that during an infection a small subset of viral structural proteins are ISGylated to disrupt the repeating structures found in viral capsids. Influenza B has evolved a mechanism to directly neutralize ISG15 with its NS1 protein 24 and coronaviruses have a papain-like protease that has deISGylase activity as a strategy to overcome ISG15, 25, 26 indicating the importance of ISG15 in the antiviral response.

In addition to its enzymatic activity, USP18 negatively regulates T1 IFN signaling. 27 USP18 is recruited by STAT2 to the type I IFN receptor subunit, IFNAR2, where it binds to IFNAR2 and prevents phosphorylation of JAK1 by blocking the interaction of JAK1 and the IFNAR2 subunit. [27] [28] [29] USP18 expression also plays a role in limiting TRAILinduced apoptosis and has also been shown to regulate the susceptibility of certain cancer cells to IFN-and drug-induced apoptosis. 30, 31 Macrophages play an important role in HIV-1 as reservoirs and can contribute directly to HIV-1 pathogenesis. 32 HIV-1 in the ART era can be seen as a chronic disease characterized by chronic immune activation and chronic inflammation with a higher risk of non-AIDS-related morbidities and mortalities. Macrophages play an important role in this process and can act as mediators of inflammation. 33 , 34 We recently reported that HIV-1 replication in macrophages requires activity of STAT1, a protein usually associated with antiviral responses. 35 The role of STAT1 was in postintegration expression of HIV-1 mRNA from the long terminal repeat (LTR) promoter. This paradoxical mechanism where HIV-1 usurps antiviral pathways as a means of driving its own replication suggests that a complex host-pathogen interplay ultimately determines if HIV-1 can efficiently replicate in macrophages. In general, the role of macrophages in the HIV-1 life cycle is important because eliminating persistently infected macrophages in addition to latently infected T cells will be necessary for a sterilizing cure to be achieved. Thus, it is critical that we understand the host-pathogen dynamics that regu- 

Lenti-X 293T cells were cultured in a T75 flask and transfected with 

Medium was removed by aspiration and 100 L of room temperature 

Monocyte-derived macrophages (MDMs) were infected with 500 TCID 50 

Human GIPZ lentiviral shRNA gene set for USP18-specific shRNA 

Differentiation was carried out as previously described 48 

Fc receptors were blocked with Fc Block (Miltenyi) and stained with antibodies in PBS with 1% human serum. For intracellular staining of CD68, the cells were fixed and permeabilized with the BD Cytofix/Cytoperm TM kit (BD Biosciences). All samples were analyzed with the BD Accuri TM C6 Cytometer (BD Biosciences).

The integration assay for detection of integrated HIV-1 proviral DNA was adapted from methods previously described. 49 

It has been demonstrated previously in vitro that T1 IFNs restrict HIV-1 replication. [1] [2] [3] [4] [5] 51 To confirm these previous findings, we pretreated TZM-bl cells with different doses of IFN-for 24 h and infected the cells with HIV-1. As previously reported, T1 IFN restricted HIV-1 replication in a dose-dependent manner (Fig. 1A) . We also tested this in MDMs and found that HIV-1 replication is potently restricted by IFN-in a dose-dependent manner (Fig. 1B) with an IC 50 of 504.7 fg/mL (± 142.1 fg/mL).

Since HIV-1 is restricted by T1 IFNs in vitro, we wanted to determine if HIV-1 was inducing an IFN response in MDMs. To test this, MDMs were infected with HIV-1 for 7 days and gene expression was analyzed by microarray ( Fig. 2A) . We observed that all genes up-regulated at least 2-fold by HIV-1 (Table 1) have been reported to be IFN-inducible in the Interferome (v2.01) database (Fig. 2B) . However, this IFN-like response is not strong enough to inhibit HIV-1 replication to the same degree as the addition of exogenous IFN (Fig. 1) . We were interested in how the IFN response was being attenuated and allowing for IFN signaling (Fig. 3) , providing evidence that HIV-1 does induce an IFN response in macrophages that has an autocrine effect.

Surprisingly, HIV-1 replication was inhibited, not enhanced, in the absence of T1 IFN signaling as measured by Gag p24 ELISA on supernatants (Fig. 3) . This finding agrees with previous work where we demonstrated the importance of STAT signaling for HIV-1 replication in MDMs. 35 These data demonstrate that HIV-1 must strike a balance between some T1 IFN signaling, which is needed for producing transcription factors such as STAT1 and STAT3, and too much IFN signaling, which allows the cells to mount an effective antiviral response. Given that USP18 attenuates the IFN response, we hypothesized that induction of USP18 allowed HIV-1 to achieve the balance needed to provide the necessary STAT signaling, without too strong an antiviral response.

USP18 is part of the normal negative feedback response that regulates T1 IFN signaling through IFNAR and JAK-STAT signaling. In the absence of USP18, signaling through the JAK-STAT pathway should be enhanced with increased levels of phosphorylated STAT1 and STAT2.

We used siRNA to knockdown USP18 expression in MDMs and treated them with IFN-for 18 hours and found increased levels of phosphorylated STAT1 and STAT2 (Fig. 4A ). (Fig. 4B) . USP18 expression was also measured to confirm knockdown of USP18 transcripts.

We next wanted to determine if USP18 expression is necessary for HIV-1 replication. Although transfecting MDMs with siRNA allowed for efficient knockdown of USP18 (Fig. 4) , it also made the cells refractory to infection even with a nontargeting control siRNA (data not shown). Instead, we utilized shRNA knockdown of USP18 in the THP-1 cell line. THP-1 cells are suspension cells that can be differentiated into adherent macrophage-like cells with PMA treatment. They are a well-established model system for studying HIV-1 infection in macrophages. 52 We generated THP-1 cell lines that constitutively express nontargeting control or USP18-specific shRNA (Supplemental Figs. S1A and S1B). After PMA differentiation, THP-1 cells expressing non-targeting control or USP18 shRNA were infected by HIV-1. We found that HIV-1 replication was significantly restricted in THP-1 cells with USP18 knockdown. There was significantly less Gag p24 detected in the supernatants and in the protein lysates of USP18 knockdown cells compared with control (Figs. 5A and 5B).

While THP-1 cells and MDMs with USP18 knockdown provide useful models for investigating the effects of USP18 deficiency on IFN signaling, it may not be the best model. THP-1 cells are transformed cells with an abnormal karyotype 53 and in our hands they do not remain differentiated and adherent in culture for more than 5 days (data not shown). 

To test the effects of USP18 deficiency in iMacs, we utilized CRISPR/Cas9 gene editing to knockout USP18 in iPSCs before differentiation to the myeloid lineage. The advantage of knocking out USP18 in iPSCs is that once knockout is achieved, we have a self- were measured by Western blot. USP18 −/− iMacs had increased and sustained phosphorylated STAT1 and phosphorylated STAT2 (Fig. 7A ).

Since USP18 −/− iMacs also had increased STAT1 and STAT2 signaling, we wanted to determine if USP18 −/− iMacs also had enhanced ISG expression after IFN-treatment. Indeed, after treatment with IFNfor 18 h, USP18 −/− iMacs had increased expression of ISGs compared with USP18 sufficient cells (Fig. 7B) . These findings are consistent with the results from siRNA knockdown in MDMs.

We next determined if iMacs can support HIV-1 replication as previously reported 48 and if USP18 is induced by HIV-1 in iMacs. Indeed, USP18 +/+ iMacs support HIV-1 replication and USP18 is induced by HIV-1 in these cells (Figs. 8A and 8B). Since USP18 is induced by HIV-1 in iMacs in a similar manner as MDMs, we concluded that iMacs are a better model for studying USP18 knockout/knockdown than THP-1 cells.

Next, we wanted to determine if USP18 knockout influenced HIV-1 replication. USP18 knockout results in an enhanced response to IFN stimulation in macrophages (Fig. 7) . Therefore, we hypothe- 

We report here that USP18 deficiency restricts HIV-1 replication in human macrophages using an iPSC model. The restriction on replication was due to an enhanced IFN response from a lack of feedback inhibition on the JAK/STAT pathway resulting in increased expression of ISGs that have antiviral effects. Previous work done by our group has shown that STAT1 and STAT3 are necessary for efficient HIV-1 replication. Blocking phosphorylation of either of these 2 molecules resulted in significant reduction in viral replication. 35 However, the current study shows that increasing the amount of phosphorylated STAT1 and STAT2 also results in a significant reduction in viral replication. Thus, we propose that USP18 acts as a tuning mechanism which prevents a robust IFN response that would restrict viral replication (Fig. 9) . Previous work has shown that Usp18 −/− mice exhibit protection from intracerebral infection by LCMV and VSV. 55 Usp18 −/− mice also The basis of antibacterial protection in USP18 deficiency can be attributed to hypersensitivity to LPS as a consequence of an overactive T1 IFN response. 56 Mechanistically, these studies showed that USP18 deficiency resulted in increased ISGylation of proteins and increased phosphorylated STAT1 levels in response to LPS treatment or viral infection. It was based on these properties that we sought to determine if human USP18 regulates HIV-1 replication. supports HIV-1 replication is a window of low level immune activation that is due, at least in part, to balanced positive and negative regulators of antiviral immunity (Fig. 9 ). Infected cells eventually become refractory to T1 IFN signaling due to induction of negative regulators such as USP18. Other negative regulators are also induced by T1 IFNs including SOCS1, SOCS3, and PIAS. 75 We focused on USP18 for this study due to its additional function as a deISGylating enzyme and its appearance in gene expression profiling experiments following HIV-1 infection. 

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CRISPR/Cas9 knockout of USP18 enhances type I IFN responsiveness and restricts HIV-1 infection in macrophages

Armitage for his assistance with CRISPR plasmid transfections and Sneha Patel for her assistance with Western blots.

The authors declare no conflicts of interest.

http://orcid.org/0000-0001-7700-530X