USP18) Expression This Information Is Current As of October 2, 2021

The Level of Hepatitis B Virus Replication Is Not Affected by Protein ISG15 Modification but Is Reduced by Inhibition of UBP43 (USP18) Expression This information is current as of October 2, 2021. Jung-Hwan Kim, Jiann-Kae Luo and Dong-Er Zhang J Immunol 2008; 181:6467-6472; ; doi: 10.4049/jimmunol.181.9.6467 http://www.jimmunol.org/content/181/9/6467 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Level of Hepatitis B Virus Replication Is Not Affected by Protein ISG15 Modiﬁcation but Is Reduced by Inhibition of UBP43 (USP18) Expression1

Jung-Hwan Kim,2* Jiann-Kae Luo,3* and Dong-Er Zhang4*†

Hepatitis B virus (HBV) causes both acute and chronic infection of the human liver and is associated with the development of liver cirrhosis and hepatocellular carcinoma. UBP43 (USP18) is known as an ISG15-deconjugating enzyme and an inhibitor of type I IFN signaling independent of its enzyme activity. In this study, we examined the role of these two previously identiﬁed functions of UBP43 in the innate immune response to HBV viral infection. As an in vivo HBV replication model system, a replication- competent DNA construct was injected hydrodynamically into the tail veins of mice. Although the lack of ISG15 conjugation in the absence of ISG15-activating enzyme UBE1L (UBA7) did not affect the level of HBV replication, the steady-state level of HBV

DNA was substantially reduced in the UBP43-deﬁcient mice in comparison to the wild-type controls. In addition, introduction of Downloaded from short hairpin RNA against UBP43 resulted in substantially lower levels of HBV DNA at day 4 postinjection and higher levels of ISG mRNAs. These results suggest that HBV infection is more rapidly cleared if UBP43 expression is reduced. Furthermore, these results illustrate the therapeutic potential of modulating UBP43 levels in treating viral infection, especially for viruses sensitive to IFN signaling. The Journal of Immunology, 2008, 181: 6467–6472.

5 epatitis B virus (HBV) causes both acute and chronic tions, including induction of the antiviral response, inhibition of http://www.jimmunol.org/ infection of the human liver. Although a vaccine is cell proliferation, and immunomodulatory activities (8–12). IFN- H available, hepatitis B remains a major health problem in ␣/␤ stimulation leads to the up-regulation of IFN-stimulated genes many countries. The number of HBV carriers worldwide is esti- (ISG). One of these ISG is ISG15, an ubiquitin-like protein that mated to be 400 million. HBV is an enveloped, partially double conjugates to cellular substrates to form ISGylated proteins (13, stranded DNA virus. The replication of the viral genome occurs 14). The conjugation is executed by an enzymatic cascade that through an RNA intermediate that requires its own reverse tran- includes an E1-activating enzyme (UBE1L, also known as UBA7; scriptase activity (1). Chronic HBV infection can lead to the de- Ref. 15), an E2-conjugating enzyme (UbcH8; Refs. 16, 17), and E3 velopment of liver cirrhosis and hepatocellular carcinoma (2, 3). ligases (18–22). The conjugation can be reversed by ubiquitin pro- Although HBV infection itself is not cytopathic to the hepatocyte, tease 43 (UBP43, also known as USP18), which is an IFN-induc- by guest on October 2, 2021 hepatitis is thought to result from the immune response to the viral ible cysteine protease (23). As expected, UBP43-deficient cells infection. show high levels of ISG15-modified proteins (24). Importantly, Host response during the early phases of viral infections are UBP43-deficient cells are hypersensitive to type I IFN and un- characterized by the production of type I IFN (IFN-␣/␤) and ac- dergo apoptosis upon IFN stimulation (25). Furthermore, lack of tivation of NK cells (4). HBV replication can be efficiently inhib- UBP43 results in enhanced and prolonged STAT1 phosphorylation ited by IFN treatment (5–7). IFN regulate diverse biological func- and increased induction of hundreds of ISG, as confirmed by gene expression microarray studies (26). Loss of UBP43 in mice results in resistance to the cytopathic effects caused by a number of vi- *Department of Molecular and Experimental Medicine, The Scripps Research Insti- tute, La Jolla, CA 92037; and †Department of Pathology, Division of Biology, and ruses including lymphocytic choriomeningitis virus (LCMV), ve- Moores University of California San Diego Cancer Center, University of California sicular stomatitis virus (VSV), and Sindbis virus (27). Interest- San Diego, La Jolla, CA 92093 ingly, UBP43 has two independent roles in the innate immune Received for publication October 10, 2007. Accepted for publication August response. First, the isopeptidase activity of UBP43 functions to 28, 2008. remove ISG15 from ISG15-conjugated proteins (23). Second, in- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance dependent of its role in protein ISGylation, UBP43 competes with with 18 U.S.C. Section 1734 solely to indicate this fact. JAK1 for binding to the IFNAR2 subunit of the IFN receptor and 1 This work was supported by National Institutes of Health Grants HL91549 and inhibits IFN-induced JAK/STAT signal transduction (28). GM066955. HBV infection is restricted to humans and chimpanzees, and the 2 Current address: Department of Surgery, University of Southern California, Los lack of a small animal model hampers the understanding of HBV Angeles, CA 90033. biology. Although several lines of transgenic mice have been es- 3 Current address: Regeneron Pharmaceuticals, Tarrytown, NY 10591. tablished, these may not fully represent the natural course of in- 4 Address correspondence and reprint requests to Dr. Dong-Er Zhang, Mail Drop fection because the production of HBV comes from an integrated 0815, Room 5328, Moores University of California San Diego Cancer Center, Uni- versity of California San Diego, 3855 Health Sciences Drive, La Jolla, CA 92093. viral genome and the mice are immunologically tolerant to viral E-mail address: [email protected] Ags (29, 30). Recently, it was found that i.v. injection of plasmid 5 Abbreviations used in this paper: HBV, hepatitis B virus; ISG, IFN-stimulated gene; DNA with acute circulatory overload leads to high levels of gene sh, short hairpin; LCMV, lymphocytic choriomeningitis virus; VSV, vesicular sto- expression in mouse liver (31, 32). A mouse model of acute HBV matitis virus. infection was established using this hydrodynamics-based gene de- Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 livery system (33). To evaluate ISG15 conjugation-dependent and www.jimmunol.org 6468 UBP43 AFFECTS THE LEVEL OF HBV REPLICATION

-independent functions of UBP43 in the innate immune response to HBV infection, we used the HBV DNA hydrodynamic injection approach and examined HBV replication in wild-type controls, UBE1LϪ/Ϫ mice that lack ISG15 conjugation upon IFN stimulation, and UBP43Ϫ/Ϫ mice (24, 34). Furthermore, we also tested the therapeutic potential of UBP43 short hairpin (sh) RNA for the treatment of HBV infection. Although there are several reports that indicate the antiviral role of ISG15 modiﬁcation (35–37), ISGyla- tion does not affect the replication of HBV in this model. However, the steady-state level of HBV DNA is substantially reduced in the absence of UBP43 or in the presence of UBP43-silencing shRNA, indicating that the anti-HBV effect of UBP43 is related to its role in modulating IFN signaling and is independent of its role in ISG15 conjugation. These results suggest UBP43 as an attractive therapeutic target for the treatment of HBV infection. Materials and Methods Plasmid constructs

pSP65-ayw1.3 containing an over-length copy of HBV was provided by Downloaded from Dr. F. V. Chisari (The Scripps Research Institute, La Jolla, CA). The FIGURE 1. The level of HBV replication obtained by hydrodynamic pGEM-ayw1.0 construct was made by excising one copy of the HBV genome from pSP65-ayw1.3 with SphI enzyme and ligation into the SphI site injection. A, Schematic diagram of ayw 1.3 and ayw 1.0 constructs. EnhI, of the pGEM-7Zf(Ϫ) vector (Promega; see Fig. 1A). Control shRNA and enhancer I; Cp, core promoter; pA, poly(A) signal; PS1p, pre-S1 promoter; mouse-specific shRNA were generated with minor modifications from pre- Sp, S promoter. B, The level of HBV replication from the ayw 1.3 con- viously established protocols (38). In brief, primers 5Ј-gggagatctc aag struct. Mice were injected hydrodynamically with 20 ␮g of the ayw 1.3 gtcgggc aggaagaggg cctatttcc-3Ј and 5Ј-ggggaattca aaaagacctc ggtgatacca construct diluted in 2 ml of saline. Mouse livers were taken 4 days after aggtctcttg aactttggta tcaccgaggt cggtgtttcg tcctttccac aagatatata a-3Ј (for injection, total DNA was isolated, digested with HindIII restriction en- http://www.jimmunol.org/ control shRNA) and primers 5Ј-gggagatctc aaggtcgggc aggaagaggg cctatt zyme, and analyzed by Southern blotting with 32P-labeled HBV probe. RC, Ј Ј tcc-3 and 5 -ggggaattca aaaaggacca gatcacggac acatctcttg aatgtgtccg Relaxed circular; SS, single-strand. Numbers on the left show DNA m.w. tgatctggtc cggtgtttcg tcctttccac aagatatata a-3Ј (for UBP43 shRNA12) were size markers in kilobases. used to PCR-amplify shRNA constructs under the control of the U6 promoter. The PCR products were digested with EcoRI/BglII and ligated into the EcoRI/BamHI site of pBluescript KS (Ϫ) (Stratagene). The insert was brane was hybridized with 32P-labeled DNA probe generated by the then excised by a two-step digestion, first with XbaI and blunted with T4 Prime-It II Random Primer Labeling Kit (Stratagene) using partial cDNA DNA polymerase, then with EcoRI. The excised construct was then ligated fragments of IFN-inducible genes as the template. The partial cDNA frag- into the HpaI/EcoRI site of pMSCVpuro (BD Clontech) and confirmed by ments were obtained by PCR amplification using a cDNA library from sequencing. Plasmid DNAs were prepared by using an EndoFree plasmid IFN-treated mouse bone marrow macrophages as a template. Primers 5Ј- by guest on October 2, 2021 maxi kit (Qiagen) according to manufacturer’s instructions. atggcctcagagatccacat-3Ј and 5Ј-ccattgactgtgatgcctcc-3Ј for Gbp1, 5Ј-atgc caatca ctcgaatgcg-3Ј and 5Ј-ctatggtgca caaggaatgg-3Ј for IRF1, 5Ј-atggct In vivo studies gaag tgaggggggt-3Ј and 5Ј-tcaaggccac tgacccaggt-3Ј for IRF7, 5Ј- Ј Ј Ј Ј Ϫ/Ϫ Ϫ/Ϫ atgaagtccg ctgttctttt-3 and 5 -ttatgtagtc ttccttgaac-3 for Cxcl9, and 5 - The generation of UBP43 and UBE1L mice was described previ- agctgaatga gggagaggag-3Ј and 5Ј-gaagaactct gaaatgaggg-3Ј for Mx2 were ously (24, 34). All animals used in the studies were handled in accordance used in amplification. with guidelines of The Scripps Research Institute, and procedures were approved by the Institutional Animal Care and Use Committee of the in- Detection of ISGylated proteins stitute. The genetic background of mice used in this study was C57BL/6, 129Sv, FVB, or mixed background of C57BL/6 and 129Sv. The pair com- Liver tissue was homogenized in radioimmune precipitation assay buffer parison is always with mice in the same strain background. (150 mM NaCl, 10 mM Tris-Cl (pH 7.2), 0.1% SDS, 1% Triton X-100, 1% deoxycholate, 5 mM EDTA), sonicated briefly, then resolved in 8–18% Hydrodynamic injection of naked plasmid DNA discontinuous gradient SDS-polyacrylamide gel. Proteins were transferred Twenty micrograms of plasmid DNA was diluted with 2.0 ml of saline and to nitrocellulose membrane (Hybond-ECL; Amersham Biosciences) and injected via the tail vein of 6–9-wk-old mice within 10 s. The livers of the detected with rabbit anti-mouse ISG15 polyclonal Ab as described previ- mice were dissected into pieces and frozen immediately in liquid nitrogen ously (39). 4 days after injection. Results Analysis of HBV replication by Southern blotting Replication of HBV in hydrodynamically injected mice A piece of liver tissue was digested overnight in lysis buffer (50 mM An ayw 1.3 super genomic DNA construct that has been shown to Tris-Cl (pH 7.4), 20 mM EDTA (pH 8.0), 100 mM NaCl, 0.5% SDS, 0.5 mg/ml proteinase K) at 55°C, and the DNA was phenol-chloroform ex- support HBV gene expression and replication in the livers of trans- tracted. Twenty micrograms of DNA was digested with HindIII, electro- genic mice (30) was injected i.v. into mice. At day 4 postinfection, phoresed in a 1% agarose gel, and transferred to nylon membrane (Hy- mice were sacrificed and total genomic DNA from liver tissue was bond-N; Amersham Biosciences). After UV cross-linking, the membrane 32 32 analyzed by Southern blotting with a P-labeled HBV DNA probe was hybridized with a P-labeled HBV DNA probe generated by the after HindIII digestion. As a negative control, a construct contain- Prime-It II Random Primer Labeling Kit (Stratagene) using the linear 3.2-kb full length HBV genome as a template. The signal was visualized by ing one copy of the HBV genome (ayw 1.0), which cannot provide exposing on an x-ray film, scanned using the VersaDoc Imaging system the appropriate transcripts of HBV, was used (Fig. 1A). As shown (Bio-Rad), and the density was quantified using Quantity One software in Fig. 1B, only the DNA sample injected with ayw 1.3, but not (Bio-Rad). with ayw 1.0, showed the conventional HBV replication signal. Northern blotting ISGylation does not affect the level of HBV replication Total RNA from liver was isolated using RNA-Bee (Tel-Test) reagent, resolved in a formaldehyde agarose gel, and transferred to nylon membrane IFN are used to treat HBV infections, and IFN stimulate the ex- (Hybond-N, Amersham Biosciences). After UV-cross-linking, the mem- pression of hundreds of ISG, including ISG15. Previous studies The Journal of Immunology 6469 Downloaded from

FIGURE 3. Absence of UBE1L does not affect the level of HBV rep- FIGURE 2. Hydrodynamic injection of naked DNA results in induction lication. A, UBE1Lϩ/ϩ (lanes 1, 3, 5, and 7) or UBE1LϪ/Ϫ (lanes 2, 4, 6, of ISGylation in the mouse liver. Liver lysate was analyzed 2 days after and 8) mice were injected with the ayw 1.3 construct, and the total DNA injection by immunoblotting using anti-mouse ISG15 Ab. A, Wild-type was analyzed by Southern blotting with an HBV probe. Relative replication mice injected with ayw 1.0 and ayw 1.3 constructs. NI, Not injected; S, was measured (see Results for details) and indicated as a percentage below ϩ/ϩ Ϫ/Ϫ saline injected; 1.0, ayw 1.0; 1.3, ayw 1.3. B, UBE1L or UBE1L each lane. B, The relative replication is summarized as a graph. The av- http://www.jimmunol.org/ mice were injected with ayw 1.3 construct. Asterisks indicate nonspeciﬁc erage HBV replication in UBE1LϪ/Ϫ mice was 98.87 Ϯ 13.87 (%) (n ϭ 4 cross-reactive bands. Ponceau staining indicates the relative amount of pairs) of that in UBE1Lϩ/ϩ mice. protein loading.

with 20 ␮g of the ayw1.3 construct. Mice were sacrificed 4 days suggested that ISG15 modification plays a role in certain antiviral later and total DNA from liver tissue was analyzed after HindIII responses. ISG15 was linked to IFN-mediated inhibition of HIV-1 digestion by Southern blotting using a 32P-labeled HBV DNA replication (40) and the NS1B protein of influenza B virus was found probe. It is known that the transfection efficiency of hepatocytes in to bind free ISG15 and possibly to inhibit its conjugation (15). A the liver by hydrodynamic injection ranges on average from 5 to by guest on October 2, 2021 more recent study suggests that ISG15 inhibits Sindbis virus in- 10% within a given experiment, depending on the efficiency of the fection in mice (35). Furthermore, ISG15 knockout mice showed plasmid injection as well as the genetic background, and replica- increased susceptibility to Sindbis, herpes, and influenza viruses tion levels can vary by as much as 5-fold between given experi- (36). However, ISG15- and ISGylation-defective mice did not ments (33). Due to this variability within and between batches of show any difference in response to VSV and LCMV infection (34, experiments, measurement of the differences in HBV replication ϩ ϩ Ϫ Ϫ 41). ISG15-deconjugating enzyme UBP43 regulates cellular levels levels between UBE1L / and UBE1L / mice was done by of ISGylated protein via its deconjugating enzyme activity and comparing, within each batch of experiments, pairs of mice of each also functions as a potent inhibitor of type I IFN signaling (23, 28). genotype that showed similar amounts of input DNA. For each pair ϩ ϩ Ϫ Ϫ UBP43 plays an important role during innate immune responses of UBE1L / and UBE1L / mice, signal intensity of the repli- (27, 42, 43). Because HBV is currently one of the major threats to cation intermediates was divided by that of the input DNA, and the Ϫ Ϫ human health, the two different roles of UBP43 in innate immune ratio for the UBE1L / mouse is expressed as a percentage of that responses were analyzed for effects on HBV. We first address the observed in the wild type. Four independent pairs are shown in Ϫ Ϫ ISG15 modification-related function of UBP43 using ISG15-acti- Fig. 3A. The relative level of HBV replication in UBE1L / mice vating enzyme UBE1L knockout mice (which lack protein ISG- was 98.87 Ϯ 13.87 (%) in comparison to the wild-type mice (Fig. ylation) and the HBV DNA hydrodynamic injection approach. 3B). Although there is a significant difference in ISGylation be- ϩ ϩ Ϫ Ϫ As a preliminary experiment, the effect of hydrodynamic injec- tween UBE1L / and UBE1L / mice (Fig. 2B), the level of tion on ISGylation was checked. C57BL/6 wild-type mice were HBV replication did not show any detectable changes. Therefore, injected with saline and 50 ␮g of either ayw1.0 or ayw1.3 con- it can be concluded that ISGylation does not affect the level of structs. Mice were sacrificed 2 days later and total liver lysate was HBV replication in this system, which implies that the enzyme analyzed by immunoblotting with an anti-mouse ISG15 Ab. Inter- activity of UBP43 has no critical influence on the level of HBV estingly, injection of any DNA construct, regardless of HBV rep- replication. lication competence (Fig. 2A, lanes 3 and 4), results in an induction of ISGylation compared with noninjected mice (Fig. 2A, lane The level of HBV replication is reduced in the absence of 1) or mice injected with an equivalent volume of saline (Fig. 2A, UBP43 lane 2). Furthermore, hydrodynamic injection of the ayw1.3 con- We next examined the effect of the absence of UBP43 on the level struct showed significant differences between UBE1Lϩ/ϩ and of HBV replication. UBP43ϩ/ϩ or UBP43Ϫ/Ϫ mice were hydro- UBE1LϪ/Ϫ mice in their ISGylation without any other treatment to dynamically injected with 20 ␮g of ayw1.3 construct and analyzed induce the IFN response (Fig. 2B). as described above. We had eight comparable pairs from four in- To check the effect of ISGylation on HBV replication, dependent injections, and four representative Southern blot results UBE1Lϩ/ϩ or UBE1LϪ/Ϫ mice were hydrodynamically injected are shown (Fig. 4A). The relative level of HBV replication in 6470 UBP43 AFFECTS THE LEVEL OF HBV REPLICATION Downloaded from

FIGURE 4. The level of HBV replication is inhibited in the absence of UBP43. A, UBP43ϩ/ϩ (lanes 1, 3, 5, and 7) or UBP43Ϫ/Ϫ (lanes 2, 4, 6, and 8) mice were injected with the ayw 1.3 construct, and total DNA was analyzed by Southern blotting with an HBV probe. Relative replication was measured as for Fig. 3 and indicated as a percentage below each lane. Four FIGURE 5. Coinjection of UBP43 shRNA with ayw 1.3 results in the http://www.jimmunol.org/ representative experiments are shown in the ﬁgure. B, The relative replication inhibition of HBV replication. A, Activity of shRNA. 293T cells were is summarized as a graph. The average HBV replication in UBP43 Ϫ/Ϫ mice cotransfected with V5-tagged mouse UBP43 and control or UBP43 shRNA was 4.01 Ϯ 2.69 (%) (n ϭ 8 pairs) of that in UBP43ϩ/ϩ mice. C, Levels of constructs. B, Coinjection of UBP43 shRNA expression constructs with Ϫ Ϫ ␮ ␮ IFN-inducible genes in UBP43 / mice. Total RNA from the liver was iso- ayw 1.3. 10 g of ayw 1.3 construct, and 10 g of shRNA constructs were lated and analyzed by Northern blotting with radiolabeled HBV (top panel), coinjected into 129sv mouse tail veins hydrodynamically. Total DNA of Cxcl9, Gbp1, IRF1, IRF7, and Mx2 probes as indicated. Equal RNA loading mouse livers were analyzed by Southern blotting as described above. was conﬁrmed by the relative amount of 18S and 28S rRNA (bottom panel).

results in a clear decrease of UBP43 levels (lane 2) compared with by guest on October 2, 2021 UBP43Ϫ/Ϫ from eight comparable pairs was 4.01 Ϯ 2.69 (%) com- that of a nonspecific control shRNA (lane 1). pared with UBP43ϩ/ϩ (Fig. 4B). In conclusion, the level of HBV The shRNAs expression vectors were coinjected with the ayw replication was reduced significantly in the absence of UBP43. 1.3 construct hydrodynamically into the tail vein of wild-type We observed previously that the lack of UBP43 results in en- mice. Mouse livers were taken 4 days after injection and total hanced and prolonged STAT1 phosphorylation (25) and increased DNA was isolated. The level of HBV replication was analyzed by induction of hundreds of ISG (26). More recently, we reported that Southern blotting. As shown in Fig. 5B, coinjection of UBP43 UBP43 specifically binds to the IFNAR2 subunit and inhibits the shRNA results in a significant decrease of HBV replication levels activity of receptor-associated JAK1 by blocking the interaction compared with that of control shRNA. between JAK and IFN receptor (28). Considering that the level of HBV replication was not affected by ISGylation (Figs. 2 and 3), Discussion this result indicates that the isopeptidase-independent activity of In UBP43 knockout mice, the steady-state level of HBV DNA was UBP43 affects the level of HBV replication. As further proof of the reduced significantly, and this result is consistent with previous effect of UBP43 on HBV, total liver RNA was analyzed by North- reports that loss of UBP43 in mice results in greater resistance to ern blotting with Cxcl9, Gbp1, IRF1, IRF7, and Mx2 probes. As viral challenge (14). It was reported previously that UBP43-defi- shown in Fig. 4C, message levels of these five IFN-inducible genes cient cells were hypersensitive to type I IFN, indicating its nega- Ϫ Ϫ were higher in UBP43 / . Furthermore, the level of HBV major tive regulatory role on IFN signaling (25). Because UBP43 is an transcripts (3.5, 2.3/2.1 kb RNA) was significantly lower in ISG15-specific protease that removes ISG15 from modified target Ϫ Ϫ UBP43 / (Fig. 4C top panel). These results suggested that in- proteins (23), it was thought that the negative regulation of JAK/ Ϫ Ϫ creased induction of ISG in UBP43 / was responsible for the STAT signaling might be achieved through controlling ISG15 con- inhibition of the steady-state levels of HBV DNA. jugation levels. However, loss of ISGylation (UBE1LϪ/Ϫ) did not affect the level of HBV replication. Together with a previous re- Coinjection of shRNA against mouse UBP43 results in reduction port (28), our results strongly suggest that UBP43 negatively reg- of HBV replication levels ulates JAK/STAT signaling by a mechanism independent of its To confirm further whether the absence of UBP43 reduces the isopeptidase activity. To further elucidate the enzyme activity-in- level of HBV replication, and to address the therapeutic potential dependent function of UBP43, we are in the process of generating of manipulating UBP43 levels for the treatment of HBV infection, mice that only express an isopeptidase inactive form of UBP43 via shRNA constructs against mouse UBP43 were generated. The ef- knockin strategy. By comparing wild-type and UBP43-mutant fective inhibition of mouse UBP43 levels was confirmed by co- mice, the two important roles of UBP43 will be further clarified. transfecting the shRNAs with V5-tagged mouse UBP43 in 293T It was reported that ISG15 knockout mice had increased sus- cells. As shown in Fig. 5A, specific UBP43 shRNA expression ceptibility to influenza, herpes, and Sindbis viruses (36). Similar to The Journal of Immunology 6471

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ISG15 modification of the eIF4E in vivo data of our current report, these results provide strong cognate 4EHP enhances cap structure-binding activity of 4EHP. Genes Dev. 21: evidence that UBP43 shRNA as a therapeutic tool can essen- 255–260. tially be applied to every viral or bacterial infection that is 23. Malakhov, M. P., O. A. Malakhova, K. I. Kim, K. J. Ritchie, and D. E. Zhang. 2002. UBP43 (USP18) specifically removes ISG15 from conjugated proteins. known to be sensitive to IFN treatment. J. Biol. Chem. 277: 9976–9981. Hydrodynamic injection of HBV DNA is a mouse model of 24. Ritchie, K. J., M. P. Malakhov, C. J. Hetherington, L. Zhou, M. T. Little, acute HBV infection. Although the current data indicate the O. A. Malakhova, J. C. Sipe, S. H. Orkin, and D. E. Zhang. 2002. Dysregulation of protein modification by ISG15 results in brain cell injury. 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