hepatitis b virus ( hbv ) is a major cause of liver disease with approximately 400 million people infected all over the world . up to one million deaths caused by liver cirrhosis and primary hepatocellular carcinoma ( hcc ) following chronic hbv infection makes it a serious public health problem . unfortunately , current type i interferon- ( ifn- ) based antiviral therapy has suboptimal antiviral effect [ 2 , 3 ] , indicating that substantial drug resistance mechanism should be explored in order to develop more effective treatment strategies . more than 300 ifn - stimulated genes ( isgs ) can be induced by type i ifns , and these isgs play different roles : direct or indirect antiviral activity , cytoskeletal remodeling , apoptosis induction , posttranscriptional and posttranslational regulation , viral molecules detection , and signaling pathways modulation [ 57 ] . isg15 , a structural homology to ubiquitin , is one of the most abundant ifn - induced isgs . similar to ubiquitin , isg15 can conjugate to target proteins , a process called isgylation , which is catalyzed by e1 activating ( ube1l ) , e2 conjugating ( ubch6 and ubch8 ) , and e3 ligase ( herc5 ) . ubiquitin - like specific protease 18 ( usp18 ) is the specific deconjugating enzyme that removes isg15 from its targets . the antiviral activity of isg15 ( and isgylation ) has been verified in a lot of studies investigating the virus and host interactions in either the isg15/herc5 overexpression / knockdown cells or isg15/usp18 deficient mice ( reviewed in [ 5 , 7 , 10 ] ) . quite interestingly , studies from our group and others demonstrated that increased hepatic baseline ( pretreatment ) expression levels of a subset of isgs , including isg15 and usp18 , were associated with treatment nonresponse to type i ifn - based therapy in both hcv [ 1113 ] and hbv   patients . isgs have been classically known as antiviral , but why increased isgs contribute to treatment failure remains obscure . however , it has been revealed that that isg15 ( and isgylation ) not only stimulated hcv production but also blunted ifn anti - hcv activity either in hcv replicon cells or in cells infected with hepatitis c virus [ 15 , 16 ] . these results indicate that hcv exploits isg15 as a host immune evasion mechanism to facilitate its replication and persistence . in this study , we set out to investigate the effect of isg15 ( and isgylation ) on hbv replication and production . isg15 ( and isgylation ) expression levels were modified by isg15 overexpression plasmid or sirna targeting ube1l . this study provides novel evidence that hbv exploits host innate immunity to facilitate its persistence . as previously described , human full - length isg15 gene was generated using potb7-isg15 plasmid dna ( mgc clones ; open biosystems ) as template , and the resulting pcr product was cloned into a pcdna4/hismax topo ta expression vector ( invitrogen , usa ) . the sequences of the primers are 5-atgggctgggacctgacggtg-3 ( forward ) and 5-ttagctccgcccgccaggctc-3 ( reverse ) , respectively . plasmid maxiprep kit ( qiagen , german ) was used to prepare plasmid dna for transfection . hepg2.2.15 cells with integrated full - length hbv genome were kindly provided by professor bo qin ( chongqing medical university , china ) , as previously described , and were cultured in dulbecco 's modified eagle 's medium ( dmem ) ( gibco , usa ) , supplemented with 10% heat - inactivated fetal bovine serum ( gibco , usa ) , 100  iu  ml penicillin ( gibco , usa ) , 100  iu  ml streptomycin ( gibco , usa ) , and 1  mg  ml g418 ( invitrogen , usa ) in 5% co2 at 37c . twenty - four hours before transfection , hepg2.2.15 cells were seeded in 6-well plates at 6  10 cells per well with antibiotics - free medium . transfection complexes were prepared in 400  l serum - free medium by mixing 4  g ( unless otherwise indicated ) plasmid dna and 12  l polyethyleneimine ( pei , 1  g  l ) and then letting the mixture stand for 15 minutes at room temperature . cells were washed three times with antibiotics - free medium and transfection mix was added into each well containing 2  ml antibiotics - free medium and the culture medium was changed 12 hours later . total rna was prepared using trizol ( invitrogen , usa ) and reverse - transcription was carried out using iscript cdna synthesis kit ( bio - rad , usa ) according to the manufacturer 's protocol . the resulting cdna was amplified by faststart universal sybr green master mix ( roche , usa ) . cycle parameters were 94c , 10  min ; ( 94c , 45  s ; 56c , 45  s ; 72c , 1  min ) 30 cycles ; 72c , 5  min . isg15 and western blot , the process described in detail previously , was also performed for further confirmation of isg15 protein expression and isgylation . sirna targeting human ube1l ( sigenome smartpool , m-019759 - 00 - 0005 ) , negative control sirna ( sigenome non - targeting sirna pool # 2 , d-001910 - 02 - 05 ) , and transfection reagent ( dharmafect 4 , t-2004 - 02 ) were purchased from ge dharmacon , usa . hepg2.2.15 cells were plated in 24-well plates at a density of 1  10 per well overnight . media were changed and the cells were transfected in solution with sirna at a final concentration of 50  nm according to the protocol . twenty - four hours later , 1.4  g per well isg15 plasmid was transfected into the cells . total rna , cellular proteins , and supernatant dna were prepared 48  h after transfection to evaluate the silencing efficiency and hbv production . for hbv production quantification , culture supernatant dna and total intracellular rna were extracted using qiaamp dna blood mini kit ( qiagen , german ) and trizol ( invitrogen , usa ) according to kit manufacturer 's instructions , respectively . real - time pcr was carried out as stated above with the hbv - specific primers ( table 1 ) . intracellular covalently closed circular dna ( cccdna ) was extracted and purified according to a previous report   measured by a real - time pcr described previously . supernatant hbsag and hbeag were detected by enzyme - linked immunosorbent assay ( elisa ) kits ( biosamite , shanghai , china ) following the manufacturer 's protocols . intracellular hbcag expression was assessed by western blot by using a polyclonal anti - hbcag antibody ( boster biological technology , wuhan , china ) and integrated densities were calculated using imagej software . as the antiviral effect of isg15 is mainly attributed to its forming isgylated proteins , we asked whether isg15 conjugation can be increased by ectopic expression of isg15 . as seen in figure 1(a ) , transfection of isg15 led to a pronounced increase in isg15 expression which was further confirmed by western blot ( figure 1(b ) ) . in addition , unlike hela cells , in which isgylation is difficult to be induced , overexpression of isg15 alone increased isgylation ( figure 1(b ) ) in hepg2.2.15 cells . to evaluate the effects of isg15/isgylation on hbv replication and production , we measured the intracellular total hbv dna , covalently closed circular dna ( cccdna ) hbv pgrna levels , and total hbv dna in the supernatant of cell cultures , following isg15 overexpression in hepg2.2.15 by real - time pcr . the results showed that although the intracellular viral cccdna ( figure 2(c ) ) , pgrna ( figure 2(d ) ) , and total hbv dna ( figure 2(e ) ) seemed not affected by isg15 overexpression and isgylation , the supernatant hbv dna was obviously increased in a isg15 dose - dependent manner ( figures 2(a ) and 2(b ) ) . since dead cells , cell debris , and apoptotic bodies may also be responsible for the elevated hbv dna in the medium , we monitored hbv replication by further measuring the supernatant hbsag ( figure 3(a ) ) and hbeag ( figure 3(b ) ) using elisa kits and cellular hbcag ( figure 3(c ) ) using western blot . however , none of these proteins expressions was significantly affected by elevated isg15 and isgylation . though mature form of isg15 conjugation to target proteins was shown to be essential for isg15-dependent antiviral effects , both free isg15 ( intracellular or extracellular ) and isgylation could be detected , indicating potential functions of free isg15 involved in viral infection . we thereby investigated whether free form isg15 or the isgylation was responsible for the increased hbv production . since ube1l catalyzes the adenylation of isg15 , inhibition of ube1l can suppress isg15 conjugation . first of all , we determined the efficiency of ube1l knockdown by real - time pcr . as shown in figure 4(a ) , ube1l mrna expression was significantly inhibited by 50  nm or 100  nm ube1l sirna . ube1l knockdown was further confirmed by western blot for isg15 ( figure 4(b ) ) . compared with the nonknockdown groups ( figure 4(b ) , lanes 4 , 6 , and 7 ) , ube1l - knockdown cells showed lower level of isg15-modified proteins ( figure 4(b ) , lane 3 ) in response to isg15 overexpression , although the levels of free isg15 experienced no apparent difference . ifn2b treated hepg2.2.15 cells were included as positive control to show the expression of isg15 and isgylation ( figure 4(b ) , lane 5 ) . in parallel with ube1l knockdown and consequent downregulation of isgylation , the supernatant hbv dna returned to baseline level ( figure 4(c ) ) . we suppressed ube1l expression in hepg2.2.15 cells without overexpression of isg15 and monitored the hbv production by measuring intracellular total hbv dna , pgrna , and hbcag , as well as hbv dna , hbsag , and hbeag in the culture medium . interestingly , hbv production seemed not affected by ube1l knockdown ( see supplementary figures 1 - 2 in supplementary material available online at http://dx.doi.org/10.1155/2016/7417648 ) . as peg - ifn is one of the efficient agents approved for the treatment of chronic hbv , we then investigated whether overexpression of isg15 boosted hbv production through blocking endogenous type i ifn production or inhibiting activation of type i ifn signaling pathway . our results showed that although elevated isg15/isgylation was parallel with increased expression of isgs such as mxa ( myxovirus resistance 1 ) in hepg2.2.15 cells , other isgs like oas3 ( 2-5-oligoadenylate synthetase 3 ) and usp18 and endogenous expressions of type in addition , hbv production in hepg2.2.15 cells was scarcely affected by short - period ( 4872  h ) ifn2b treatment ( our unpublished data ) . these results suggested that isg15 may be involved in hbv production independent of type i ifn signaling pathway . although isg15 exerts its function in three different forms including free intracellular isg15 , conjugated isg15 , and extracellular isg15 as a cytokine , isgylation is the undoubtedly important one . isg15 is able to conjugate with over 300 cellular proteins [ 2123 ] and many viral proteins { e.g. , influenza a virus ns1 protein   and human papillomavirus ( hpv ) l1 capsid protein } , directly altering the functions of the modified proteins or disrupting the ubiquitination process to prevent degradation of the conjugated proteins . in our study , blocking isgylation abrogated the increased production of hbv , indicating that isgylation is the predominant form of isg15 to boost hbv production in hepg2.2.15 cells . due to this fact , we also checked the antiviral effects of ube1l by suppressing ube1l alone ( without isg15 overexpression ) in hepg2.2.15 cells . unfortunately , no significant anti - hbv effect was observed . since isg15 is an ifn - stimulated gene which is mainly induced by ifn stimulation , the baseline expression of isg15 in hepg2.2.15 is limited . this may help us to explain , at least partly , why knockdown ube1l could not significantly inhibit hbv production in hepg2.2.15 . although isg15/isgylation has been proposed as an efficient host defense response to various viral infections , increasing evidence implied this antiviral mechanism may be virus - specific and isg15/isgylation may be exploited by some viruses to facilitate viral replication and production by either negatively regulating host immunity [ 26 , 27 ] or directly promoting viral replication through blunting ifn antiviral activity [ 15 , 28 ] . in many respects , isgylation / deisgylation process resembles ubiquitination / deubiquitination system which is involved in most physiological and pathological processes , suggesting the potential regulation activity of isg15/isgylation in biology , especially in innate immunity . rna helicase enzymes retinoic acid inducible gene i ( rig - i ) is a pattern recognition receptor ( prr ) which can be activated by viral infection , resulting in endogenous type i ifn expression . kim et al .   demonstrated that isgylation of rig - i can reduce both basal and virus - induced ifn promoter activity at the cellular level in a negative feedback manner . jeon et al .   found that isgylation of filamin b antagonizes the type i ifn - induced jun n - terminal kinase ( jnk ) signaling pathway . filamin b acts as a scaffold that tethers the cascade members in type i ifns activated jnk - specific signaling cascade which is composed of rac1 , mekk1 , mkk4 , and jnk . isg15 conjugation of filamin b leads to the release of rac1 , mekk1 , and mkk4 from the scaffold protein , interdicting the signaling . consequently , it is easy to understand why decreased isgylation results in an increased phenotypic sensitivity to ifn- in several hepatoma derived cell lines or even nonhepatic cell lines [ 16 , 30 ] . our previous work   also indicated that isgylation promotes hcv production partly by decreasing the anti - hcv effect of ifn-. we therefore explored the expressions of type i interferon and ifn pathway activation in hepg2.2.15 cells . consistent with previous studies [ 31 , 32 ] that have demonstrated that isg15 could increase phosphorylation of jak2 , stat1 , and irf-3 , promoting type i ifn signaling pathway , our results also revealed an activated type i ifn signaling characterized by amplified expressions of some isgs . however , no significant change of ifn or ifn expression was observed following isg15 overexpression . although it has been reported   that hbv replication can be significantly suppressed in hepg2.2.15 cells with the 9-day treatment of novel liver - targeting interferon 2b ( ifn - csp ) , our data ( unpublished ) showed that hbv production in hepg2.2.15 cells is not sensitive to short - period ifn2b treatment ( e.g. , 2472  h treatment ) , which indicated that the effects of transient isg15 upregulation ( and isgylation ) on hbv production might be independent of type i ifn . stable transfected hepg2.2.15 cells may be employed to investigate the effect of isg15 ( and isgylation ) on type i ifn signaling pathway in future study . on the other hand , considering the apparent antiviral role of isg15 , it is not surprising that many viruses have developed immune evasion tactics during evolution to avoid isgylation by either inhibiting the formation of conjugation or disrupting the formed modification . as reported by yuan and krug , ns1 of influenza b virus can noncovalently bind to isg15 and inhibit interaction between isg15 and ube1l , thereby inhibiting isgylation . guerra et al .   also observed that vaccinia virus e3 protein can bind to isg15 through its c - terminal domain and inhibit isg15 conjugation . some other viruses , like dugbe virus ( dugv ) and sars coronavirus , can even hydrolyze isg15 from target proteins by their ovarian tumor domain- ( otu- ) containing proteases   or papain - like proteases . moreover , in the present study , it seems that hbv has evolved to make use of isgylation to promote its production , indicating a novel mechanism for hbv persistence . in order to explore the detailed mechanism , we used real - time pcr ( as described in [ 3841 ] ) to test the expression levels of sodium taurocholate cotransporting polypeptide ( ntcp ) , vsp4b , and 78  kda glucose - regulated protein ( grp78 ) , all of which have been identified as essential factors in hbv life cycle [ 42 , 43 ] . however , none of these genes experienced conspicuous changes in mrna expressions following overexpression of isg15 ( data not shown ) . therefore , we hypothesize that , instead of regulation of gene expression , isgylation may primarily alter the function or the stability of cellular or viral proteins involved in hbv production , especially in hbv secretion considering the unaffected intracellular pgrna , cccdna , and total hbv dna . our study reveals the complexity and multiple roles of isgylation , and further work will be needed to determine the precise mechanism , so that therapies targeting these pathways or targets can be developed in the future . more interestingly , the function of isg15 ( and isgylation ) during viral infection seems to be restricted to specific hosts . kim et al .   reported that hbv replication was not affected in isgylation - deficient mice . they constructed a hbv replication model by injecting a replication - competent dna construct hydrodynamically into the mice . although there is a significant difference in isg15 conjugation between ube1l knockout mice and wild mice , hbv replication was not affected . studies of influenza b virus may provide some implication to explain the difference ; although ns1 of influenza b virus can protect virus from isgylation by binding isg15 in human primates , it can not bind to mouse isg15 , indicating the species - specific feature of isg15/isgylation . in conclusion , in this current study we reported an important and novel role of isg15 and isgylation in regulating hbv life cycle in hepg2.2.15 cell model stably expressing hbv . results from this study provide a rational explanation for drug ( especially type i ifn ) resistance and persistent infection of hbv , making isg15 a possible predictor to traditional ifn therapy and a potential antiviral target for hbv treatment .
hepatitis b virus ( hbv ) is an important account of infectious hepatitis and interferon ( ifn ) remains one of the best treatment options . 
 activation of type i ifn signaling pathway leads to expressions of ifn - stimulated genes ( isgs ) which play important roles in antiviral and immunomodulatory responses to hbv or hepatitis c virus ( hcv ) infection . 
 our previous studies indicated that isg15 and its conjugation ( isgylation ) were exploited by hcv to benefit its replication and persistent infection . 
 this study was designed to assess the role of isg15 and isgylation in hbv infection in vitro . 
 the levels of isg15 and isgylation were upregulated by isg15 plasmid transfection into hepg2.2.15 cells . decreased isgylation 
 was achieved by sirna targeting ube1l , the only e1 activating enzyme for isgylation . 
 overexpression of isg15 and subsequent isgylation significantly increased the levels of hbv dna in the culture supernatants although the intracellular viral replication remained unaffected . silencing ube1l , with 
 decreased isgylation achieved , abrogated this isgylation - mediated promoting effect . 
 our data indicated that overexpression of isg15 stimulated hbv production in an isgylation - dependent manner . 
 identification of isg15-conjugated proteins ( either hbv viral or host proteins ) may reveal promising candidates for further antiviral drug development .