fact, while it is remarkable that desensitization can be achieved by constitutive USP18 expression in na¨ıve cells, it is also conceivable that the native protein acquires distinctive biochemical properties - in terms of stability, partners and/or substrates - when it gradually accumulates in IFN-stimulated cells, along with the ISGylation manchinery. On that line, additional work is required to understand the functional link between USP18, ISG15 and the ISGylation machinery in desensitization. Interestingly, several features of the ISG15 system are closely related to the ubiquitin system [20]. Notably, ISG15 is the only ubiquitin-like molecule whose C-terminal residues (LRLRGG) are identical to those of ubiquitin. These similarities suggest functional or regulatory overlap between the two pathways and indeed, in vitro, murine USP18 can remove ubiquitin from substrates [21,22]. From in vitro studies, it is known that the assembly of the IFN a2- receptor complex on artificial membranes is conditioned by the IFNAR1 concentration, whereas IFN b recruits IFNAR1 even if present at very low concentration [6]. Indeed, in cells the surface level of IFNAR1 is critical for the intensity of IFN a signaling [5,23,24]. Importantly and in accordance with other reports [13,25], we found that the presence of USP18 has no effect on the global cell surface level of IFNAR2 and IFNAR1. Nonetheless, our study shows that binding of IFN a is impaired in cells expressing USP18, whether IFN-primed or USP18-transfected. Overall, we favor a model whereby the interaction of USP18 with IFNAR2 ([13] and our data) may lead to a re-organization of the architecture of the type I IFN receptor. A change in lateral mobility of the receptor chains, in their localisation in membrane microdomains or their physical preassociation could weaken assembling and signaling of IFN a2. Conversely, owing to its higher affinity for the receptor, IFN b would retain activity on USP18-expressing cells. It is remarkable that, at least in humans, the 13 a IFNs exhibit non-optimal affinity to the receptor chains and it is precisely this weakness that allows a/b differential bioactivities and desensitiza- tion [6]. Thus, in a viral infection, abundant IFN a is likely to be induced from the multiple genes and limits the spread of the virus by exerting potent antiviral action in a timely regulated mode on cells that will then be desensitized. On the other hand, the single IFN b - that is induced alone or, in response to viral infection, co-induced with IFN a [26] - is optimized to bind the receptor chains with high affinity and retains activity on cells desensitized for IFN a. This exclusive property of IFN b may be critical for the stimulation of adaptive immune responses necessary to eradicate the virus. Type I IFNs and type III IFNs are induced by similar stimuli, exhibit common bioactivities and synergyze in antiviral activity towards several viruses, including HCV [27,28,29]. Their functional overlap was somehow expected given the activation, through different receptors, of the same transcriptional factor ISGF3 [30]. This is the first report of an inhibitory effect exerted by IFN l upon IFN a activities. One particular context where cellular desensitization to IFN a could be relevant is the therapeutical control of chronic HCV infection. The current standard therapy is pegylated-IFN a2 and ribavirin, whose success is influenced by the virus genotype and multiple host factors. Among the strongest predictive factors of treatment outcome is the expression level of ISGs in liver tissue. Indeed, high baseline ISG expression in hepatocytes has been consistently associated with poor response to therapy [14,15,31,32,33]. Intrahepatic differenc- es in ISG expression may reflect differences in host innate antiviral responses before and/or during the chronic phase. The ISG «signature» is likely to be driven and maintained by local innate cytokines, such as IFNs, and may ultimately result in failure to respond to therapeutic IFN. Differential Desensitization to Type I Interferons Interestingly, USP18 is a component of the gene signature predictive of poor treatment response [15,32,33]. Moreover, the knockdown of USP18 in hepatoma cells was shown to potentiate the anti-HCV effect of IFN a [11]. Here, we provide evidence that primary human hepatocytes respond to IFN l and, when primed with it, they express USP18 and become desensitized to IFN a2. Thus, it is tempting to speculate that this USP18-mediated refractoriness to IFN a could contribute, at least in part, to lower the effectiveness of an IFN a-based therapy. In that event, IFN b or l would represent alternative therapeutic approaches. Another strong predictive factor of successful treatment of chronically HCV infected patients (and spontaneous viral clearance) is the IFN l3 (IL28B) genotype. Paradoxically, the good response IFN l variant, ie predicting higher success rate of IFN a-based therapy, was found to be associated with higher viral load [34,35,36,37]. These consistent observations have spurred intensive studies to try to relate the IFN l3 (IL28B) genotype with the level of hepatic ISG[33,38,39,40]. To date contradictory conclusions have been reported that do not yet provide a clear picture. Likewise, we are still missing consistent analyses of which of the variants, if any, alters expression level and/or potency of IFN l. In view of these and our present data, one can speculate that a patient with the hapless genotype may induce IFN l inappropri- ately (e.g. altered level, potency or timing) upon HCV infection. On the one hand, this will lower the viral load without however clearing the virus and, on the other hand, will maintain a high level of ISGs, including USP18. Sustained level of USP18 may contribute, at least in part, to desensitize liver cells to administered IFN a. Materials and Methods Cells HLLR1-1.4 cells are described in [16]. HLLR1-1.4 and derived clones were cultured in DMEM with 10% fetal calf serum (FCS), hypoxanthine, thymidine and aminopterin (HAT) and 400 mg/ml G418. To obtain HU and HUS clones, HLLR1-1.4 cells were co- transfected with pSVpuro and pMet7 empty vector or pMet7 encoding USP18 using FuGENE6 (Roche Applied Science). Colonies selected in 0.4 mg/ml puromycin were analysed by immunoblot for USP18 level as compared to primed HLLR1-1.4 cells. Primary hepatocytes were isolated as described [41] from a human liver sample obtained from a 51 y-o female with intrahepatic lithiasis. The French National Ethics Committee has authorized the use of these samples for research. The patient was free of any HCV, HBV and HIV markers at the time of surgery. Hepatocytes were plated at confluence in a 12-well plates at 106 cell/well precoated with collagen in culture medium consisting of Williams’ E and Ham’s F-12 (Sigma) (1/1 in volume). For the first 24 h, 5% FCS (Gibco) was added to favor cell attachment. The standard medium was then replaced with 1 ml of serum-free medium as described [41]. Cultures were incubated at 37uC and 5% CO2. Plasmids and reagents USP18 cDNA was cloned by PCR using as template the cDNA prepared from HLLR1-1.4 cells stimulated with IFN b-treated for 6 hr and as primers: forward 59TTTGATATCCTGGGGGTT- TTGGAGTGA39 and reverse 59TAGACCGGTCTGAAGG- TTTTGGGCATTTC 39. The PCR product was subcloned in pMET7 vector. The catalytic activity of USP18 was assessed by comparing the global protein ISGylation level in 293T cells transiently transfected with ISG15, E1, E2 and E3 enzymes of the PLoS ONE | www.plosone.org 10 July 2011 | Volume 6 | Issue 7 | e22200