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The Journal of Immunology, 2008, 181, 6467 -6472
Copyright © 2008 by The American Association of Immunologists, Inc.
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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 Kim2,*, Jiann-Kae Luo3,* and Dong-Er Zhang4,*,{dagger}

* Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037; and {dagger} Department of Pathology, Division of Biology, and Moores University of California San Diego Cancer Center, University of California San Diego, La Jolla, CA 92093


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Hepatitis B virus (HBV)5 causes both acute and chronic infection of the human liver. Although a vaccine is available, hepatitis B remains a major health problem in many countries. The number of HBV carriers worldwide is estimated to be 400 million. HBV is an enveloped, partially double stranded DNA virus. The replication of the viral genome occurs through an RNA intermediate that requires its own reverse transcriptase activity (1). Chronic HBV infection can lead to the development of liver cirrhosis and hepatocellular carcinoma (2, 3). Although HBV infection itself is not cytopathic to the hepatocyte, hepatitis is thought to result from the immune response to the viral infection.

Host response during the early phases of viral infections are characterized by the production of type I IFN (IFN-{alpha}/β) and activation of NK cells (4). HBV replication can be efficiently inhibited by IFN treatment (5, 6, 7). IFN regulate diverse biological functions, including induction of the antiviral response, inhibition of cell proliferation, and immunomodulatory activities (8, 9, 10, 11, 12). IFN-{alpha}/β stimulation leads to the up-regulation of IFN-stimulated genes (ISG). One of these ISG is ISG15, an ubiquitin-like protein that conjugates to cellular substrates to form ISGylated proteins (13, 14). The conjugation is executed by an enzymatic cascade that includes an E1-activating enzyme (UBE1L, also known as UBA7; Ref. 15), an E2-conjugating enzyme (UbcH8; Refs. 16 , 17), and E3 ligases (18, 19, 20, 21, 22). The conjugation can be reversed by ubiquitin protease 43 (UBP43, also known as USP18), which is an IFN-inducible cysteine protease (23). As expected, UBP43-deficient cells show high levels of ISG15-modified proteins (24). Importantly, UBP43-deficient cells are hypersensitive to type I IFN and undergo apoptosis upon IFN stimulation (25). Furthermore, lack of UBP43 results in enhanced and prolonged STAT1 phosphorylation 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 viruses including lymphocytic choriomeningitis virus (LCMV), vesicular stomatitis virus (VSV), and Sindbis virus (27). Interestingly, UBP43 has two independent roles in the innate immune response. First, the isopeptidase activity of UBP43 functions to remove ISG15 from ISG15-conjugated proteins (23). Second, independent of its role in protein ISGylation, UBP43 competes with JAK1 for binding to the IFNAR2 subunit of the IFN receptor and inhibits IFN-induced JAK/STAT signal transduction (28).

HBV infection is restricted to humans and chimpanzees, and the lack of a small animal model hampers the understanding of HBV biology. Although several lines of transgenic mice have been established, these may not fully represent the natural course of infection because the production of HBV comes from an integrated viral genome and the mice are immunologically tolerant to viral Ags (29, 30). Recently, it was found that i.v. injection of plasmid DNA with acute circulatory overload leads to high levels of gene expression in mouse liver (31, 32). A mouse model of acute HBV infection was established using this hydrodynamics-based gene delivery system (33). To evaluate ISG15 conjugation-dependent and -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, UBE1L–/– mice that lack ISG15 conjugation upon IFN stimulation, and UBP43–/– 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, 36, 37), ISGylation 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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Plasmid constructs

pSP65-ayw1.3 containing an over-length copy of HBV was provided by Dr. F. V. Chisari (The Scripps Research Institute, La Jolla, CA). The 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 of the pGEM-7Zf(–) vector (Promega; see Fig. 1A). Control shRNA and mouse-specific shRNA were generated with minor modifications from previously established protocols (38). In brief, primers 5'-gggagatctc aaggtcgggc aggaagaggg cctatttcc-3' and 5'-ggggaattca aaaagacctc ggtgatacca aggtctcttg aactttggta tcaccgaggt cggtgtttcg tcctttccac aagatatata a-3' (for control shRNA) and primers 5'-gggagatctc aaggtcgggc aggaagaggg cctatttcc-3' and 5'-ggggaattca aaaaggacca gatcacggac acatctcttg aatgtgtccg tgatctggtc cggtgtttcg tcctttccac aagatatata a-3' (for UBP43 shRNA12) were 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 (–) (Stratagene). The insert was then excised by a two-step digestion, first with XbaI and blunted with T4 DNA polymerase, then with EcoRI. The excised construct was then ligated into the HpaI/EcoRI site of pMSCVpuro (BD Clontech) and confirmed by sequencing. Plasmid DNAs were prepared by using an EndoFree plasmid maxi kit (Qiagen) according to manufacturer’s instructions.


Figure 1
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  		FIGURE 1. The level of HBV replication obtained by hydrodynamic injection. A, Schematic diagram of ayw 1.3 and ayw 1.0 constructs. EnhI, enhancer I; Cp, core promoter; pA, poly(A) signal; PS1p, pre-S1 promoter; Sp, S promoter. B, The level of HBV replication from the ayw 1.3 construct. Mice were injected hydrodynamically with 20 µg of the ayw 1.3 construct diluted in 2 ml of saline. Mouse livers were taken 4 days after injection, total DNA was isolated, digested with HindIII restriction enzyme, and analyzed by Southern blotting with 32P-labeled HBV probe. RC, Relaxed circular; SS, single-strand. Numbers on the left show DNA m.w. size markers in kilobases.

 
In vivo studies

The generation of UBP43–/– and UBE1L–/– mice was described previously (24, 34). All animals used in the studies were handled in accordance with guidelines of The Scripps Research Institute, and procedures were approved by the Institutional Animal Care and Use Committee of the institute. The genetic background of mice used in this study was C57BL/6, 129Sv, FVB, or mixed background of C57BL/6 and 129Sv. The pair comparison is always with mice in the same strain background.

Hydrodynamic injection of naked plasmid DNA

Twenty micrograms of plasmid DNA was diluted with 2.0 ml of saline and injected via the tail vein of 6–9-wk-old mice within 10 s. The livers of the mice were dissected into pieces and frozen immediately in liquid nitrogen 4 days after injection.

Analysis of HBV replication by Southern blotting

A piece of liver tissue was digested overnight in lysis buffer (50 mM 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 extracted. Twenty micrograms of DNA was digested with HindIII, electrophoresed in a 1% agarose gel, and transferred to nylon membrane (Hybond-N; Amersham Biosciences). After UV cross-linking, the membrane was hybridized with a 32P-labeled HBV DNA probe generated by the 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 exposing on an x-ray film, scanned using the VersaDoc Imaging system (Bio-Rad), and the density was quantified using Quantity One software (Bio-Rad).

Northern blotting

Total RNA from liver was isolated using RNA-Bee (Tel-Test) reagent, resolved in a formaldehyde agarose gel, and transferred to nylon membrane (Hybond-N, Amersham Biosciences). After UV-cross-linking, the membrane was hybridized with 32P-labeled DNA probe generated by the Prime-It II Random Primer Labeling Kit (Stratagene) using partial cDNA fragments of IFN-inducible genes as the template. The partial cDNA fragments were obtained by PCR amplification using a cDNA library from IFN-treated mouse bone marrow macrophages as a template. Primers 5'-atggcctcagagatccacat-3' and 5'-ccattgactgtgatgcctcc-3' for Gbp1, 5'-atgccaatca ctcgaatgcg-3' and 5'-ctatggtgca caaggaatgg-3' for IRF1, 5'-atggctgaag tgaggggggt-3' and 5'-tcaaggccac tgacccaggt-3' for IRF7, 5'-atgaagtccg ctgttctttt-3' and 5'-ttatgtagtc ttccttgaac-3' for Cxcl9, and 5'-agctgaatga gggagaggag-3' and 5'-gaagaactct gaaatgaggg-3' for Mx2 were used in amplification.

Detection of ISGylated proteins

Liver tissue was homogenized in radioimmune precipitation assay buffer (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% discontinuous gradient SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose membrane (Hybond-ECL; Amersham Biosciences) and detected with rabbit anti-mouse ISG15 polyclonal Ab as described previously (39).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Replication of HBV in hydrodynamically injected mice

An ayw 1.3 super genomic DNA construct that has been shown to support HBV gene expression and replication in the livers of transgenic mice (30) was injected i.v. into mice. At day 4 postinfection, mice were sacrificed and total genomic DNA from liver tissue was analyzed by Southern blotting with a 32P-labeled HBV DNA probe after HindIII digestion. As a negative control, a construct containing one copy of the HBV genome (ayw 1.0), which cannot provide the appropriate transcripts of HBV, was used (Fig. 1A). As shown in Fig. 1B, only the DNA sample injected with ayw 1.3, but not with ayw 1.0, showed the conventional HBV replication signal.

ISGylation does not affect the level of HBV replication

IFN are used to treat HBV infections, and IFN stimulate the expression of hundreds of ISG, including ISG15. Previous studies suggested that ISG15 modification plays a role in certain antiviral responses. ISG15 was linked to IFN-mediated inhibition of HIV-1 replication (40) and the NS1B protein of influenza B virus was found to bind free ISG15 and possibly to inhibit its conjugation (15). A more recent study suggests that ISG15 inhibits Sindbis virus infection in mice (35). Furthermore, ISG15 knockout mice showed increased susceptibility to Sindbis, herpes, and influenza viruses (36). However, ISG15- and ISGylation-defective mice did not show any difference in response to VSV and LCMV infection (34, 41). ISG15-deconjugating enzyme UBP43 regulates cellular levels of ISGylated protein via its deconjugating enzyme activity and also functions as a potent inhibitor of type I IFN signaling (23, 28). UBP43 plays an important role during innate immune responses (27, 42, 43). Because HBV is currently one of the major threats to human health, the two different roles of UBP43 in innate immune responses were analyzed for effects on HBV. We first address the ISG15 modification-related function of UBP43 using ISG15-activating enzyme UBE1L knockout mice (which lack protein ISGylation) and the HBV DNA hydrodynamic injection approach.

As a preliminary experiment, the effect of hydrodynamic injection on ISGylation was checked. C57BL/6 wild-type mice were injected with saline and 50 µg of either ayw1.0 or ayw1.3 constructs. Mice were sacrificed 2 days later and total liver lysate was analyzed by immunoblotting with an anti-mouse ISG15 Ab. Interestingly, injection of any DNA construct, regardless of HBV replication competence (Fig. 2A, lanes 3 and 4), results in an induction of ISGylation compared with noninjected mice (Fig. 2A, lane 1) or mice injected with an equivalent volume of saline (Fig. 2A, lane 2). Furthermore, hydrodynamic injection of the ayw1.3 construct showed significant differences between UBE1L+/+ and UBE1L–/– mice in their ISGylation without any other treatment to induce the IFN response (Fig. 2B).


Figure 2
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  		FIGURE 2. Hydrodynamic injection of naked DNA results in induction of ISGylation in the mouse liver. Liver lysate was analyzed 2 days after injection by immunoblotting using anti-mouse ISG15 Ab. A, Wild-type mice injected with ayw 1.0 and ayw 1.3 constructs. NI, Not injected; S, saline injected; 1.0, ayw 1.0; 1.3, ayw 1.3. B, UBE1L+/+ or UBE1L–/– mice were injected with ayw 1.3 construct. Asterisks indicate nonspecific cross-reactive bands. Ponceau staining indicates the relative amount of protein loading.

 
To check the effect of ISGylation on HBV replication, UBE1L+/+ or UBE1L–/– mice were hydrodynamically injected with 20 µg of the ayw1.3 construct. Mice were sacrificed 4 days later and total DNA from liver tissue was analyzed after HindIII digestion by Southern blotting using a 32P-labeled HBV DNA probe. It is known that the transfection efficiency of hepatocytes in the liver by hydrodynamic injection ranges on average from 5 to 10% within a given experiment, depending on the efficiency of the plasmid injection as well as the genetic background, and replication levels can vary by as much as 5-fold between given experiments (33). Due to this variability within and between batches of experiments, measurement of the differences in HBV replication levels between UBE1L+/+ and UBE1L–/– mice was done by comparing, within each batch of experiments, pairs of mice of each genotype that showed similar amounts of input DNA. For each pair of UBE1L+/+ and UBE1L–/– mice, signal intensity of the replication intermediates was divided by that of the input DNA, and the ratio for the UBE1L–/– mouse is expressed as a percentage of that observed in the wild type. Four independent pairs are shown in Fig. 3A. The relative level of HBV replication in UBE1L–/– mice was 98.87 ± 13.87 (%) in comparison to the wild-type mice (Fig. 3B). Although there is a significant difference in ISGylation between UBE1L+/+ and UBE1L–/– mice (Fig. 2B), the level of HBV replication did not show any detectable changes. Therefore, it can be concluded that ISGylation does not affect the level of HBV replication in this system, which implies that the enzyme activity of UBP43 has no critical influence on the level of HBV replication.


Figure 3
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  		FIGURE 3. Absence of UBE1L does not affect the level of HBV replication. A, UBE1L+/+ (lanes 1, 3, 5, and 7) or UBE1L–/– (lanes 2, 4, 6, and 8) mice were injected with the ayw 1.3 construct, and the total DNA was analyzed by Southern blotting with an HBV probe. Relative replication was measured (see Results for details) and indicated as a percentage below each lane. B, The relative replication is summarized as a graph. The average HBV replication in UBE1L–/– mice was 98.87 ± 13.87 (%) (n = 4 pairs) of that in UBE1L+/+ mice.

 
The level of HBV replication is reduced in the absence of UBP43

We next examined the effect of the absence of UBP43 on the level of HBV replication. UBP43+/+ or UBP43–/– mice were hydrodynamically injected with 20 µg of ayw1.3 construct and analyzed as described above. We had eight comparable pairs from four independent injections, and four representative Southern blot results are shown (Fig. 4A). The relative level of HBV replication in UBP43–/– from eight comparable pairs was 4.01 ± 2.69 (%) compared with UBP43+/+ (Fig. 4B). In conclusion, the level of HBV replication was reduced significantly in the absence of UBP43.


Figure 4
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  		FIGURE 4. The level of HBV replication is inhibited in the absence of UBP43. A, UBP43+/+ (lanes 1, 3, 5, and 7) or UBP43–/– (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 representative experiments are shown in the figure. B, The relative replication is summarized as a graph. The average HBV replication in UBP43 –/– mice was 4.01 ± 2.69 (%) (n = 8 pairs) of that in UBP43+/+ mice. C, Levels of IFN-inducible genes in UBP43–/– mice. Total RNA from the liver was isolated and analyzed by Northern blotting with radiolabeled HBV (top panel), Cxcl9, Gbp1, IRF1, IRF7, and Mx2 probes as indicated. Equal RNA loading was confirmed by the relative amount of 18S and 28S rRNA (bottom panel).

 
We observed previously that the lack of UBP43 results in enhanced and prolonged STAT1 phosphorylation (25) and increased induction of hundreds of ISG (26). More recently, we reported that UBP43 specifically binds to the IFNAR2 subunit and inhibits the activity of receptor-associated JAK1 by blocking the interaction between JAK and IFN receptor (28). Considering that the level of HBV replication was not affected by ISGylation (Figs. 2 and 3), this result indicates that the isopeptidase-independent activity of UBP43 affects the level of HBV replication. As further proof of the effect of UBP43 on HBV, total liver RNA was analyzed by Northern blotting with Cxcl9, Gbp1, IRF1, IRF7, and Mx2 probes. As shown in Fig. 4C, message levels of these five IFN-inducible genes were higher in UBP43–/–. Furthermore, the level of HBV major transcripts (3.5, 2.3/2.1 kb RNA) was significantly lower in UBP43–/– (Fig. 4C top panel). These results suggested that increased induction of ISG in UBP43–/– was responsible for the inhibition of the steady-state levels of HBV DNA.

Coinjection of shRNA against mouse UBP43 results in reduction of HBV replication levels

To confirm further whether the absence of UBP43 reduces the level of HBV replication, and to address the therapeutic potential of manipulating UBP43 levels for the treatment of HBV infection, shRNA constructs against mouse UBP43 were generated. The effective inhibition of mouse UBP43 levels was confirmed by cotransfecting the shRNAs with V5-tagged mouse UBP43 in 293T cells. As shown in Fig. 5A, specific UBP43 shRNA expression results in a clear decrease of UBP43 levels (lane 2) compared with that of a nonspecific control shRNA (lane 1).


Figure 5
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  		FIGURE 5. Coinjection of UBP43 shRNA with ayw 1.3 results in the inhibition of HBV replication. A, Activity of shRNA. 293T cells were cotransfected with V5-tagged mouse UBP43 and control or UBP43 shRNA constructs. B, Coinjection of UBP43 shRNA expression constructs with ayw 1.3. 10 µg of ayw 1.3 construct, and 10 µg of shRNA constructs were coinjected into 129sv mouse tail veins hydrodynamically. Total DNA of mouse livers were analyzed by Southern blotting as described above.

 
The shRNAs expression vectors were coinjected with the ayw 1.3 construct hydrodynamically into the tail vein of wild-type mice. Mouse livers were taken 4 days after injection and total DNA was isolated. The level of HBV replication was analyzed by Southern blotting. As shown in Fig. 5B, coinjection of UBP43 shRNA results in a significant decrease of HBV replication levels compared with that of control shRNA.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In UBP43 knockout mice, the steady-state level of HBV DNA was reduced significantly, and this result is consistent with previous reports that loss of UBP43 in mice results in greater resistance to viral challenge (14). It was reported previously that UBP43-deficient cells were hypersensitive to type I IFN, indicating its negative regulatory role on IFN signaling (25). Because UBP43 is an ISG15-specific protease that removes ISG15 from modified target proteins (23), it was thought that the negative regulation of JAK/STAT signaling might be achieved through controlling ISG15 conjugation levels. However, loss of ISGylation (UBE1L–/–) did not affect the level of HBV replication. Together with a previous report (28), our results strongly suggest that UBP43 negatively regulates JAK/STAT signaling by a mechanism independent of its isopeptidase activity. To further elucidate the enzyme activity-independent function of UBP43, we are in the process of generating mice that only express an isopeptidase inactive form of UBP43 via knockin strategy. By comparing wild-type and UBP43-mutant mice, the two important roles of UBP43 will be further clarified.

It was reported that ISG15 knockout mice had increased susceptibility to influenza, herpes, and Sindbis viruses (36). Similar to ISG15 knockout mice, UBE1L knockout mice also had increased susceptibility to influenza B virus infection (D. Lenschow, manuscript in preparation). However, we could not observe any effect on HBV replication in UBE1L knockout mice. This correlates with our recent study that demonstrated that UBE1L knockout mice also exhibited normal antiviral responses against VSV and LCMV infections (34). It seems likely that the antiviral activity of ISG15 or ISGylation might be restricted to specific viruses.

Changes caused by hydrodynamic injection with naked DNA within mice were reported previously (31). Serum biochemistry including major ion concentration (Na+, K+, and Cl–), major protein concentration (albumin and the total protein), and the concentration of liver-specific enzymes including alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase and total bilirubin were checked. All the biochemical parameters evaluated were in the normal range with the exception of alanine aminotransferase on day 1. In this study, we could observe induction of ISGylation as shown in Fig. 2, A and B. These results indicate that hydrodynamic injection of DNA itself, regardless of HBV replication, may induce the IFN response, although further research is required to confirm this.

In this study, by coinjecting shRNA against UBP43 together with ayw 1.3 into wild-type mice, we showed that UBP43 plays an important role in regulating HBV replication. In a stable transfection study, inhibition of UBP43 expression levels by shRNA resulted in prolonged STAT1 activation (phosphorylation) (28). Consistent with this fact, expression of several IFN-inducible genes including Cxcl9, Gbp1, IRF1, IRF7, and Mx2 are up-regulated in the liver tissue of UBP43 KO mice (Fig. 4C). The inhibition of UBP43 expression by shRNA results in hypersensitivity to IFN. In other words, the decrease in UBP43 levels results in a strengthened immune response. As further evidence, it has been recently shown that UBP43 expression is up-regulated in patients that are nonresponsive to IFN treatment (44). Most recently, Randall et al. reported that knockdown of UBP43 was effective in inhibiting HCV replication and viral production in an in vitro cell line model (45). Together with the in vivo data of our current report, these results provide strong evidence that UBP43 shRNA as a therapeutic tool can essentially be applied to every viral or bacterial infection that is known to be sensitive to IFN treatment.

Hydrodynamic injection of HBV DNA is a mouse model of acute HBV infection. Although the current data indicate the potential use of UBP43 shRNA as an anti-HBV therapy, we have not yet provided evidence for effects on chronic HBV infection. Additional studies, such as hydrodynamic injection of UBP43 shRNA into HBV transgenic mice, would be a good approach to address this issue. Furthermore, mouse models for various infectious diseases could be examined with UBP43 shRNA to provide further evidences of the therapeutic value of targeting UBP43 expression.


   Acknowledgments
 
We thank Dr. F. V. Chisari for providing the pSP65-ayw1.3 construct, Drs. A. Boyapati and J. R. Biggs for editing the manuscript, and members of the Zhang laboratory for valuable discussions. This is manuscript 18109 from The Scripps Research Institute.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants HL91549 and GM066955. Back

2 Current address: Department of Surgery, University of Southern California, Los Angeles, CA 90033. Back

3 Current address: Regeneron Pharmaceuticals, Tarrytown, NY 10591. Back

4 Address correspondence and reprint requests to Dr. Dong-Er Zhang, Mail Drop 0815, Room 5328, Moores University of California San Diego Cancer Center, University of California San Diego, 3855 Health Sciences Drive, La Jolla, CA 92093. E-mail address: d7zhang{at}ucsd.edu Back

5 Abbreviations used in this paper: HBV, hepatitis B virus; ISG, IFN-stimulated gene; sh, short hairpin; LCMV, lymphocytic choriomeningitis virus; VSV, vesicular stomatitis virus. Back

Received for publication October 10, 2007. Accepted for publication August 28, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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